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Sample records for intracellular pathogen selective

  1. Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium.

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    Emboulé, Loïc; Daigle, France; Meyer, Damien F; Mari, Bernard; Pinarello, Valérie; Sheikboudou, Christian; Magnone, Virginie; Frutos, Roger; Viari, Alain; Barbry, Pascal; Martinez, Dominique; Lefrançois, Thierry; Vachiéry, Nathalie

    2009-12-24

    Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.

  2. Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium

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    Lefrançois Thierry

    2009-12-01

    Full Text Available Abstract Background Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER, the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. Results We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7. Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. Conclusions We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation

  3. Strategies of Intracellular Pathogens for Obtaining Iron from the Environment

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    Nidia Leon-Sicairos

    2015-01-01

    Full Text Available Most microorganisms are destroyed by the host tissues through processes that usually involve phagocytosis and lysosomal disruption. However, some organisms, called intracellular pathogens, are capable of avoiding destruction by growing inside macrophages or other cells. During infection with intracellular pathogenic microorganisms, the element iron is required by both the host cell and the pathogen that inhabits the host cell. This minireview focuses on how intracellular pathogens use multiple strategies to obtain nutritional iron from the intracellular environment in order to use this element for replication. Additionally, the implications of these mechanisms for iron acquisition in the pathogen-host relationship are discussed.

  4. The Role of Autophagy in Intracellular Pathogen Nutrient Acquisition

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    Shaun eSteele

    2015-06-01

    Full Text Available Following entry into host cells intracellular pathogens must simultaneously evade innate host defense mechanisms and acquire energy and anabolic substrates from the nutrient-limited intracellular environment. Most of the potential intracellular nutrient sources are stored within complex macromolecules that are not immediately accessible by intracellular pathogens. To obtain nutrients for proliferation, intracellular pathogens must compete with the host cell for newly-imported simple nutrients or degrade host nutrient storage structures into their constituent components (fatty acids, carbohydrates and amino acids. It is becoming increasingly evident that intracellular pathogens have evolved a wide variety of strategies to accomplish this task. One recurrent microbial strategy is to exploit host degradative processes that break down host macromolecules into simple nutrients that the microbe can use. Herein we focus on how a subset of bacterial, viral and eukaryotic pathogens leverage the host process of autophagy to acquire nutrients that support their growth within infected cells

  5. Innate host defense against intracellular pathogens

    NARCIS (Netherlands)

    Vaart, Michiel van der

    2013-01-01

    This thesis focuses on the recognition of pathogenic bacteria and the defense mechanisms that are activated during the innate immune response to infection. Detection of pathogens, such as bacteria, viruses, and parasites, depends on receptors that bind to evolutionary conserved structures on their

  6. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms.

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    Pareja, Maria Eugenia Mansilla; Colombo, Maria I

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.

  7. Advances in genetic manipulation of obligate intracellular bacterial pathogens

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    Paul eBeare

    2011-05-01

    Full Text Available Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

  8. Manipulation of costimulatory molecules by intracellular pathogens: veni, vidi, vici!!

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    Nargis Khan

    Full Text Available Some of the most successful pathogens of human, such as Mycobacterium tuberculosis (Mtb, HIV, and Leishmania donovani not only establish chronic infections but also remain a grave global threat. These pathogens have developed innovative strategies to evade immune responses such as antigenic shift and drift, interference with antigen processing/presentation, subversion of phagocytosis, induction of immune regulatory pathways, and manipulation of the costimulatory molecules. Costimulatory molecules expressed on the surface of various cells play a decisive role in the initiation and sustenance of immunity. Exploitation of the "code of conduct" of costimulation pathways provides evolutionary incentive to the pathogens and thereby abates the functioning of the immune system. Here we review how Mtb, HIV, Leishmania sp., and other pathogens manipulate costimulatory molecules to establish chronic infection. Impairment by pathogens in the signaling events delivered by costimulatory molecules may be responsible for defective T-cell responses; consequently organisms grow unhindered in the host cells. This review summarizes the convergent devices that pathogens employ to tune and tame the immune system using costimulatory molecules. Studying host-pathogen interaction in context with costimulatory signals may unveil the molecular mechanism that will help in understanding the survival/death of the pathogens. We emphasize that the very same pathways can potentially be exploited to develop immunotherapeutic strategies to eliminate intracellular pathogens.

  9. Metabolic host responses to infection by intracellular bacterial pathogens

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    Wolfgang eEisenreich

    2013-07-01

    Full Text Available The interaction of bacterial pathogens with mammalian hosts leads to a variety of physiological responses of the interacting partners aimed at an adaptation to the new situation. These responses include multiple metabolic changes in the affected host cells which are most obvious when the pathogen replicates within host cells as in case of intracellular bacterial pathogens. While the pathogen tries to deprive nutrients from the host cell, the host cell in return takes various metabolic countermeasures against the nutrient theft. During this conflicting interaction, the pathogen triggers metabolic host cell responses by means of common cell envelope components and specific virulence-associated factors. These host reactions generally promote replication of the pathogen. There is growing evidence that pathogen-specific factors may interfere in different ways with the complex regulatory network that controls the carbon and nitrogen metabolism of mammalian cells. The host cell defence answers include general metabolic reactions, like the generation of oxygen- and/or nitrogen-reactive species, and more specific measures aimed to prevent access to essential nutrients for the respective pathogen. Accurate results on metabolic host cell responses are often hampered by the use of cancer cell lines that already exhibit various de-regulated reactions in the primary carbon metabolism. Hence, there is an urgent need for cellular models that more closely reflect the in vivo infection conditions. The exact knowledge of the metabolic host cell responses may provide new interesting concepts for antibacterial therapies.

  10. The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens.

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    Voigt, Anja; Schöfl, Gerhard; Saluz, Hans Peter

    2012-01-01

    Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis. A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins. This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions.

  11. The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens.

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    Anja Voigt

    Full Text Available Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins.This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions.

  12. Mechanisms of Defense against Intracellular Pathogens Mediated by Human Macrophages.

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    Bloom, Barry R; Modlin, Robert L

    2016-06-01

    The key question our work has sought to address has been, "What are the necessary and sufficient conditions that engender protection from intracellular pathogens in the human host?" The origins of this work derive from a long-standing interest in the mechanisms of protection against two such paradigmatic intracellular pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, that have brilliantly adapted to the human host. It was obvious that these pathogens, which cause chronic diseases and persist in macrophages, must have acquired subtle strategies to resist host microbicidal mechanisms, yet since the vast majority of individuals infected with M. tuberculosis do not develop disease, there must be some potent human antimicrobial mechanisms. What follows is not a comprehensive review of the vast literature on the role of human macrophages in protection against infectious disease, but a summary of the research in our two laboratories with collaborators that we hope has contributed to some understanding of mechanisms of resistance and pathogenesis. While mouse models revealed some necessary conditions for protection, e.g., innate immunity, Th1 cells and their cytokines, and major histocompatibility complex class I-restricted T cells, here we emphasize multiple antimicrobial mechanisms that exist in human macrophages that differ from those of most experimental animals. Prominent here is the vitamin D-dependent antimicrobial pathway common to human macrophages activated by innate and acquired immune responses, mediated by antimicrobial peptides, e.g., cathelicidin, through an interleukin-15- and interleukin-32-dependent common pathway that is necessary for macrophage killing of M. tuberculosis in vitro.

  13. Superdiffusion dominates intracellular particle motion in the supercrowded cytoplasm of pathogenic Acanthamoeba castellanii

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    Reverey, Julia F.; Jeon, Jae-Hyung; Bao, Han; Leippe, Matthias; Metzler, Ralf; Selhuber-Unkel, Christine

    2015-06-01

    Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements, and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin, and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved by actively driven motion, while purely thermally driven diffusion is negligible.

  14. Host-directed antimicrobial drugs with broad-spectrum efficacy against intracellular bacterial pathogens.

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    Czyż, Daniel M; Potluri, Lakshmi-Prasad; Jain-Gupta, Neeta; Riley, Sean P; Martinez, Juan J; Steck, Theodore L; Crosson, Sean; Shuman, Howard A; Gabay, Joëlle E

    2014-07-29

    We sought a new approach to treating infections by intracellular bacteria, namely, by altering host cell functions that support their growth. We screened a library of 640 Food and Drug Administration (FDA)-approved compounds for agents that render THP-1 cells resistant to infection by four intracellular pathogens. We identified numerous drugs that are not antibiotics but were highly effective in inhibiting intracellular bacterial growth with limited toxicity to host cells. These compounds are likely to target three kinds of host functions: (i) G protein-coupled receptors, (ii) intracellular calcium signals, and (iii) membrane cholesterol distribution. The compounds that targeted G protein receptor signaling and calcium fluxes broadly inhibited Coxiella burnetii, Legionella pneumophila, Brucella abortus, and Rickettsia conorii, while those directed against cholesterol traffic strongly attenuated the intracellular growth of C. burnetii and L. pneumophila. These pathways probably support intracellular pathogen growth so that drugs that perturb them may be therapeutic candidates. Combining host- and pathogen-directed treatments is a strategy to decrease the emergence of drug-resistant intracellular bacterial pathogens. Importance: Although antibiotic treatment is often successful, it is becoming clear that alternatives to conventional pathogen-directed therapy must be developed in the face of increasing antibiotic resistance. Moreover, the costs and timing associated with the development of novel antimicrobials make repurposed FDA-approved drugs attractive host-targeted therapeutics. This paper describes a novel approach of identifying such host-targeted therapeutics against intracellular bacterial pathogens. We identified several FDA-approved drugs that inhibit the growth of intracellular bacteria, thereby implicating host intracellular pathways presumably utilized by bacteria during infection. Copyright © 2014 Czyż et al.

  15. Modeling the intracellular pathogen-immune interaction with cure rate

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    Dubey, Balram; Dubey, Preeti; Dubey, Uma S.

    2016-09-01

    Many common and emergent infectious diseases like Influenza, SARS, Hepatitis, Ebola etc. are caused by viral pathogens. These infections can be controlled or prevented by understanding the dynamics of pathogen-immune interaction in vivo. In this paper, interaction of pathogens with uninfected and infected cells in presence or absence of immune response are considered in four different cases. In the first case, the model considers the saturated nonlinear infection rate and linear cure rate without absorption of pathogens into uninfected cells and without immune response. The next model considers the effect of absorption of pathogens into uninfected cells while all other terms are same as in the first case. The third model incorporates innate immune response, humoral immune response and Cytotoxic T lymphocytes (CTL) mediated immune response with cure rate and without absorption of pathogens into uninfected cells. The last model is an extension of the third model in which the effect of absorption of pathogens into uninfected cells has been considered. Positivity and boundedness of solutions are established to ensure the well-posedness of the problem. It has been found that all the four models have two equilibria, namely, pathogen-free equilibrium point and pathogen-present equilibrium point. In each case, stability analysis of each equilibrium point is investigated. Pathogen-free equilibrium is globally asymptotically stable when basic reproduction number is less or equal to unity. This implies that control or prevention of infection is independent of initial concentration of uninfected cells, infected cells, pathogens and immune responses in the body. The proposed models show that introduction of immune response and cure rate strongly affects the stability behavior of the system. Further, on computing basic reproduction number, it has been found to be minimum for the fourth model vis-a-vis other models. The analytical findings of each model have been exemplified by

  16. Intracellular phase for an extracellular bacterial pathogen: MgtC shows the way

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    Audrey Bernut

    2015-08-01

    Full Text Available Pseudomonas aeruginosa is an extracellular pathogen known to impair host phagocytic functions. However, our recent results identify MgtC as a novel actor in P. aeruginosa virulence, which plays a role in an intramacrophage phase of this pathogen. In agreement with its intracellular function, P. aeruginosa mgtC gene expression is strongly induced when the bacteria reside within macrophages. MgtC was previously known as a horizontally-acquired virulence factor important for multiplication inside macrophages in several intracellular bacterial pathogens. MgtC thus provides a singular example of a virulence determinant that subverts macrophages both in intracellular and extracellular pathogens. Moreover, we demonstrate that P. aeru-ginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosaMgtC prevents biofilm formation. We propose that MgtC has a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to the host in relation to the different bacterial lifestyles. MgtC thus appears as an attractive target for antivirulence strategies and our work provides a natural peptide as MgtC antagonist, which paves the way for the development of MgtC inhibitors.

  17. Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

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    Bouwer, H. G. Archie; Alberti-Segui, Christine; Montfort, Megan J.; Berkowitz, Nathan D.; Higgins, Darren E.

    2006-03-01

    We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8+ effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8+ effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens. CD8+ T cell | replication-deficient | Listeria monocytogenes

  18. Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

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    Mellouk, Nora; Enninga, Jost

    2016-01-01

    Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

  19. Cytosolic access of intracellular bacterial pathogens: the Shigella paradigm

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    Nora eMellouk

    2016-04-01

    Full Text Available Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

  20. Host-Pathogen Checkpoints and Population Bottlenecks in Persistent and Intracellular Uropathogenic E. coli Bladder Infection

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    Hannan, Thomas J.; Totsika, Makrina; Mansfield, Kylie J.; Moore, Kate H.; Schembri, Mark A.; Hultgren, Scott J.

    2013-01-01

    Bladder infections affect millions of people yearly, and recurrent symptomatic infections (cystitis) are very common. The rapid increase in infections caused by multi-drug resistant uropathogens threatens to make recurrent cystitis an increasingly troubling public health concern. Uropathogenic E. coli (UPEC) cause the vast majority of bladder infections. Upon entry into the lower urinary tract, UPEC face obstacles to colonization that constitute population bottlenecks, reducing diversity and selecting for fit clones. A critical mucosal barrier to bladder infection is the epithelium (urothelium). UPEC bypass this barrier when they invade urothelial cells and form intracellular bacterial communities (IBCs), a process which requires type 1 pili. IBCs are transient in nature, occurring primarily during acute infection. Chronic bladder infection is common and can be either latent, in the form of the Quiescent Intracellular Reservoir (QIR), or active, in the form of asymptomatic bacteriuria (ASB/ABU) or chronic cystitis. In mice, the fate of bladder infection: QIR, ASB, or chronic cystitis, is determined within the first 24 hours of infection and constitutes a putative host-pathogen mucosal checkpoint that contributes to susceptibility to recurrent cystitis. Knowledge of these checkpoints and bottlenecks is critical for our understanding of bladder infection and efforts to devise novel therapeutic strategies. PMID:22404313

  1. Hijacking host cell highways: manipulation of the host actin cytoskeleton by obligate intracellular bacterial pathogens

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    Punsiri M Colonne

    2016-09-01

    Full Text Available Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, subversion of cell intrinsic immunity, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions.

  2. Outrunning the Red Queen: bystander activation as a means of outpacing innate immune subversion by intracellular pathogens.

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    Holmgren, Alicia M; McConkey, Cameron A; Shin, Sunny

    2017-01-01

    Originally described by the late evolutionary biologist Leigh Van Valen, the Red Queen hypothesis posits that the evolutionary arms race between hosts and their pathogens selects for discrete, genetically encoded events that lead to competitive advantages over the other species. Examples of immune evasion strategies are seen throughout the co-evolution of the mammalian immune system and pathogens, such as the enzymatic inactivation of nuclear factor-κB signaling or host translation by pathogen-encoded virulence factors. Such immunoevasive maneuvers would be expected to select for the evolution of innate immune counterstrategies. Recent advances in our understanding of host immunity and microbial pathogenesis have provided insight into a particular innate immune adaptation, termed bystander activation. Bystander activation occurs as a consequence of infected cells alerting and instructing neighboring uninfected cells to produce inflammatory mediators, either through direct cell contact or paracrine signals. Thus, bystander activation can allow the immune system to overcome the ability of pathogens to disarm immune signaling in directly infected cells. This review presents an overview of the general hallmarks of bystander activation and their emerging role in innate immunity to intracellular pathogens, as well as examples of recent mechanistic discoveries relating to the bystander activation during infection with specific pathogens relevant to human health and disease.

  3. Thioredoxin 80-Activated-Monocytes (TAMs) Inhibit the Replication of Intracellular Pathogens

    DEFF Research Database (Denmark)

    Cortes-Bratti, Ximena; Brasseres, Eugenie; Herrera-Rodriquez, Fabiola

    2011-01-01

    for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L....... monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria...

  4. The role of phagocytes and specific antibodies in gamma irradiated mice infected by intracellular bacterial pathogens

    International Nuclear Information System (INIS)

    Kovarova, H.; Stulik, J.; Ledvina, M.

    1987-01-01

    The activation of oxygen metabolism in peritoneal macrophages during the defence against Francisella tularensis infection was inhibited by gamma irradiation of mice with 4.0 Gy. The application of specific antibodies protected the irradiated mice from the lethal infection without reactivation of oxygen metabolism in mononuclear phagocytes. These results demonstrated that the protecting function of the specific antibodies in the defence system against intracellular bacterial pathogens will be mediated by the oxygen-independent mechanisms. (author)

  5. ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

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    Wu, Yu; Pons, Valérie; Goudet, Amélie; Panigai, Laetitia; Fischer, Annette; Herweg, Jo-Ana; Kali, Sabrina; Davey, Robert A; Laporte, Jérôme; Bouclier, Céline; Yousfi, Rahima; Aubenque, Céline; Merer, Goulven; Gobbo, Emilie; Lopez, Roman; Gillet, Cynthia; Cojean, Sandrine; Popoff, Michel R; Clayette, Pascal; Le Grand, Roger; Boulogne, Claire; Tordo, Noël; Lemichez, Emmanuel; Loiseau, Philippe M; Rudel, Thomas; Sauvaire, Didier; Cintrat, Jean-Christophe; Gillet, Daniel; Barbier, Julien

    2017-11-14

    Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

  6. Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum.

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    Jose C Garcia-Garcia

    2009-06-01

    Full Text Available Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1 expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.

  7. Breaking fat! How mycobacteria and other intracellular pathogens manipulate host lipid droplets.

    Science.gov (United States)

    Barisch, Caroline; Soldati, Thierry

    2017-10-01

    Tuberculosis (Tb) is a lung infection caused by Mycobacterium tuberculosis (Mtb). With one third of the world population latently infected, it represents the most prevalent bacterial infectious diseases worldwide. Typically, persistence is linked to so-called "dormant" slow-growing bacteria, which have a low metabolic rate and a reduced response to antibiotic treatments. However, dormant bacteria regain growth and virulence when the immune system is weakened, leading again to the active form of the disease. Fatty acids (FAs) released from host triacylglycerols (TAGs) and sterols are proposed to serve as sole carbon sources during infection. The metabolism of FAs requires beta-oxidation as well as gluconeogenesis and the glyoxylate shunt. Interestingly, the Mtb genome encodes more than hundred proteins involved in the five reactions of beta-oxidation, clearly demonstrating the importance of lipids as energy source. FAs have also been proposed to play a role during resuscitation, the resumption of replicative activities from dormancy. Lipid droplets (LDs) are energy and carbon reservoirs and have been described in all domains. TAGs and sterol esters (SEs) are stored in their hydrophobic core, surrounded by a phospholipid monolayer. Importantly, host LDs have been described as crucial for several intracellular bacterial pathogens and viruses and specifically translocate to the pathogen-containing vacuole (PVC) during mycobacteria infection. FAs released from host LDs are used by the pathogen as energy source and as building blocks for membrane synthesis. Despite their essential role, the mechanisms by which pathogenic mycobacteria induce the cellular redistribution of LDs and gain access to the stored lipids are still poorly understood. This review describes recent evidence about the dual interaction of mycobacteria with host LDs and membrane phospholipids and integrates them in a broader view of the underlying cellular processes manipulated by various intracellular

  8. Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies.

    Science.gov (United States)

    Chirichella, Michele; Lisi, Simonetta; Fantini, Marco; Goracci, Martina; Calvello, Mariantonietta; Brandi, Rossella; Arisi, Ivan; D'Onofrio, Mara; Di Primio, Cristina; Cattaneo, Antonino

    2017-03-01

    The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells.

  9. Dormant intracellular Salmonella enterica serovar Typhimurium discriminates among Salmonella pathogenicity island 2 effectors to persist inside fibroblasts.

    Science.gov (United States)

    Núñez-Hernández, Cristina; Alonso, Ana; Pucciarelli, M Graciela; Casadesús, Josep; García-del Portillo, Francisco

    2014-01-01

    Salmonella enterica uses effector proteins delivered by type III secretion systems (TTSS) to colonize eukaryotic cells. Recent in vivo studies have shown that intracellular bacteria activate the TTSS encoded by Salmonella pathogenicity island-2 (SPI-2) to restrain growth inside phagocytes. Growth attenuation is also observed in vivo in bacteria colonizing nonphagocytic stromal cells of the intestinal lamina propria and in cultured fibroblasts. SPI-2 is required for survival of nongrowing bacteria persisting inside fibroblasts, but its induction mode and the effectors involved remain unknown. Here, we show that nongrowing dormant intracellular bacteria use the two-component system OmpR-EnvZ to induce SPI-2 expression and the PhoP-PhoQ system to regulate the time at which induction takes place, 2 h postentry. Dormant bacteria were shown to discriminate the usage of SPI-2 effectors. Among the effectors tested, SseF, SseG, and SseJ were required for survival, while others, such as SifA and SifB, were not. SifA and SifB dispensability correlated with the inability of intracellular bacteria to secrete these effectors even when overexpressed. Conversely, SseJ overproduction resulted in augmented secretion and exacerbated bacterial growth. Dormant bacteria produced other effectors, such as PipB and PipB2, that, unlike what was reported for epithelial cells, did not to traffic outside the phagosomal compartment. Therefore, permissiveness for secreting only a subset of SPI-2 effectors may be instrumental for dormancy. We propose that the S. enterica serovar Typhimurium nonproliferative intracellular lifestyle is sustained by selection of SPI-2 effectors that are produced in tightly defined amounts and delivered to phagosome-confined locations.

  10. Brucella abortus Induces a Warburg Shift in Host Metabolism That Is Linked to Enhanced Intracellular Survival of the Pathogen.

    Science.gov (United States)

    Czyż, Daniel M; Willett, Jonathan W; Crosson, Sean

    2017-08-01

    Intracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected with Brucella abortus undergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well described in cancer cells and also occurs in activated inflammatory cells. B. abortus efficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affect in vitro growth of B. abortus in axenic culture but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, and B. abortus uses this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens. IMPORTANCE Brucella spp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected with Brucella abortus undergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism

  11. Epigenetic Regulation of the Nitrosative Stress Response and Intracellular Macrophage Survival by Extraintestinal Pathogenic Escherichia coli

    Science.gov (United States)

    Bateman, Stacey L.; Seed, Patrick C.

    2013-01-01

    Summary Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection, and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor IHF. In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNI), primarily through de-repression of hmpA, encoding a nitric oxide detoxifying flavohemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX. PMID:22221182

  12. Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli.

    Science.gov (United States)

    Bateman, Stacey L; Seed, Patrick C

    2012-03-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor (IHF). In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNIs), primarily through de-repression of hmpA, encoding a nitric oxide-detoxifying flavohaemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX. © 2012 Blackwell Publishing Ltd.

  13. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

    Science.gov (United States)

    Furuse, Yuki; Finethy, Ryan; Saka, Hector A; Xet-Mull, Ana M; Sisk, Dana M; Smith, Kristen L Jurcic; Lee, Sunhee; Coers, Jörn; Valdivia, Raphael H; Tobin, David M; Cullen, Bryan R

    2014-01-01

    MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

  14. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

    Directory of Open Access Journals (Sweden)

    Yuki Furuse

    Full Text Available MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

  15. Approach to discover T- and B-cell antigens of intracellular pathogens applied to the design of Chlamydia trachomatis vaccines

    Science.gov (United States)

    Finco, Oretta; Frigimelica, Elisabetta; Buricchi, Francesca; Petracca, Roberto; Galli, Giuliano; Faenzi, Elisa; Meoni, Eva; Bonci, Alessandra; Agnusdei, Mauro; Nardelli, Filomena; Bartolini, Erika; Scarselli, Maria; Caproni, Elena; Laera, Donatello; Zedda, Luisanna; Skibinski, David; Giovinazzi, Serena; Bastone, Riccardo; Ianni, Elvira; Cevenini, Roberto; Grandi, Guido; Grifantini, Renata

    2011-01-01

    Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis. We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis-infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis-infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4+/IFN-γ+ T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4+/IFN-γ+–inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4+ T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti-Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens. PMID:21628568

  16. Neutrophils exert a suppressive effect on Th1 responses to intracellular pathogen Brucella abortus.

    Directory of Open Access Journals (Sweden)

    Elías Barquero-Calvo

    2013-02-01

    Full Text Available Polymorphonuclear neutrophils (PMNs are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i comparatively reduced spleen swelling; ii augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs; iii higher recruitment of monocytes and monocyte/DCs phenotype; iv significant activation of B and T lymphocytes, and v increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.

  17. Dormant intracellular salmonella enterica serovar typhimurium discriminates among salmonella pathogenicity island 2 effectors to persist inside fibroblasts

    OpenAIRE

    Núñez Hernández, Cristina; Alonso, Ana; Pucciarelli, María Graciela; Casadesús Pursals, Josep; García del Portillo, Francisco

    2014-01-01

    Salmonella enterica uses effector proteins delivered by type III secretion systems (TTSS) to colonize eukaryotic cells. Recent in vivo studies have shown that intracellular bacteria activate the TTSS encoded by Salmonella pathogenicity island-2 (SPI-2) to restrain growth inside phagocytes. Growth attenuation is also observed in vivo in bacteria colonizing nonphagocytic stromal cells of the intestinal lamina propria and in cultured fibroblasts. SPI-2 is required for survival of nongrowing bact...

  18. To Eat and to Be Eaten: Mutual Metabolic Adaptations of Immune Cells and Intracellular Bacterial Pathogens upon Infection

    Science.gov (United States)

    Eisenreich, Wolfgang; Rudel, Thomas; Heesemann, Jürgen; Goebel, Werner

    2017-01-01

    Intracellular bacterial pathogens (IBPs) invade and replicate in different cell types including immune cells, in particular of the innate immune system (IIS) during infection in the acute phase. However, immune cells primarily function as essential players in the highly effective and integrated host defense systems comprising the IIS and the adaptive immune system (AIS), which cooperatively protect the host against invading microbes including IBPs. As countermeasures, the bacterial pathogens (and in particular the IBPs) have developed strategies to evade or reprogram the IIS at various steps. The intracellular replication capacity and the anti-immune defense responses of the IBP's as well as the specific antimicrobial responses of the immune cells of the innate and the AIS depend on specific metabolic programs of the IBPs and their host cells. The metabolic programs of the immune cells supporting or counteracting replication of the IBPs appear to be mutually exclusive. Indeed, recent studies show that upon interaction of naïve, metabolically quiescent immune cells with IBPs, different metabolic activation processes occur which may result in the provision of a survival and replication niche for the pathogen or its eradication. It is therefore likely that within a possible host cell population subsets exist that are metabolically programmed for pro- or anti-microbial conditions. These metabolic programs may be triggered by the interactions between different bacterial agonistic components and host cell receptors. In this review, we summarize the current status in the field and discuss metabolic adaptation processes within immune cells of the IIS and the IBPs that support or restrict the intracellular replication of the pathogens. PMID:28752080

  19. Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters In Vivo during Interaction of the Pathogenic Fungus Aspergillus fumigatus with Human Monocytes.

    Directory of Open Access Journals (Sweden)

    Sara Mohebbi

    Full Text Available Hyperspectral imaging (HSI is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus Aspergillus fumigatus conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised A. fumigatus conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of A. fumigatus is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell.

  20. Small non-coding RNAs: new insights in modulation of host immune response by intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Waqas Ahmed

    2016-10-01

    Full Text Available Pathogenic bacteria possess intricate regulatory networks that temporally control the production of virulence factors, and enable the bacteria to survive and proliferate within host cell. Small non-coding RNAs (sRNAs have been identified as important regulators of gene expression in diverse biological contexts. Recent research has shown bacterial sRNAs involved in growth and development, cell proliferation, differentiation, metabolism, cell signaling and immune response through regulating protein–protein interactions or via their ability to base pair with RNA and DNA. In this review, we provide a brief overview of mechanism of action employed by immune-related sRNAs, their known functions in immunity, and how they can be integrated into regulatory circuits that govern virulence, which will facilitates to understand pathogenesis and the development of novel, more effective therapeutic approaches to treat infections caused by intracellular bacterial pathogens.

  1. Pathogen-mediated selection for MHC variability in wild zebrafish

    Czech Academy of Sciences Publication Activity Database

    Smith, C.; Ondračková, Markéta; Spence, R.; Adams, S.; Betts, D. S.; Mallon, E.

    2011-01-01

    Roč. 13, č. 6 (2011), s. 589-605 ISSN 1522-0613 Institutional support: RVO:68081766 Keywords : digenean * frequency-dependent selection * heterozygote advantage * major histocompatibility complex * metazoan parasite * pathogen-driven selection Subject RIV: EG - Zoology Impact factor: 1.029, year: 2011

  2. An optimal method of iron starvation of the obligate intracellular pathogen, Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Christopher C. Thompson

    2011-02-01

    Full Text Available Iron is an essential cofactor in a number of critical biochemical reactions, and as such, its acquisition, storage, and metabolism is highly regulated in most organisms. The obligate intracellular bacterium, Chlamydia trachomatis experiences a developmental arrest when iron within the host is depleted. The nature of the iron starvation response in Chlamydia is relatively uncharacterized because of the likely inefficient method of iron depletion, which currently relies on the compound deferoxamine mesylate (DFO. Inefficient induction of the iron starvation response precludes the identification of iron-regulated genes. This report evaluated DFO with another iron chelator, 2,2’-bipyridyl (Bpdl and presented a systematic comparison of the two across a range of criteria in a single-treatment time-of-infection regimen. We demonstrate that the membrane permeable Bpdl was superior to DFO in the inhibition of chlamydia development, the induction of aberrant morphology, and the induction of an iron starvation transcriptional response in both host and bacteria. Furthermore, iron starvation using Bpdl identified the periplasmic iron binding protein-encoding ytgA gene as iron- responsive. Overall, the data present a compelling argument for the use of Bpdl, rather than DFO, in future iron starvation studies of chlamydia and other intracellular bacteria.

  3. Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Ogata

    2006-05-01

    Full Text Available The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair-long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic "melting pot" where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.

  4. Host Actin Polymerization Tunes the Cell Division Cycle of an Intracellular Pathogen

    Directory of Open Access Journals (Sweden)

    M. Sloan Siegrist

    2015-04-01

    Full Text Available Growth and division are two of the most fundamental capabilities of a bacterial cell. While they are well described for model organisms growing in broth culture, very little is known about the cell division cycle of bacteria replicating in more complex environments. Using a D-alanine reporter strategy, we found that intracellular Listeria monocytogenes (Lm spend a smaller proportion of their cell cycle dividing compared to Lm growing in broth culture. This alteration to the cell division cycle is independent of bacterial doubling time. Instead, polymerization of host-derived actin at the bacterial cell surface extends the non-dividing elongation period and compresses the division period. By decreasing the relative proportion of dividing Lm, actin polymerization biases the population toward cells with the highest propensity to form actin tails. Thus, there is a positive-feedback loop between the Lm cell division cycle and a physical interaction with the host cytoskeleton.

  5. Intracellular persisting Staphylococcus aureus is the major pathogen in recurrent tonsillitis.

    Directory of Open Access Journals (Sweden)

    Andreas E Zautner

    Full Text Available BACKGROUND: The two major indications for tonsillectomy are recurrent tonsillitis (RT and peritonsillar abscess (PTA. Unlike PTAs, which are primarily treated surgically, RT is often cured by tonsillectomy only after a series of failed drug therapy attempts. Although the bacteriological background of RT has been studied, the reason for the lack of success of conservative therapeutic approaches is not well understood. METHODS: In a prospective study, tonsil specimens from 130 RT patients and 124 PTA patients were examined for the presence of extra- and intracellular bacteria using antibiotic protection assays. Staphylococcus aureus isolates from RT patients were characterized by pulsed-field gel electrophoresis (PFGE, spa-typing and MSCRAMM-gene-PCR. Their ability for biofilm formation was tested and their cell invasiveness was confirmed by a flow cytometric invasion assay (FACS, fluorescent in situ hybridization (FISH and immunohistochemistry. FINDINGS: S. aureus was the predominant species (57.7% in RT patients, whereas Streptococcus pyogenes was most prevalent (20.2% in PTA patients. Three different assays (FACS, FISH, antibiotic protection assay showed that nearly all RT-associated S. aureus strains were located inside tonsillar cells. Correspondingly, the results of the MSCRAMM-gene-PCRs confirmed that 87% of these S. aureus isolates were invasive strains and not mere colonizers. Based upon PFGE analyses of genomic DNA and on spa-gene typing the vast majority of the S. aureus isolates belonged to different clonal lineages. CONCLUSIONS: Our results demonstrate that intracellular residing S. aureus is the most common cause of RT and indicate that S. aureus uses this location to survive the effects of antibiotics and the host immune response. A German translation of the Abstract is provided as supplementary material (Abstract S1.

  6. The pathogen transmission avoidance theory of sexual selection

    Energy Technology Data Exchange (ETDEWEB)

    Loehle, C.

    1997-08-01

    The current theory that sexual selection results from female preference for males with good genes suffers from several problems. An alternative explanation, the pathogen transmission avoidance hypothesis, argues that the primary function of showy traits is to provide a reliable signal of current disease status, so that sick individuals can be avoided during mating. This study shows that a significant risk of pathogen transmission occurs during mating and that showy traits are reliable indicators of current disease status. The origin of female choosiness is argued to lie in a general tendency to avoid sick individuals, even in the absence of showy traits, which originate as exaggerations of normal traits that are indicative of good health (bright feathers, vigorous movement, large size). Thus, in this new model the origins of both showy traits and female choosiness are not problematic and there is no threshold effect. This model predicts that when the possession of male showy traits does not help to reduce disease in the female, showy traits are unlikely to occur. This case corresponds to thorough exposure of every animal to all group pathogens, on average, in large groups. Such species are shown with a large data set on birds to be less likely to exhibit showy traits. The good-genes model does not make this prediction. The pathogen transmission avoidance model can also lead to the evolution of showy traits even when selection is not effective against a given pathogen (e.g., when there is no heritable variation for resistance), but can result in selection for resistance if such genes are present. Monogamy is argued to reduce selection pressures for showy traits; data show monogamous species to be both less parasitized and less showy. In the context of reduction of pathogen transmission rates in showy populations, selection pressure becomes inversely frequency-dependent, which makes showy traits likely to be self-limiting rather than runaway.

  7. Surface adhesins and exopolymers of selected foodborne pathogens

    DEFF Research Database (Denmark)

    Jaglic, Zoran; Desvaux, Mickaël; Weiss, Agnes

    2014-01-01

    of bacterial surface structures are involved in this process and these promote bacterial adhesion in a more or less specific manner. In this review, we will focus on those surface adhesins and exopolymers in selected foodborne pathogens that are involved mainly in primary adhesion. Their role in biofilm...

  8. Host actin polymerization tunes the cell division cycle of an intracellular pathogen.

    Science.gov (United States)

    Siegrist, M Sloan; Aditham, Arjun K; Espaillat, Akbar; Cameron, Todd A; Whiteside, Sarah A; Cava, Felipe; Portnoy, Daniel A; Bertozzi, Carolyn R

    2015-04-28

    Growth and division are two of the most fundamental capabilities of a bacterial cell. While they are well described for model organisms growing in broth culture, very little is known about the cell division cycle of bacteria replicating in more complex environments. Using a D-alanine reporter strategy, we found that intracellular Listeria monocytogenes (Lm) spend a smaller proportion of their cell cycle dividing compared to Lm growing in broth culture. This alteration to the cell division cycle is independent of bacterial doubling time. Instead, polymerization of host-derived actin at the bacterial cell surface extends the non-dividing elongation period and compresses the division period. By decreasing the relative proportion of dividing Lm, actin polymerization biases the population toward cells with the highest propensity to form actin tails. Thus, there is a positive-feedback loop between the Lm cell division cycle and a physical interaction with the host cytoskeleton. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

    International Nuclear Information System (INIS)

    Whittingham, Jean L.; Blagova, Elena V.; Finn, Ciaran E.; Luo, Haixia; Miranda-CasoLuengo, Raúl; Turkenburg, Johan P.; Leech, Andrew P.; Walton, Paul H.; Murzin, Alexey G.; Meijer, Wim G.; Wilkinson, Anthony J.

    2014-01-01

    VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded β-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins

  10. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

    Energy Technology Data Exchange (ETDEWEB)

    Whittingham, Jean L.; Blagova, Elena V. [University of York, Heslington, York YO10 5DD (United Kingdom); Finn, Ciaran E.; Luo, Haixia; Miranda-CasoLuengo, Raúl [University College Dublin, Dublin (Ireland); Turkenburg, Johan P.; Leech, Andrew P.; Walton, Paul H. [University of York, Heslington, York YO10 5DD (United Kingdom); Murzin, Alexey G. [MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH (United Kingdom); Meijer, Wim G. [University College Dublin, Dublin (Ireland); Wilkinson, Anthony J., E-mail: tony.wilkinson@york.ac.uk [University of York, Heslington, York YO10 5DD (United Kingdom)

    2014-08-01

    VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded β-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

  11. Sexual selection as a consequence of pathogen avoidance behaviors

    Energy Technology Data Exchange (ETDEWEB)

    Loehle, C. [Argonne National Lab., IL (United States); Logofet, D.O. [Russian Academy of Sciences, Moscow (Russian Federation). Laboratory of Mathematical Ecology

    1997-08-01

    The current theory that sexual selection results from female choice for good genes suffers from several problems. An alternative explanation is proposed. The pathogen avoidance hypothesis argues that the primary function of showy traits is to provide a reliable signal of current disease status so that sick individuals may be avoided during mating. Our studies shown that a significant risk of pathogen transmission occurs during mating and that showy traits are reliable indicators of current disease status. The origin of female choosiness is argued to lie in a general tendency to avoid sick individuals, even in the absence of showy traits. The showy traits are argued to originate as simple exaggerations of normal traits that are indicative of good health (bright feathers; vigorous movement; large size). Thus the origins of both showy traits and female choosiness are not problematic in this theory. A game theory analysis is employed to formalize the theory. Results of the game theory model support the theory. In particular, when the possession of male showy traits does not help reduce disease in the female, then showy traits are unlikely to occur. This case corresponds to the situation in large flocks or herds in which every animal is thoroughly exposed to all group pathogens on average. Such species do not exhibit showy traits. The good genes model does not make this prediction. The pathogen avoidance model can also lead to the evolution of showy traits even when selection is not effective against a given pathogen (e.g., when there is no heritable variation for resistance) but will lead to selection for resistance if such genes are present. Overall, the pathogen avoidance hypothesis provides a complete alternative to the good genes theory.

  12. Antibody-mediated immunity to the obligate intracellular bacterial pathogen Coxiella burnetii is Fc receptor- and complement-independent

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    Heinzen Robert A

    2009-05-01

    Full Text Available Abstract Background The obligate intracellular bacterial pathogen Coxiella burnetii causes the zoonosis Q fever. The intracellular niche of C. burnetii has led to the assumption that cell-mediated immunity is the most important immune component for protection against this pathogen. However, passive immunization with immune serum can protect naïve animals from challenge with virulent C. burnetii, indicating a role for antibody (Ab in protection. The mechanism of this Ab-mediated protection is unknown. Therefore, we conducted a study to determine whether Fc receptors (FcR or complement contribute to Ab-mediated immunity (AMI to C. burnetii. Results Virulent C. burnetii infects and replicates within human dendritic cells (DC without inducing their maturation or activation. We investigated the effects of Ab opsonized C. burnetii on human monocyte-derived and murine bone marrow-derived DC. Infection of DC with Ab-opsonized C. burnetii resulted in increased expression of maturation markers and inflammatory cytokine production. Bacteria that had been incubated with naïve serum had minimal effect on DC, similar to virulent C. burnetii alone. The effect of Ab opsonized C. burnetii on DC was FcR dependent as evidenced by a reduced response of DC from FcR knockout (FcR k/o compared to C57Bl/6 (B6 mice. To address the potential role of FcR in Ab-mediated protection in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, we found that FcR are not essential for AMI to C. burnetii in vivo. We subsequently examined the role of complement in AMI by passively immunizing and challenging several different strains of complement-deficient mice and found that AMI to C. burnetii is also complement-independent. Conclusion Despite our data showing FcR-dependent stimulation of DC in vitro, Ab-mediated immunity to C. burnetii in vivo is FcR-independent. We also found that passive immunity to this pathogen is independent of

  13. NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens.

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    Samar Habib

    Full Text Available Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE, which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8-10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia.

  14. Modulation of Stat-1 in Human Macrophages Infected with Different Species of Intracellular Pathogenic Bacteria

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    Giuditta Fiorella Schiavano

    2016-01-01

    Full Text Available The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701 after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages.

  15. Selection of pathogen-resistant mutants in rapeseed

    International Nuclear Information System (INIS)

    Spanier, A.; Roebbelen, G.

    1990-01-01

    Full text: Significant yield reductions are due to Phoma lingam and Alternaria brassicae. Toxin containing culture filtrates of the pathogens as well as concentrated toxins of Phoma (Sirodesmins) are used for resistance selections in in-vitro cultures of haploid rapeseed materials. A few days after transfer of the in-vitro materials to the selective media the inhibitory effect of both the culture filtrates as well as the Sirodesmins was apparent. Clones were obtained, surviving several transfers onto toxin containing media. Further experiments shall clarify whether the toxin tolerance, selected in vitro at the cell level, is expressed in the differentiated plant in the greenhouse and finally in the field. (author)

  16. Pathogen-Driven Selection in the Human Genome

    Directory of Open Access Journals (Sweden)

    Rachele Cagliani

    2013-01-01

    Full Text Available Infectious diseases and epidemics have always accompanied and characterized human history, representing one of the main causes of death. Even today, despite progress in sanitation and medical research, infections are estimated to account for about 15% of deaths. The hypothesis whereby infectious diseases have been acting as a powerful selective pressure was formulated long ago, but it was not until the availability of large-scale genetic data and the development of novel methods to study molecular evolution that we could assess how pervasively infectious agents have shaped human genetic diversity. Indeed, recent evidences indicated that among the diverse environmental factors that acted as selective pressures during the evolution of our species, pathogen load had the strongest influence. Beside the textbook example of the major histocompatibility complex, selection signatures left by pathogen-exerted pressure can be identified at several human loci, including genes not directly involved in immune response. In the future, high-throughput technologies and the availability of genetic data from different populations are likely to provide novel insights into the evolutionary relationships between the human host and its pathogens. Hopefully, this will help identify the genetic determinants modulating the susceptibility to infectious diseases and will translate into new treatment strategies.

  17. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major.

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    Rafael Ricci-Azevedo

    2016-04-01

    Full Text Available ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1 immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS, form neutrophil extracellular traps (NETs and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF and interleukin-1beta (IL-1β release; otherwise, transforming growth factor-beta (TGF-β production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be

  18. Intracellular Bacterial Pathogens Trigger the Formation of U Small Nuclear RNA Bodies (U Bodies) through Metabolic Stress Induction.

    Science.gov (United States)

    Tsalikis, Jessica; Tattoli, Ivan; Ling, Arthur; Sorbara, Matthew T; Croitoru, David O; Philpott, Dana J; Girardin, Stephen E

    2015-08-21

    Invasive bacterial pathogens induce an amino acid starvation (AAS) response in infected host cells that controls host defense in part by promoting autophagy. However, whether AAS has additional significant effects on the host response to intracellular bacteria remains poorly characterized. Here we showed that Shigella, Salmonella, and Listeria interfere with spliceosomal U snRNA maturation in the cytosol. Bacterial infection resulted in the rerouting of U snRNAs and their cytoplasmic escort, the survival motor neuron (SMN) complex, to processing bodies, thus forming U snRNA bodies (U bodies). This process likely contributes to the decline in the cytosolic levels of U snRNAs and of the SMN complex proteins SMN and DDX20 that we observed in infected cells. U body formation was triggered by membrane damage in infected cells and was associated with the induction of metabolic stresses, such as AAS or endoplasmic reticulum stress. Mechanistically, targeting of U snRNAs to U bodies was regulated by translation initiation inhibition and the ATF4/ATF3 pathway, and U bodies rapidly disappeared upon removal of the stress, suggesting that their accumulation represented an adaptive response to metabolic stress. Importantly, this process likely contributed to shape the host response to invasive bacteria because down-regulation of DDX20 expression using short hairpin RNA (shRNA) amplified ATF3- and NF-κB-dependent signaling. Together, these results identify a critical role for metabolic stress and invasive bacterial pathogens in U body formation and suggest that this process contributes to host defense. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Aggressive chemotherapy and the selection of drug resistant pathogens.

    Science.gov (United States)

    Huijben, Silvie; Bell, Andrew S; Sim, Derek G; Tomasello, Danielle; Mideo, Nicole; Day, Troy; Read, Andrew F

    2013-09-01

    Drug resistant pathogens are one of the key public health challenges of the 21st century. There is a widespread belief that resistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize the probability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor of resistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generates opposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatment regimens which best retard resistance evolution while maximizing health gains and minimizing disease transmission requires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes and infectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimens substantially reduced the probability of onward transmission of resistance (by >150-fold), without compromising health outcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectively with treatment regimens other than those which reduce pathogen burdens as fast as possible.

  20. Aggressive chemotherapy and the selection of drug resistant pathogens.

    Directory of Open Access Journals (Sweden)

    Silvie Huijben

    2013-09-01

    Full Text Available Drug resistant pathogens are one of the key public health challenges of the 21st century. There is a widespread belief that resistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize the probability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor of resistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generates opposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatment regimens which best retard resistance evolution while maximizing health gains and minimizing disease transmission requires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes and infectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimens substantially reduced the probability of onward transmission of resistance (by >150-fold, without compromising health outcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectively with treatment regimens other than those which reduce pathogen burdens as fast as possible.

  1. An Oxygen-Sensing Two-Component System in the Burkholderia cepacia Complex Regulates Biofilm, Intracellular Invasion, and Pathogenicity.

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    Matthew M Schaefers

    2017-01-01

    Full Text Available Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC, which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence.

  2. Priority setting of foodborne pathogens: disease burden and costs of selected enteric pathogens

    NARCIS (Netherlands)

    Kemmeren JM; Mangen MJJ; Duynhoven YTHP van; Havelaar AH; MGB

    2006-01-01

    Toxoplasmosis causes the highest disease burden among seven evaluated foodborne pathogens. This is the preliminary conclusion of a major study of the disease burden and related costs of foodborne pathogens. The other micro-organisms that were studied are Campylobacter spp., Salmonella spp.,

  3. Non-recessive Bt toxin resistance conferred by an intracellular cadherin mutation in field-selected populations of cotton bollworm.

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    Haonan Zhang

    Full Text Available Transgenic crops producing Bacillus thuringiensis (Bt toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.

  4. Impact of electromagnetic field on the pathogenicity of selected ...

    African Journals Online (AJOL)

    Rhipicephalus decoloratus) to variable intensities of electromagnetic field for different periods of time was examined on their pathogenicity on tick. Some bacterial isolates from the macerate of tick cadavers were used in the infection of healthy engorged ...

  5. Triclosan Enhances the Clearing of Pathogenic IntracellularSalmonellaorCandida albicansbut Disturbs the Intestinal Microbiota through mTOR-Independent Autophagy.

    Science.gov (United States)

    Wang, Chao; Yu, Zhongyang; Shi, Xiaochen; Tang, Xudong; Wang, Yang; Wang, Xueyan; An, Yanan; Li, Shulin; Li, Yan; Wang, Xuefei; Luan, Wenjing; Chen, Zhaobin; Liu, Mingyuan; Yu, Lu

    2018-01-01

    Triclosan (TCS) is a broad-spectrum antimicrobial agent, whose well-known antibacterial mechanism is inhibiting lipid synthesis. Autophagy, an innate immune response, is an intracellular process that delivers the cargo including pathogens to lysosomes for degradation. In this study, we first demonstrated that TCS induced autophagy in a dose-dependent manner in non-phagocytic cells (HeLa) and in macrophages (Raw264.7) and in vivo . The western blot results also revealed that TCS induced autophagy via the AMPK/ULK1 and JNK/ERK/p38 pathways independent of mTOR. The immunofluorescence results indicated that TCS up-regulated the expression of the ubiquitin receptors NDP52 and p62 and strengthened the co-localization of these receptors with Salmonella enterica Typhimurium ( S . typhimurium) or Candida albicans ( C. albicans ) in infected MΦ cells. In addition, sub-lethal concentrations of TCS enhanced the clearing of the pathogens S . typhimurium or C. albicans in infected MΦ and in corresponding mouse infection models in vivo . Specifically, we found that a sub-inhibitory concentration of TCS induced autophagy, leading to an imbalance of the intestinal microflora in mice through the analysis of 16s rRNA Sequencing. Together, these results demonstrated that TCS induced autophagy, which enhanced the killing against pathogenic S . typhimurium or C. albicans within mammal cells but broke the balance of the intestinal microflora.

  6. Triclosan Enhances the Clearing of Pathogenic Intracellular Salmonella or Candida albicans but Disturbs the Intestinal Microbiota through mTOR-Independent Autophagy

    Directory of Open Access Journals (Sweden)

    Chao Wang

    2018-02-01

    Full Text Available Triclosan (TCS is a broad-spectrum antimicrobial agent, whose well-known antibacterial mechanism is inhibiting lipid synthesis. Autophagy, an innate immune response, is an intracellular process that delivers the cargo including pathogens to lysosomes for degradation. In this study, we first demonstrated that TCS induced autophagy in a dose-dependent manner in non-phagocytic cells (HeLa and in macrophages (Raw264.7 and in vivo. The western blot results also revealed that TCS induced autophagy via the AMPK/ULK1 and JNK/ERK/p38 pathways independent of mTOR. The immunofluorescence results indicated that TCS up-regulated the expression of the ubiquitin receptors NDP52 and p62 and strengthened the co-localization of these receptors with Salmonella enterica Typhimurium (S. typhimurium or Candida albicans (C. albicans in infected MΦ cells. In addition, sub-lethal concentrations of TCS enhanced the clearing of the pathogens S. typhimurium or C. albicans in infected MΦ and in corresponding mouse infection models in vivo. Specifically, we found that a sub-inhibitory concentration of TCS induced autophagy, leading to an imbalance of the intestinal microflora in mice through the analysis of 16s rRNA Sequencing. Together, these results demonstrated that TCS induced autophagy, which enhanced the killing against pathogenic S. typhimurium or C. albicans within mammal cells but broke the balance of the intestinal microflora.

  7. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

    Science.gov (United States)

    Raymond, Frédéric; Boisvert, Sébastien; Roy, Gaétan; Ritt, Jean-François; Légaré, Danielle; Isnard, Amandine; Stanke, Mario; Olivier, Martin; Tremblay, Michel J.; Papadopoulou, Barbara; Ouellette, Marc; Corbeil, Jacques

    2012-01-01

    The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage. PMID:21998295

  8. Disease burden of selected gastrointestinal pathogens in Australia, 2010

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    Katherine B. Gibney

    2014-11-01

    Conclusions: The pathogen causing the greatest disease burden varied according to the metric used, however DALYs are considered most useful given the incorporation of morbidity, mortality, and sequelae. These results can be used to prioritize public health interventions toward Salmonella and Campylobacter infections and to measure the impact of these interventions.

  9. The compact genome of the plant pathogen Plasmodiophora brassicae is adapted to intracellular interactions with host Brassica spp.

    Science.gov (United States)

    Rolfe, Stephen A; Strelkov, Stephen E; Links, Matthew G; Clarke, Wayne E; Robinson, Stephen J; Djavaheri, Mohammad; Malinowski, Robert; Haddadi, Parham; Kagale, Sateesh; Parkin, Isobel A P; Taheri, Ali; Borhan, M Hossein

    2016-03-31

    The protist Plasmodiophora brassicae is a soil-borne pathogen of cruciferous species and the causal agent of clubroot disease of Brassicas including agriculturally important crops such as canola/rapeseed (Brassica napus). P. brassicae has remained an enigmatic plant pathogen and is a rare example of an obligate biotroph that resides entirely inside the host plant cell. The pathogen is the cause of severe yield losses and can render infested fields unsuitable for Brassica crop growth due to the persistence of resting spores in the soil for up to 20 years. To provide insight into the biology of the pathogen and its interaction with its primary host B. napus, we produced a draft genome of P. brassicae pathotypes 3 and 6 (Pb3 and Pb6) that differ in their host range. Pb3 is highly virulent on B. napus (but also infects other Brassica species) while Pb6 infects only vegetable Brassica crops. Both the Pb3 and Pb6 genomes are highly compact, each with a total size of 24.2 Mb, and contain less than 2 % repetitive DNA. Clustering of genome-wide single nucleotide polymorphisms (SNP) of Pb3, Pb6 and three additional re-sequenced pathotypes (Pb2, Pb5 and Pb8) shows a high degree of correlation of cluster grouping with host range. The Pb3 genome features significant reduction of intergenic space with multiple examples of overlapping untranslated regions (UTRs). Dependency on the host for essential nutrients is evident from the loss of genes for the biosynthesis of thiamine and some amino acids and the presence of a wide range of transport proteins, including some unique to P. brassicae. The annotated genes of Pb3 include those with a potential role in the regulation of the plant growth hormones cytokinin and auxin. The expression profile of Pb3 genes, including putative effectors, during infection and their potential role in manipulation of host defence is discussed. The P. brassicae genome sequence reveals a compact genome, a dependency of the pathogen on its host for some

  10. Comparative Proteomics of Purified Pathogen Vacuoles Correlates Intracellular Replication of Legionella pneumophila with the Small GTPase Ras-related protein 1 (Rap1).

    Science.gov (United States)

    Schmölders, Johanna; Manske, Christian; Otto, Andreas; Hoffmann, Christine; Steiner, Bernhard; Welin, Amanda; Becher, Dörte; Hilbi, Hubert

    2017-04-01

    Legionella pneumophila is an opportunistic bacterial pathogen that causes a severe lung infection termed "Legionnaires' disease." The pathogen replicates in environmental protozoa as well as in macrophages within a unique membrane-bound compartment, the Legionella -containing-vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates ca. 300 "effector proteins" into host cells, where they target distinct host factors. The L. pneumophila "pentuple" mutant (Δpentuple) lacks 5 gene clusters (31% of the effector proteins) and replicates in macrophages but not in Dictyostelium discoideum amoeba. To elucidate the host factors defining a replication-permissive compartment, we compare here the proteomes of intact LCVs isolated from D. discoideum or macrophages infected with Δpentuple or the parental strain Lp02. This analysis revealed that the majority of host proteins are shared in D. discoideum or macrophage LCVs containing the mutant or the parental strain, respectively, whereas some proteins preferentially localize to distinct LCVs. The small GTPase Rap1 was identified on D. discoideum LCVs containing strain Lp02 but not the Δpentuple mutant and on macrophage LCVs containing either strain. The localization pattern of active Rap1 on D. discoideum or macrophage LCVs was confirmed by fluorescence microscopy and imaging flow cytometry, and the depletion of Rap1 by RNA interference significantly reduced the intracellular growth of L. pneumophila Thus, comparative proteomics identified Rap1 as a novel LCV host component implicated in intracellular replication of L. pneumophila . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The Steroid Catabolic Pathway of the Intracellular Pathogen Rhodococcus equi Is Important for Pathogenesis and a Target for Vaccine Development

    Science.gov (United States)

    van der Geize, R.; Grommen, A. W. F.; Hessels, G. I.; Jacobs, A. A. C.; Dijkhuizen, L.

    2011-01-01

    Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551), ipdB (rv3552), fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD) and 3aα-H-4α(3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP). Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections. PMID:21901092

  12. The steroid catabolic pathway of the intracellular pathogen Rhodococcus equi is important for pathogenesis and a target for vaccine development.

    Directory of Open Access Journals (Sweden)

    R van der Geize

    2011-08-01

    Full Text Available Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551, ipdB (rv3552, fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD and 3aα-H-4α(3'-propionic acid-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP. Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections.

  13. Development of a Selective Medium for the Fungal Pathogen Fusarium graminearum Using Toxoflavin Produced by the Bacterial Pathogen Burkholderia glumae

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    Boknam Jung

    2013-12-01

    Full Text Available The ascomycete fungus Fusarium graminearum is a major causal agent for Fusarium head blight in cereals and produces mycotoxins such as trichothecenes and zearalenone. Isolation of the fungal strains from air or cereals can be hampered by various other airborne fungal pathogens and saprophytic fungi. In this study, we developed a selective medium specific to F. graminearum using toxoflavin produced by the bacterial pathogen Burkholderia glumae. F. graminearum was resistant to toxoflavin, while other fungi were sensitive to this toxin. Supplementing toxoflavin into medium enhanced the isolation of F. graminearum from rice grains by suppressing the growth of saprophytic fungal species. In addition, a medium with or without toxoflavin exposed to wheat fields for 1 h had 84% or 25%, respectively, of colonies identified as F. graminearum. This selection medium provides an efficient tool for isolating F. graminearum, and can be adopted by research groups working on genetics and disease forecasting.

  14. TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

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    Kwang-Hyung Kim

    2009-11-01

    Full Text Available The regulation of intracellular levels of reactive oxygen species (ROS is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we

  15. Tick-Borne Encephalitis Virus Replication, Intracellular Trafficking, and Pathogenicity in Human Intestinal Caco-2 Cell Monolayers

    Science.gov (United States)

    Möller, Lars; Schulzke, Joerg D.; Niedrig, Matthias; Bücker, Roland

    2014-01-01

    Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route. PMID

  16. Mannose-binding lectin and l-ficolin polymorphisms in patients with community-acquired pneumonia caused by intracellular pathogens.

    Science.gov (United States)

    van Kempen, Gijs; Meijvis, Sabine; Endeman, Henrik; Vlaminckx, Bart; Meek, Bob; de Jong, Ben; Rijkers, Ger; Bos, Willem Jan

    2017-05-01

    Community-acquired pneumonia (CAP) is the leading infectious disease requiring hospitalization in the western world. Genetic variability affecting the host response to infection may play a role in susceptibility and outcome in patients with CAP. Mannose-binding lectin (MBL) and l-ficolin (l-FCN) are two important activators of the complement system and they can enhance phagocytosis by opsonization. In a prospective cohort of 505 Dutch patients with CAP and 227 control participants we studied whether polymorphisms in the MBL (MBL2) and FCN (FCN2) genes influenced susceptibility and outcome. No difference in frequency of these genotypes was found between patients with CAP in general and controls. However, the +6424G>T single nucleotide polymorphism (SNP) in FCN2 was more common in patients with a Coxiella burnetii pneumonia (P = 0·014). Moreover, the haplotypes coding for the highest MBL serum levels (YA/YA and YA/XA) predisposed to atypical pneumonia (C. burnetii, Legionella or Chlamydia species or Mycoplasma pneumoniae) compared with controls (P = 0·016). Furthermore, patients with these haplotypes were more often bacteraemic (P = 0·019). It can therefore be concluded that MBL2 and FCN2 polymorphisms are not major risk factors for CAP in general, but that the +6424G>T SNP in the FCN2 gene predisposes to C. burnetii pneumonia. In addition, patients with genotypes corresponding with high serum MBL levels are at risk for atypical pneumonia, possibly caused by enhanced phagocytosis, thereby promoting cell entry of these intracellular bacteria. © 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.

  17. Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.

    Directory of Open Access Journals (Sweden)

    Chao Yu

    Full Text Available Tick-borne encephalitis virus (TBEV is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin and microtubules (Nocodazole are important for PI3K-dependent (LY294002 virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1 as well as with sorting nexin-5 (SNX5, pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary

  18. Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Yu, Chao; Achazi, Katharina; Möller, Lars; Schulzke, Joerg D; Niedrig, Matthias; Bücker, Roland

    2014-01-01

    Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.

  19. Serosurvey for selected pathogens in Iberian roe deer

    Directory of Open Access Journals (Sweden)

    Oleaga Álvaro

    2010-11-01

    Full Text Available Abstract Background The roe deer is the most abundant and widespread wild Eurasian cervid. Its populations are expanding and increasingly in contact with livestock. This may affect the distribution of infectious diseases shared with other wild and domestic ungulates. Methods We investigated the antibody seroprevalence against Pestivirus, Herpesvirus, Bluetongue (BT virus, M. avium paratuberculosis (MAP, and Brucella sp. in 519 roe deer from different regions in Spain, south-western Europe. Results No antibodies were detected against BT and Brucella sp. However, antibodies were detected against Pestivirus (1.5%, Herpesvirus (0.2% and MAP (9.2%. MAP antibodies were detected in seven of the eight populations (range 5-16.4%. Conclusions The detection of MAP antibodies in samples from most roe deer populations suggests that contact with MAP is widespread in this wildlife species. The highest prevalence was detected in sites with abundant dairy cattle and frequent use of liquid manure on pastures. Considering the results obtained regarding exposure to different pathogens, we suggest that antibody prevalences in this non-gregarious browser are largely determined by environmental factors, potentially modulating vector populations or pathogen survival in the environment.

  20. Survival of selected foodborne pathogens on dry cured pork loins.

    Science.gov (United States)

    Morales-Partera, Ángela M; Cardoso-Toset, Fernando; Jurado-Martos, Francisco; Astorga, Rafael J; Huerta, Belén; Luque, Inmaculada; Tarradas, Carmen; Gómez-Laguna, Jaime

    2017-10-03

    The safety of ready-to-eat products such as cured pork loins must be guaranteed by the food industry. In the present study, the efficacy of the dry curing process of pork loins obtained from free-range pigs in the reduction of three of the most important foodborne pathogens is analysed. A total of 28 pork loin segments, with an average weight of 0.57±0.12kg, were divided into four groups with three being inoculated by immersion with 7logCFU/ml of either Salmonella Typhimurium, Campylobacter coli or Listeria innocua and the last one inoculated by immersion with sterile medium (control group). The loin segments were treated with a seasoning mixture of curing agents and spices, packed in a synthetic sausage casing and cured for 64days. Microbiological analysis, pH and water activity (a w ) were assessed at four stages. The values of pH and a w decreased with curing time as expected. S. Typhimurium and C. coli dropped significantly (3.28 and 2.14 log units, respectively), but limited reduction of L. innocua (0.84 log unit) was observed along the curing process. In our study, three factors were considered critical: the initial concentration of the bacteria, the progressive reduction of pH and the reduction of a w values. Our results encourage performing periodic analysis at different stages of the manufacturing of dry cured pork loins to ensure the absence of the three evaluated foodborne pathogens. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Selective Killing of Pathogenic Bacteria by Antimicrobial Silver Nanoparticle - Cell Wall Binding Domain (CBD) Conjugates.

    Science.gov (United States)

    Kim, Domyoung; Kwon, Seok Joon; Wu, Xia; Sauve, Jessica; Lee, Inseon; Nam, Jahyun; Kim, Jungbae; Dordick, Jonathan S

    2018-04-05

    Broad-spectrum antibiotics indiscriminately kill bacteria, removing non-pathogenic microorganisms and leading to evolution of antibiotic resistant strains. Specific antimicrobials that could selectively kill pathogenic bacteria without targeting other bacteria in the natural microbial community or microbiome may be able to address this concern. In this work, we demonstrate that silver nanoparticles, suitably conjugated to a selective cell wall binding domain (CBD), can efficiently target and selectively kill bacteria. As a relevant example, CBDBA from Bacillus anthracis selectively bound to B. anthracis in a mixture with B. subtilis, as well in a mixture with Staphylococcus aureus. This new biologically-assisted hybrid strategy, therefore, has the potential to provide selective decontamination of pathogenic bacteria with minimal impact on normal microflora.

  2. Exposure to selected Pathogens in to selected pathogens in Geoffroy's cats and domestic carnivores from central Argentina.

    Science.gov (United States)

    Uhart, Marcela M; Rago, M Virginia; Marull, Carolina A; Ferreyra, Hebe del Valle; Pereira, Javier A

    2012-10-01

    Wild carnivores share a high percentage of parasites and viruses with closely related domestic carnivores. Because of increased overlap and potential contact with domestic species, we conducted a retrospective serosurvey for 11 common carnivore pathogens in 40 Geoffroy's cats (Leopardus geoffroyi) sampled between 2000 and 2008 within or near two protected areas in central Argentina (Lihué Calel National Park, La Pampa, and Campos del Tuyú National Park, Buenos Aires), as well as five domestic cats and 11 domestic dogs from catde ranches adjacent to Lihué Calel Park. Geoffroy's cats had detectable antibody to canine distemper virus (CDV), feline calicivirus (FCV), feline coronavirus, feline panleukopenia virus (FPV), Toxoplasma gondii, Leptospira interrogans (serovars Ictero/Icter and Ballum), and Dirofilaria immitis. None of the wild cats had antibodies to feline herpesvirus, feline immunodeficiency virus (FIV), feline leukemia virus, or rabies virus. Domestic dogs had antibodies to CDV, canine adenovirus, canine herpesvirus, and canine parvovirus. Antibodies to FPV, FCV, FIV, and T. gondii were found in domestic cats. We provide the first data on exposure of free-ranging Geoffroy's cats to pathogens at two sites within the core area of the species distribution range, including the first report of antibodies to CDV in this species. We encourage continued monitoring for diseases in wild and domestic carnivores as well as preventive health care for domestic animals, particularly in park buffer zones where overlap is greatest.

  3. High temperature and bacteriophages can indirectly select for bacterial pathogenicity in environmental reservoirs.

    Directory of Open Access Journals (Sweden)

    Ville-Petri Friman

    2011-03-01

    Full Text Available The coincidental evolution hypothesis predicts that traits connected to bacterial pathogenicity could be indirectly selected outside the host as a correlated response to abiotic environmental conditions or different biotic species interactions. To investigate this, an opportunistic bacterial pathogen, Serratia marcescens, was cultured in the absence and presence of the lytic bacteriophage PPV (Podoviridae at 25°C and 37°C for four weeks (N = 5. At the end, we measured changes in bacterial phage-resistance and potential virulence traits, and determined the pathogenicity of all bacterial selection lines in the Parasemia plantaginis insect model in vivo. Selection at 37°C increased bacterial motility and pathogenicity but only in the absence of phages. Exposure to phages increased the phage-resistance of bacteria, and this was costly in terms of decreased maximum population size in the absence of phages. However, this small-magnitude growth cost was not greater with bacteria that had evolved in high temperature regime, and no trade-off was found between phage-resistance and growth rate. As a result, phages constrained the evolution of a temperature-mediated increase in bacterial pathogenicity presumably by preferably infecting the highly motile and virulent bacteria. In more general perspective, our results suggest that the traits connected to bacterial pathogenicity could be indirectly selected as a correlated response by abiotic and biotic factors in environmental reservoirs.

  4. Economic values and expected effect of selection index for pathogen-specific mastitis under Danish conditions

    DEFF Research Database (Denmark)

    Sørensen, Lars Peter; Mark, Thomas; Sørensen, M.K.

    2010-01-01

    goal was identical to selection index 3. Mastitis data from primiparous cows calving from 1998 to 2008 were used to estimate genetic parameters of the mastitis traits using linear models and AI-REML algorithm. These parameters were used for construction of the selection index equations......The objectives of this study were 1) to estimate costs related to 5 different pathogen-specific mastitis traits (susceptibility to different pathogens causing mastitis in dairy cattle) and unspecific mastitis, and 2) to compare selection differentials for an udder health index consisting of 5...... to 570 euro per mastitis case and were highest for contagious pathogens such as Staph. aureus and coagulase-negative staphylococci and lowest for Strep. dysgalactiae and Strep. uberis. The value for unspecific mastitis was 231 euro per case. Selection differentials (in euro) were estimated for 4...

  5. Intracellular Diagnostics: Hunting for the Mode of Action of Redox-Modulating Selenium Compounds in Selected Model Systems

    Directory of Open Access Journals (Sweden)

    Dominika Mániková

    2014-08-01

    Full Text Available Redox-modulating compounds derived from natural sources, such as redox active secondary metabolites, are currently of considerable interest in the field of chemoprevention, drug and phytoprotectant development. Unfortunately, the exact and occasionally even selective activity of such products, and the underlying (bio-chemical causes thereof, are often only poorly understood. A combination of the nematode- and yeast-based assays provides a powerful platform to investigate a possible biological activity of a new compound and also to explore the “redox link” which may exist between its activity on the one side and its chemistry on the other. Here, we will demonstrate the usefulness of this platform for screening several selenium and tellurium compounds for their activity and action. We will also show how the nematode-based assay can be used to obtain information on compound uptake and distribution inside a multicellular organism, whilst the yeast-based system can be employed to explore possible intracellular mechanisms via chemogenetic screening and intracellular diagnostics. Whilst none of these simple and easy-to-use assays can ultimately substitute for in-depth studies in human cells and animals, these methods nonetheless provide a first glimpse on the possible biological activities of new compounds and offer direction for more complicated future investigations. They may also uncover some rather unpleasant biochemical actions of certain compounds, such as the ability of the trace element supplement selenite to induce DNA strand breaks.

  6. Positive Selection Acting on a Surface Membrane Protein of the Plant-Pathogenic Phytoplasmas†

    OpenAIRE

    Kakizawa, Shigeyuki; Oshima, Kenro; Jung, Hee-Young; Suzuki, Shiho; Nishigawa, Hisashi; Arashida, Ryo; Miyata, Shin-ichi; Ugaki, Masashi; Kishino, Hirohisa; Namba, Shigetou

    2006-01-01

    Phytoplasmas are plant-pathogenic bacteria that cause numerous diseases. This study shows a strong positive selection on the phytoplasma antigenic membrane protein (Amp). The ratio of nonsynonymous to synonymous substitutions was >1 with all the methods we tested. The clear positive selections imply an important biological role for Amp in host-bacterium interactions.

  7. Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens.

    Science.gov (United States)

    Singh, Tejinder P; Kaur, Gurpreet; Kapila, Suman; Malik, Ravinder K

    2017-01-01

    Adhesion ability of probiotics is the key factor that decides their colonization in the gastrointestinal tract and potential to inhibit pathogens. Therefore, adhesion ability can be considered as a key determinant for probiotic efficacy. Presents study documents the antagonistic activity of viable/untreated, Lithium chloride (LiCl) treated or heat-killed forms of eight probiotic Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. All strains investigated were able to adhere to Caco-2 cells. L. reuteri strains tested were able to inhibit and displace ( P strain L. reuteri LR6 showed the strongest adhesion and pathogen inhibition ability among the eight L. reuteri strains tested. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting the involvement of various mechanisms. The adhesion and antagonistic potential of the probiotic strains were significantly decreased upon exposure to 5 M LiCl, showing that surface molecules, proteinaceous in nature, are involved. The heat-killed forms of the probiotic L. reuteri strains also inhibited the attachment of selected pathogens to Caco-2 cells. In conclusion, in vitro assays showed that L. reuteri strains, as viable or heat-killed forms, are adherent to Caco-2 cells and are highly antagonistic to pathogens tested in which surface associated proteins play an important role.

  8. Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens

    OpenAIRE

    Singh, Tejinder P.; Kaur, Gurpreet; Kapila, Suman; Malik, Ravinder K.

    2017-01-01

    Adhesion ability of probiotics is the key factor that decides their colonization in the gastrointestinal tract and potential to inhibit pathogens. Therefore, adhesion ability can be considered as a key determinant for probiotic efficacy. Presents study documents the antagonistic activity of viable/untreated, Lithium chloride (LiCl) treated or heat-killed forms of eight probiotic Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. All strains investigated were ...

  9. A selectivity filter at the intracellular end of the acid-sensing ion channel pore

    DEFF Research Database (Denmark)

    Lynagh, Timothy; Flood, Emelie; Boiteux, Céline

    2017-01-01

    Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification...

  10. Ortholog-based screening and identification of genes related to intracellular survival.

    Science.gov (United States)

    Yang, Xiaowen; Wang, Jiawei; Bing, Guoxia; Bie, Pengfei; De, Yanyan; Lyu, Yanli; Wu, Qingmin

    2018-04-20

    Bioinformatics and comparative genomics analysis methods were used to predict unknown pathogen genes based on homology with identified or functionally clustered genes. In this study, the genes of common pathogens were analyzed to screen and identify genes associated with intracellular survival through sequence similarity, phylogenetic tree analysis and the λ-Red recombination system test method. The total 38,952 protein-coding genes of common pathogens were divided into 19,775 clusters. As demonstrated through a COG analysis, information storage and processing genes might play an important role intracellular survival. Only 19 clusters were present in facultative intracellular pathogens, and not all were present in extracellular pathogens. Construction of a phylogenetic tree selected 18 of these 19 clusters. Comparisons with the DEG database and previous research revealed that seven other clusters are considered essential gene clusters and that seven other clusters are associated with intracellular survival. Moreover, this study confirmed that clusters screened by orthologs with similar function could be replaced with an approved uvrY gene and its orthologs, and the results revealed that the usg gene is associated with intracellular survival. The study improves the current understanding of intracellular pathogens characteristics and allows further exploration of the intracellular survival-related gene modules in these pathogens. Copyright © 2018. Published by Elsevier B.V.

  11. Disease resistance in Atlantic salmon (Salmo salar: coinfection of the intracellular bacterial pathogen Piscirickettsia salmonis and the sea louse Caligus rogercresseyi.

    Directory of Open Access Journals (Sweden)

    Jean Paul Lhorente

    Full Text Available BACKGROUND: Naturally occurring coinfections of pathogens have been reported in salmonids, but their consequences on disease resistance are unclear. We hypothesized that 1 coinfection of Caligus rogercresseyi reduces the resistance of Atlantic salmon to Piscirickettsia salmonis; and 2 coinfection resistance is a heritable trait that does not correlate with resistance to a single infection. METHODOLOGY: In total, 1,634 pedigreed Atlantic salmon were exposed to a single infection (SI of P. salmonis (primary pathogen or coinfection with C. rogercresseyi (secondary pathogen. Low and high level of coinfection were evaluated (LC = 44 copepodites per fish; HC = 88 copepodites per fish. Survival and quantitative genetic analyses were performed to determine the resistance to the single infection and coinfections. MAIN FINDINGS: C. rogercresseyi significantly increased the mortality in fish infected with P. salmonis (SI mortality = 251/545; LC mortality = 544/544 and HC mortality = 545/545. Heritability estimates for resistance to P. salmonis were similar and of medium magnitude in all treatments (h2SI = 0.23 ± 0.07; h2LC = 0.17 ± 0.08; h2HC = 0.24 ± 0.07. A large and significant genetic correlation with regard to resistance was observed between coinfection treatments (rg LC-HC = 0.99 ± 0.01 but not between the single and coinfection treatments (rg SI-LC = -0.14 ± 0.33; rg SI-HC = 0.32 ± 0.34. CONCLUSIONS/SIGNIFICANCE: C. rogercresseyi, as a secondary pathogen, reduces the resistance of Atlantic salmon to the pathogen P. salmonis. Resistance to coinfection of Piscirickettsia salmonis and Caligus rogercresseyi in Atlantic salmon is a heritable trait. The absence of a genetic correlation between resistance to a single infection and resistance to coinfection indicates that different genes control these processes. Coinfection of different pathogens and resistance to coinfection needs to be considered in future

  12. Disease resistance in Atlantic salmon (Salmo salar): coinfection of the intracellular bacterial pathogen Piscirickettsia salmonis and the sea louse Caligus rogercresseyi.

    Science.gov (United States)

    Lhorente, Jean Paul; Gallardo, José A; Villanueva, Beatriz; Carabaño, María J; Neira, Roberto

    2014-01-01

    Naturally occurring coinfections of pathogens have been reported in salmonids, but their consequences on disease resistance are unclear. We hypothesized that 1) coinfection of Caligus rogercresseyi reduces the resistance of Atlantic salmon to Piscirickettsia salmonis; and 2) coinfection resistance is a heritable trait that does not correlate with resistance to a single infection. In total, 1,634 pedigreed Atlantic salmon were exposed to a single infection (SI) of P. salmonis (primary pathogen) or coinfection with C. rogercresseyi (secondary pathogen). Low and high level of coinfection were evaluated (LC = 44 copepodites per fish; HC = 88 copepodites per fish). Survival and quantitative genetic analyses were performed to determine the resistance to the single infection and coinfections. C. rogercresseyi significantly increased the mortality in fish infected with P. salmonis (SI mortality = 251/545; LC mortality = 544/544 and HC mortality = 545/545). Heritability estimates for resistance to P. salmonis were similar and of medium magnitude in all treatments (h2SI = 0.23 ± 0.07; h2LC = 0.17 ± 0.08; h2HC = 0.24 ± 0.07). A large and significant genetic correlation with regard to resistance was observed between coinfection treatments (rg LC-HC = 0.99 ± 0.01) but not between the single and coinfection treatments (rg SI-LC = -0.14 ± 0.33; rg SI-HC = 0.32 ± 0.34). C. rogercresseyi, as a secondary pathogen, reduces the resistance of Atlantic salmon to the pathogen P. salmonis. Resistance to coinfection of Piscirickettsia salmonis and Caligus rogercresseyi in Atlantic salmon is a heritable trait. The absence of a genetic correlation between resistance to a single infection and resistance to coinfection indicates that different genes control these processes. Coinfection of different pathogens and resistance to coinfection needs to be considered in future research on salmon farming, selective breeding and conservation.

  13. Major histocompatibility complex selection dynamics in pathogen-infected túngara frog (Physalaemus pustulosus) populations.

    Science.gov (United States)

    Kosch, Tiffany A; Bataille, Arnaud; Didinger, Chelsea; Eimes, John A; Rodríguez-Brenes, Sofia; Ryan, Michael J; Waldman, Bruce

    2016-08-01

    Pathogen-driven selection can favour major histocompatibility complex (MHC) alleles that confer immunological resistance to specific diseases. However, strong directional selection should deplete genetic variation necessary for robust immune function in the absence of balancing selection or challenges presented by other pathogens. We examined selection dynamics at one MHC class II (MHC-II) locus across Panamanian populations of the túngara frog, Physalaemus pustulosus, infected by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We compared MHC-II diversity in highland túngara frog populations, where amphibian communities have experienced declines owing to Bd, with those in the lowland region that have shown no evidence of decline. Highland region frogs had MHC variants that confer resistance to Bd. Variant fixation appeared to occur by directional selection rather than inbreeding, as overall genetic variation persisted in populations. In Bd-infected lowland sites, however, selective advantage may accrue to individuals with only one Bd-resistance allele, which were more frequent. Environmental conditions in lowlands should be less favourable for Bd infection, which may reduce selection for specific Bd resistance in hosts. Our results suggest that MHC selection dynamics fluctuate in túngara frog populations as a function of the favourability of habitat to pathogen spread and the vulnerability of hosts to infection. © 2016 The Author(s).

  14. Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

    Science.gov (United States)

    Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

    2009-01-01

    Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

  15. Multiple scales of selection influence the evolutionary emergence of novel pathogens.

    Science.gov (United States)

    Park, Miran; Loverdo, Claude; Schreiber, Sebastian J; Lloyd-Smith, James O

    2013-03-19

    When pathogens encounter a novel environment, such as a new host species or treatment with an antimicrobial drug, their fitness may be reduced so that adaptation is necessary to avoid extinction. Evolutionary emergence is the process by which new pathogen strains arise in response to such selective pressures. Theoretical studies over the last decade have clarified some determinants of emergence risk, but have neglected the influence of fitness on evolutionary rates and have not accounted for the multiple scales at which pathogens must compete successfully. We present a cross-scale theory for evolutionary emergence, which embeds a mechanistic model of within-host selection into a stochastic model for emergence at the population scale. We explore how fitness landscapes at within-host and between-host scales can interact to influence the probability that a pathogen lineage will emerge successfully. Results show that positive correlations between fitnesses across scales can greatly facilitate emergence, while cross-scale conflicts in selection can lead to evolutionary dead ends. The local genotype space of the initial strain of a pathogen can have disproportionate influence on emergence probability. Our cross-scale model represents a step towards integrating laboratory experiments with field surveillance data to create a rational framework to assess emergence risk.

  16. Sensitive and Selective Ratiometric Fluorescence Probes for Detection of Intracellular Endogenous Monoamine Oxidase A.

    Science.gov (United States)

    Wu, Xiaofeng; Li, Lihong; Shi, Wen; Gong, Qiuyu; Li, Xiaohua; Ma, Huimin

    2016-01-19

    Monoamine oxidase A (MAO-A) is known to widely exist in most cell lines in the body, and its dysfunction (unusually high or low levels of MAO-A) is thought to be responsible for several psychiatric and neurological disorders. Thus, a sensitive and selective method for evaluating the relative MAO-A levels in different live cells is urgently needed to better understand the function of MAO-A, but to our knowledge such a method is still lacking. Herein, we rationally design two new ratiometric fluorescence probes (1 and 2) that can sensitively and selectively detect MAO-A. The probes are constructed by incorporating a recognition group of propylamine into the fluorescent skeleton of 1,8-naphthalimide, and the detection mechanism is based on amine oxidation and β-elimination to release the fluorophore (4-hydroxy-N-butyl-1,8-naphthalimide), which is verified by HPLC analysis. Reaction of the probes with MAO-A produces a remarkable fluorescence change from blue to green, and the ratio of fluorescence intensity at 550 and 454 nm is directly proportional to the concentration of MAO-A in the ranges of 0.5-1.5 and 0.5-2.5 μg/mL with detection limits of 1.1 and 10 ng/mL (k = 3) for probes 1 and 2, respectively. Surprisingly, these probes show strong fluorescence responses to MAO-A but almost none to MAO-B (one of two isoforms of MAO), indicating superior ability to distinguish MAO-A from MAO-B. The high specificity of the probes for MAO-A over MAO-B is further supported by different inhibitor experiments. Moreover, probe 1 displays higher sensitivity than probe 2 and is thus investigated to image the relative MAO-A levels in different live cells, such as HeLa and NIH-3T3 cells. It is found that the concentration of endogenous MAO-A in HeLa cells is approximately 1.8 times higher than that in NIH-3T3 cells, which is validated by the result from an ELISA kit. Additionally, the proposed probes may find more uses in the specific detection of MAO-A between the two isoforms of MAO

  17. Evolution of a higher intracellular oxidizing environment in Caenorhabditis elegans under relaxed selection.

    Directory of Open Access Journals (Sweden)

    Joanna Joyner-Matos

    Full Text Available We explored the relationship between relaxed selection, oxidative stress, and spontaneous mutation in a set of mutation-accumulation (MA lines of the nematode Caenorhabditis elegans and in their common ancestor. We measured steady-state levels of free radicals and oxidatively damaged guanosine nucleosides in the somatic tissues of five MA lines for which nuclear genome base substitution and GC-TA transversion frequencies are known. The two markers of oxidative stress are highly correlated and are elevated in the MA lines relative to the ancestor; point estimates of the per-generation rate of mutational decay (ΔM of these measures of oxidative stress are similar to those reported for fitness-related traits. Conversely, there is no significant relationship between either marker of oxidative stress and the per-generation frequencies of base substitution or GC-TA transversion. Although these results provide no direct evidence for a causative relationship between oxidative damage and base substitution mutations, to the extent that oxidative damage may be weakly mutagenic in the germline, the case for condition-dependent mutation is advanced.

  18. The Arginine/Lysine-Rich Element within the DNA-Binding Domain Is Essential for Nuclear Localization and Function of the Intracellular Pathogen Resistance 1.

    Science.gov (United States)

    Yao, Kezhen; Wu, Yongyan; Chen, Qi; Zhang, Zihan; Chen, Xin; Zhang, Yong

    2016-01-01

    The mouse intracellular pathogen resistance 1 (Ipr1) gene plays important roles in mediating host immunity and previous work showed that it enhances macrophage apoptosis upon mycobacterium infection. However, to date, little is known about the regulation pattern of Ipr1 action. Recent studies have investigated the protein-coding genes and microRNAs regulated by Ipr1 in mouse macrophages, but the structure and the functional motif of the Ipr1 protein have yet to be explored. In this study, we analyzed the domains and functional motif of the Ipr1 protein. The resulting data reveal that Ipr1 protein forms a homodimer and that the Sp100-like domain mediates the targeting of Ipr1 protein to nuclear dots (NDs). Moreover, we found that an Ipr1 mutant lacking the classic nuclear localization signal (cNLS) also translocated into the nuclei, suggesting that the cNLS is not the only factor that directs Ipr1 nuclear localization. Additionally, mechanistic studies revealed that an arginine/lysine-rich element within the DNA-binding domain (SAND domain) is critical for Ipr1 binding to the importin protein receptor NPI-1, demonstrating that this element plays an essential role in mediating the nuclear localization of Ipr1 protein. Furthermore, our results show that this arginine/lysine-rich element contributes to the transcriptional regulation and apoptotic activity of Ipr1. These findings highlight the structural foundations of Ipr1 action and provide new insights into the mechanism of Ipr1-mediated resistance to mycobacterium.

  19. Depletion of autophagy-related genes ATG3 and ATG5 in Tenebrio molitor leads to decreased survivability against an intracellular pathogen, Listeria monocytogenes.

    Science.gov (United States)

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Noh, Mi Young; Kim, Dong Hyun; Kim, Iksoo; Han, Yeon Soo; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung

    2015-01-01

    Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model. © 2014 Wiley Periodicals, Inc.

  20. Selective effect of hydroxyapatite nanoparticles on osteoporotic and healthy bone formation correlates with intracellular calcium homeostasis regulation.

    Science.gov (United States)

    Zhao, Rui; Xie, Pengfei; Zhang, Kun; Tang, Zhurong; Chen, Xuening; Zhu, Xiangdong; Fan, Yujiang; Yang, Xiao; Zhang, Xingdong

    2017-09-01

    Adequate bone substitutes osseointegration has been difficult to achieve in osteoporosis. Hydroxyapatite of the osteoporotic bone, secreted by pathologic osteoblasts, had a smaller crystal size and lower crystallinity than that of the normal. To date, little is known regarding the interaction of synthetic hydroxyapatite nanoparticles (HANPs) with osteoblasts born in bone rarefaction. The present study investigated the biological effects of HANPs on osteoblastic cells derived from osteoporotic rat bone (OVX-OB), in comparison with the healthy ones (SHM-OB). A selective effect of different concentrations of HANPs on the two cell lines was observed that the osteoporotic osteoblasts had a higher tolerance. Reductions in cell proliferation, ALP activity, collagen secretion and osteoblastic gene expressions were found in the SHM-OB when administered with HANPs concentration higher than 25µg/ml. In contrast, those of the OVX-OB suffered no depression but benefited from 25 to 250µg/ml HANPs in a dose-dependent manner. We demonstrated that the different effects of HANPs on osteoblasts were associated with the intracellular calcium influx into the endoplasmic reticulum. The in vivo bone defect model further confirmed that, with a critical HANPs concentration administration, the osteoporotic rats had more and mechanically matured new bone formation than the non-treated ones, whilst the sham rats healed no better than the natural healing control. Collectively, the observed epigenetic regulation of osteoblastic cell function by HANPs has significant implication on defining design parameters for a potential therapeutic use of nanomaterials. In this study, we investigated the biological effects of hydroxyapatite nanoparticles (HANPs) on osteoporotic rat bone and the derived osteoblast. Our findings revealed a previously unrecognized phenomenon that the osteoporotic individuals could benefit from higher concentrations of HANPs, as compared with the healthy individuals. The in

  1. Signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution.

    Directory of Open Access Journals (Sweden)

    Matteo Fumagalli

    2011-11-01

    Full Text Available Previous genome-wide scans of positive natural selection in humans have identified a number of non-neutrally evolving genes that play important roles in skin pigmentation, metabolism, or immune function. Recent studies have also shown that a genome-wide pattern of local adaptation can be detected by identifying correlations between patterns of allele frequencies and environmental variables. Despite these observations, the degree to which natural selection is primarily driven by adaptation to local environments, and the role of pathogens or other ecological factors as selective agents, is still under debate. To address this issue, we correlated the spatial allele frequency distribution of a large sample of SNPs from 55 distinct human populations to a set of environmental factors that describe local geographical features such as climate, diet regimes, and pathogen loads. In concordance with previous studies, we detected a significant enrichment of genic SNPs, and particularly non-synonymous SNPs associated with local adaptation. Furthermore, we show that the diversity of the local pathogenic environment is the predominant driver of local adaptation, and that climate, at least as measured here, only plays a relatively minor role. While background demography by far makes the strongest contribution in explaining the genetic variance among populations, we detected about 100 genes which show an unexpectedly strong correlation between allele frequencies and pathogenic environment, after correcting for demography. Conversely, for diet regimes and climatic conditions, no genes show a similar correlation between the environmental factor and allele frequencies. This result is validated using low-coverage sequencing data for multiple populations. Among the loci targeted by pathogen-driven selection, we found an enrichment of genes associated to autoimmune diseases, such as celiac disease, type 1 diabetes, and multiples sclerosis, which lends credence to the

  2. Signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution.

    Science.gov (United States)

    Fumagalli, Matteo; Sironi, Manuela; Pozzoli, Uberto; Ferrer-Admetlla, Anna; Ferrer-Admettla, Anna; Pattini, Linda; Nielsen, Rasmus

    2011-11-01

    Previous genome-wide scans of positive natural selection in humans have identified a number of non-neutrally evolving genes that play important roles in skin pigmentation, metabolism, or immune function. Recent studies have also shown that a genome-wide pattern of local adaptation can be detected by identifying correlations between patterns of allele frequencies and environmental variables. Despite these observations, the degree to which natural selection is primarily driven by adaptation to local environments, and the role of pathogens or other ecological factors as selective agents, is still under debate. To address this issue, we correlated the spatial allele frequency distribution of a large sample of SNPs from 55 distinct human populations to a set of environmental factors that describe local geographical features such as climate, diet regimes, and pathogen loads. In concordance with previous studies, we detected a significant enrichment of genic SNPs, and particularly non-synonymous SNPs associated with local adaptation. Furthermore, we show that the diversity of the local pathogenic environment is the predominant driver of local adaptation, and that climate, at least as measured here, only plays a relatively minor role. While background demography by far makes the strongest contribution in explaining the genetic variance among populations, we detected about 100 genes which show an unexpectedly strong correlation between allele frequencies and pathogenic environment, after correcting for demography. Conversely, for diet regimes and climatic conditions, no genes show a similar correlation between the environmental factor and allele frequencies. This result is validated using low-coverage sequencing data for multiple populations. Among the loci targeted by pathogen-driven selection, we found an enrichment of genes associated to autoimmune diseases, such as celiac disease, type 1 diabetes, and multiples sclerosis, which lends credence to the hypothesis that some

  3. Rapidly evolving genes in pathogens: methods for detecting positive selection and examples among fungi, bacteria, viruses and protists.

    Science.gov (United States)

    Aguileta, Gabriela; Refrégier, Guislaine; Yockteng, Roxana; Fournier, Elisabeth; Giraud, Tatiana

    2009-07-01

    The ongoing coevolutionary struggle between hosts and pathogens, with hosts evolving to escape pathogen infection and pathogens evolving to escape host defences, can generate an 'arms race', i.e., the occurrence of recurrent selective sweeps that each favours a novel resistance or virulence allele that goes to fixation. Host-pathogen coevolution can alternatively lead to a 'trench warfare', i.e., balancing selection, maintaining certain alleles at loci involved in host-pathogen recognition over long time scales. Recently, technological and methodological progress has enabled detection of footprints of selection directly on genes, which can provide useful insights into the processes of coevolution. This knowledge can also have practical applications, for instance development of vaccines or drugs. Here we review the methods for detecting genes under positive selection using divergence data (i.e., the ratio of nonsynonymous to synonymous substitution rates, d(N)/d(S)). We also review methods for detecting selection using polymorphisms, such as methods based on F(ST) measures, frequency spectrum, linkage disequilibrium and haplotype structure. In the second part, we review examples where targets of selection have been identified in pathogens using these tests. Genes under positive selection in pathogens have mostly been sought among viruses, bacteria and protists, because of their paramount importance for human health. Another focus is on fungal pathogens owing to their agronomic importance. We finally discuss promising directions in pathogen studies, such as detecting selection in non-coding regions.

  4. The influence of genetic drift and selection on quantitative traits in a plant pathogenic fungus.

    Science.gov (United States)

    Stefansson, Tryggvi S; McDonald, Bruce A; Willi, Yvonne

    2014-01-01

    Genetic drift and selection are ubiquitous evolutionary forces acting to shape genetic variation in populations. While their relative importance has been well studied in plants and animals, less is known about their relative importance in fungal pathogens. Because agro-ecosystems are more homogeneous environments than natural ecosystems, stabilizing selection may play a stronger role than genetic drift or diversifying selection in shaping genetic variation among populations of fungal pathogens in agro-ecosystems. We tested this hypothesis by conducting a QST/FST analysis using agricultural populations of the barley pathogen Rhynchosporium commune. Population divergence for eight quantitative traits (QST) was compared with divergence at eight neutral microsatellite loci (FST) for 126 pathogen strains originating from nine globally distributed field populations to infer the effects of genetic drift and types of selection acting on each trait. Our analyses indicated that five of the eight traits had QST values significantly lower than FST, consistent with stabilizing selection, whereas one trait, growth under heat stress (22°C), showed evidence of diversifying selection and local adaptation (QST>FST). Estimates of heritability were high for all traits (means ranging between 0.55-0.84), and average heritability across traits was negatively correlated with microsatellite gene diversity. Some trait pairs were genetically correlated and there was significant evidence for a trade-off between spore size and spore number, and between melanization and growth under benign temperature. Our findings indicate that many ecologically and agriculturally important traits are under stabilizing selection in R. commune and that high within-population genetic variation is maintained for these traits.

  5. Transport of selected bacterial pathogens in agricultural soil and quartz sand.

    Science.gov (United States)

    Schinner, Tim; Letzner, Adrian; Liedtke, Stefan; Castro, Felipe D; Eydelnant, Irwin A; Tufenkji, Nathalie

    2010-02-01

    The protection of groundwater supplies from microbial contamination necessitates a solid understanding of the key factors controlling the migration and retention of pathogenic organisms through the subsurface environment. The transport behavior of five waterborne pathogens was examined using laboratory-scale columns packed with clean quartz at two solution ionic strengths (10 mM and 30 mM). Escherichia coli O157:H7 and Yersinia enterocolitica were selected as representative Gram-negative pathogens, Enterococcus faecalis was selected as a representative Gram-positive organism, and two cyanobacteria (Microcystis aeruginosa and Anabaena flos-aquae) were also studied. The five organisms exhibit differing attachment efficiencies to the quartz sand. The surface (zeta) potential of the microorganisms was characterized over a broad range of pH values (2-8) at two ionic strengths (10 mM and 30 mM). These measurements are used to evaluate the observed attachment behavior within the context of the DLVO theory of colloidal stability. To better understand the possible link between bacterial transport in model quartz sand systems and natural soil matrices, additional experiments were conducted with two of the selected organisms using columns packed with loamy sand obtained from an agricultural field. This investigation highlights the need for further characterization of waterborne pathogen surface properties and transport behavior over a broader range of environmentally relevant conditions. Copyright 2008 Elsevier Ltd. All rights reserved.

  6. Positive Selection Pressure Drives Variation on the Surface-Exposed Variable Proteins of the Pathogenic Neisseria.

    Science.gov (United States)

    Wachter, Jenny; Hill, Stuart

    2016-01-01

    Pathogenic species of Neisseria utilize variable outer membrane proteins to facilitate infection and proliferation within the human host. However, the mechanisms behind the evolution of these variable alleles remain largely unknown due to analysis of previously limited datasets. In this study, we have expanded upon the previous analyses to substantially increase the number of analyzed sequences by including multiple diverse strains, from various geographic locations, to determine whether positive selective pressure is exerted on the evolution of these variable genes. Although Neisseria are naturally competent, this analysis indicates that only intrastrain horizontal gene transfer among the pathogenic Neisseria principally account for these genes exhibiting linkage equilibrium which drives the polymorphisms evidenced within these alleles. As the majority of polymorphisms occur across species, the divergence of these variable genes is dependent upon the species and is independent of geographical location, disease severity, or serogroup. Tests of neutrality were able to detect strong selection pressures acting upon both the opa and pil gene families, and were able to locate the majority of these sites within the exposed variable regions of the encoded proteins. Evidence of positive selection acting upon the hypervariable domains of Opa contradicts previous beliefs and provides evidence for selection of receptor binding. As the pathogenic Neisseria reside exclusively within the human host, the strong selection pressures acting upon both the opa and pil gene families provide support for host immune system pressure driving sequence polymorphisms within these variable genes.

  7. Antibacterial activity of some medicinal plants against selected human pathogenic bacteria.

    Science.gov (United States)

    Khan, Usman Ali; Rahman, Hazir; Niaz, Zeeshan; Qasim, Muhammad; Khan, Jafar; Tayyaba; Rehman, Bushra

    2013-12-01

    Medicinal plants are traditionally used for the treatment of human infections. The present study was undertaken to investigate Bergenia ciliata, Jasminum officinale, and Santalum album for their potential activity against human bacterial pathogens. B. ciliata, J. officinale, and S. album extracts were prepared in cold and hot water. The activity of plant extracts and selected antibiotics was evaluated against five bacterial pathogens including Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli using agar well diffusion method. Among the three medicinal plants, B. ciliata extracts displayed potential activity against bacterial pathogens. Cold water extract of Bergenia ciliate showed the highest activity against B. subtilis, which is comparable with a zone of inhibition exhibited by ceftriaxone and erythromycin. J. officinale and S. album extracts demonstrated variable antibacterial activity. Further studies are needed to explore the novel antibacterial bioactive molecules.

  8. Exposure of extensively farmed wild boars (Sus scrofa scrofa) to selected pig pathogens in Greece.

    Science.gov (United States)

    Marinou, K A; Papatsiros, V G; Gkotsopoulos, E K; Odatzoglou, P K; Athanasiou, L V

    2015-06-01

    Increased density and distribution of wild boar populations are likely to promote interactions and transmission of certain pathogens, not only among wild boar but also from wild boar to livestock or humans and vice versa. The purpose of this study was to determine seroprevalence against seven selected pathogens in wild boar living in four different areas in Greece. In total, 359 serum samples were collected from extensively farmed wild boar (Sus scrofa scrofa) originating from four distinct geographical areas throughout Greece from April 2012 to August 2013. Samples were tested for antibodies to Actinobacillus pleuropneumoniae, African swine fever virus (ASFV), Aujeszky's disease virus (ADV), classical swine fever virus (CSFV), Erysipelothrix rhusiopathiae, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). Prevalence was compared among the four regions using Fisher's exact test. Low overall seropositivities of 2.4% and 5.6% were detected for E. rhusiopathiae and PRRSV, respectively, higher ones for ADV (32.0%) and the highest (72.5% and 90.5%) for M. hyopneumoniae and A. pleuropneumoniae, respectively. All sera tested were found negative for antibodies directed against CSFV and ASFV. This is the first report of exposure of wild boars to selected pig pathogens in Greece. These results are indicative of the circulation of these pathogens in Greece with the exception of CSFV and ASFV and suggestive of the potential role of wild boars on their maintenance and transmission to their domestic counterparts and vice versa.

  9. Effect of phenolic compounds on the growth of selected probiotic and pathogenic bacteria.

    Science.gov (United States)

    Pacheco-Ordaz, R; Wall-Medrano, A; Goñi, M G; Ramos-Clamont-Montfort, G; Ayala-Zavala, J F; González-Aguilar, G A

    2018-01-01

    Fruit extracts from different tissues (pulp, seed and peel) have shown antimicrobial and prebiotic activities related to their phenolic profile, although structure-specific evaluations have not been reported yet. The effect of five phenolic compounds (catechin and gallic, vanillic, ferulic and protocatechuic acids) identified in different fruits, particularly in mango, was evaluated on the growth of two probiotic (Lactobacillus rhamnosusGG ATCC 53103 and Lactobacillus acidophilusNRRLB 4495) and two pathogenic (Escherichia coli 0157:H7 ATCC 43890 and Salmonella enterica serovar Typhimurium ATCC 14028) bacteria. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of phenolic acids ranged from 15-20 mmol l -1 and 20-30 mmol l -1 against E. coli and S. Typhimurium, respectively. For catechin, the MIC and MBC were 35 mmol l -1 and >35 mmol l -1 against E. coli and S. Typhimurium, respectively. The presence of catechin and gallic, protocatechuic and vanillic acids in MRS broth without dextrose allowed the growth of lactobacilli. Catechin combined with protocatechuic or vanillic acid mildly allowed the growth of both probiotics. In conclusion, phenolic compounds can selectively inhibit the growth of pathogenic bacteria without affecting the viability of probiotics. This study provides relevant information about the effects of phenolic compounds commonly present in fruit and vegetables on the growth of probiotic and pathogenic bacteria. The compounds selectively allowed the growth of probiotic lactobacilli (Lactobacillus rhamnosus GG and Lactobacillus acidophilus) and inhibited pathogenic bacteria (Escherichia coli and Salmonella Typhimurium) at the same concentration (20 mmol l -1 ). These findings can contribute to the formulation of nutraceutical products, such as synbiotics, that can restore or maintain an optimal composition of human microbiota, potentially improving the overall health of the consumer. © 2017 The

  10. Mycelial growth rate and toxin production in the seed pathogen Pyrenophora semeniperda: Resource trade-offs and temporally varying selection

    Science.gov (United States)

    S. E. Meyer; M. Masi; S. Clement; T. L. Davis; J. Beckstead

    2015-01-01

    Pyrenophora semeniperda, an important pathogen in Bromus tectorum seed banks in semi-arid western North America, exhibits >4-fold variation in mycelial growth rate. Host seeds exhibit seasonal changes in dormancy that affect the risk of pathogen-caused mortality. The hypothesis tested is that contrasting seed dormancy phenotypes select for contrasting strategies...

  11. Genome-Wide Survey of Genes Under Positive Selection in Avian Pathogenic Escherichia coli Strains.

    Science.gov (United States)

    Rojas, Thaís Cabrera Galvão; Lobo, Francisco Pereira; Hongo, Jorge Augusto; Vicentini, Renato; Verma, Renu; Maluta, Renato Pariz; da Silveira, Wanderley Dias

    2017-05-01

    The ability to obtain bacterial genomes from the same host has allowed for comparative studies that help in the understanding of the molecular evolution of specific pathotypes. Avian pathogenic Escherichia coli (APEC) is a group of extraintestinal strains responsible for causing colibacillosis in birds. APEC is also suggested to possess a role as a zoonotic agent. Despite its importance, APEC pathogenesis still has several cryptic pathogenic processes that need to be better understood. In this work, a genome-wide survey of eight APEC strains for genes with evidence of recombination revealed that ∼14% of the homologous groups evaluated present signs of recombination. Enrichment analyses revealed that nine Gene Ontology (GO) terms were significantly more represented in recombinant genes. Among these GO terms, several were noted to be ATP-related categories. The search for positive selection in these APEC genomes revealed 32 groups of homologous genes with evidence of positive selection. Among these groups, we found several related to cell metabolism, as well as several uncharacterized genes, beyond the well-known virulence factors ompC, lamB, waaW, waaL, and fliC. A GO term enrichment test showed a prevalence of terms related to bacterial cell contact with the external environment (e.g., viral entry into host cell, detection of virus, pore complex, bacterial-type flagellum filament C, and porin activity). Finally, the genes with evidence of positive selection were retrieved from genomes of non-APEC strains and tested as were done for APEC strains. The result revealed that none of the groups of genes presented evidence of positive selection, confirming that the analysis was effective in inferring positive selection for APEC and not for E. coli in general, which means that the study of the genes with evidence of positive selection identified in this study can contribute for the better understanding of APEC pathogenesis processes.

  12. [Detection of biofilm formation by selected pathogens relevant to the food industry].

    Science.gov (United States)

    Šilhová-Hrušková, L; Moťková, P; Šilha, D; Vytřasová, J

    2015-09-01

    Detection of biofilm formation by microbial pathogens relevant to the food industry and comparison of biofilm formation under different conditions of culture. The following microorganisms were selected for the study: Staphylococcus aureus, Listeria innocua, Listeria ivanovii, Cronobacter sakazakii, Cronobacter muytjensii, Arcobacter butzleri, Arcobacter cryaerophilus, Campylobacter jejuni, and Campylobacter coli. To detect biofilm formation the microtiter plate assay, as described by Christensen and culture on stainless steel coupons were used. The biofilm forming capacity was confirmed in all microorganisms tested, both on the microtiter plates and stainless steel coupons. Biofilm formation was influenced by the culture medium, material used, and culture duration as well as by the test microorganism. It was found that different species and strains of the same genus differ in biofilm formation. Differences were also found between the collection strains and isolates from the environment. Some bacteria tended to form biofilm more readily on the surface of the polyethylene microtiter plates and less readily on stainless steel coupons while others appeared to have an opposite tendency. Some pathogens were able to increase the planktonic cell density in the initial suspension even by three orders of magnitude within 72 hours while producing plenty of biofilm. The study of biofilm formation by high risk pathogens is of utmost importance, not only to the food industry. From the obtained results, it is evident that bacterial biofilms form rapidly (within 24 hours in the present study). Due to their architecture, these biofilms are difficult to eradicate, and therefore, it is crucial to prevent biofilm formation.

  13. Evaluation of antimicrobial efficacy of nano coated silver-titania metallic plates against selective pathogens

    Directory of Open Access Journals (Sweden)

    Mohamad, S.M.

    2012-01-01

    Full Text Available Aim: Nanotechnology is an increasingly growing field with its current application in Science and Technology for the purpose of manufacture of novel materials at the nanoscale level. Silver-Titania nanoparticles (AgTiO2-NPs have been known to have inhibitory and bactericidal effects.Methodology and Results: In the present study, stable silver-titania nanoparticles coated metallic blocks were prepared for testing their efficacy against selected bacterial pathogens like Escherichia coli and Staphylococcus aureus. In the experimental part, the bacterial pathogens were inoculated on silver-titania nanoparticle coated blocks and the treatment was carried out in „0‟ time and „24‟ h interval and were enumerated.Conclusion, significance and impact of study:The results were compared with the control (uncoated metallic blocks and analyzed by using Japanese Industrial Standard (JIS Z2801:2000 method. From this study, it was concluded that silver-titania nanoparticles has inhibitory effect on bacterial pathogen tested.

  14. In vitro effects of food extracts on selected probiotic and pathogenic bacteria.

    Science.gov (United States)

    Sutherland, Juliet; Miles, Michelle; Hedderley, Duncan; Li, Jessie; Devoy, Sarah; Sutton, Kevin; Lauren, Denis

    2009-12-01

    A panel of 148 extracts from 37 food products was prepared using organic and aqueous solvents and both neutral and acidic conditions. The panel of food products tested included fruits, vegetables, grains, herbs and spices, most of which are common in a normal European-style diet. The impact of these extracts on the growth of selected probiotic bacteria (Lactobacillus reuteri, Lactobacillus rhamnosus, Bifidobacteria lactis) and pathogenic bacteria (Escherichia coli 0157:H7 and Escherichia coli LF82) was assessed using a standard minimum inhibitory concentration method. The results showed that aqueous extractions of garlic and black peppercorns significantly enhanced the growth of one strain of probiotic bacteria (L. reuteri) whilst inhibiting both pathogenic strains of E. coli at a 1:50 dilution. Aqueous extracts of banana, apple and orange all enhanced the growth of the three probiotic strains significantly, and inhibited the pathogens to approximately 80% of the controls (not significant). Both aqueous and organic extractions of ginger significantly inhibited the growth of one or both E. coli strains, respectively (also at the 1:50 dilution).

  15. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

    OpenAIRE

    Raymond, Fr?d?ric; Boisvert, S?bastien; Roy, Ga?tan; Ritt, Jean-Fran?ois; L?gar?, Danielle; Isnard, Amandine; Stanke, Mario; Olivier, Martin; Tremblay, Michel J.; Papadopoulou, Barbara; Ouellette, Marc; Corbeil, Jacques

    2011-01-01

    The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae a...

  16. Finding candidate genes under positive selection in Non-model species: examples of genes involved in host specialization in pathogens.

    Science.gov (United States)

    Aguileta, G; Lengelle, J; Marthey, S; Chiapello, H; Rodolphe, F; Gendrault, A; Yockteng, R; Vercken, E; Devier, B; Fontaine, M C; Wincker, P; Dossat, C; Cruaud, C; Couloux, A; Giraud, T

    2010-01-01

    Numerous genes in diverse organisms have been shown to be under positive selection, especially genes involved in reproduction, adaptation to contrasting environments, hybrid inviability, and host-pathogen interactions. Looking for genes under positive selection in pathogens has been a priority in efforts to investigate coevolution dynamics and to develop vaccines or drugs. To elucidate the functions involved in host specialization, here we aimed at identifying candidate sequences that could have evolved under positive selection among closely related pathogens specialized on different hosts. For this goal, we sequenced c. 17,000-32,000 ESTs from each of four Microbotryum species, which are fungal pathogens responsible for anther smut disease on host plants in the Caryophyllaceae. Forty-two of the 372 predicted orthologous genes showed significant signal of positive selection, which represents a good number of candidate genes for further investigation. Sequencing 16 of these genes in 9 additional Microbotryum species confirmed that they have indeed been rapidly evolving in the pathogen species specialized on different hosts. The genes showing significant signals of positive selection were putatively involved in nutrient uptake from the host, secondary metabolite synthesis and secretion, respiration under stressful conditions and stress response, hyphal growth and differentiation, and regulation of expression by other genes. Many of these genes had transmembrane domains and may therefore also be involved in pathogen recognition by the host. Our approach thus revealed fruitful and should be feasible for many non-model organisms for which candidate genes for diversifying selection are needed.

  17. Synergistically enhanced selective intracellular uptake of anticancer drug carrier comprising folic acid-conjugated hydrogels containing magnetite nanoparticles

    Science.gov (United States)

    Kim, Haneul; Jo, Ara; Baek, Seulgi; Lim, Daeun; Park, Soon-Yong; Cho, Soo Kyung; Chung, Jin Woong; Yoon, Jinhwan

    2017-01-01

    Targeted drug delivery has long been extensively researched since drug delivery and release at the diseased site with minimum dosage realizes the effective therapy without adverse side effects. In this work, to achieve enhanced intracellular uptake of anticancer drug carriers for efficient chemo-therapy, we have designed targeted multifunctional anticancer drug carrier hydrogels. Temperature-responsive poly(N-isopropylacrylamide) (PNIPAm) hydrogel core containing superparamagnetic magnetite nanoparticles (MNP) were prepared using precipitation polymerization, and further polymerized with amine-functionalized copolymer shell to facilitate the conjugation of targeting ligand. Then, folic acid, specific targeting ligand for cervical cancer cell line (HeLa), was conjugated on the hydrogel surface, yielding the ligand conjugated hybrid hydrogels. We revealed that enhanced intracellular uptake by HeLa cells in vitro was enabled by both magnetic attraction and receptor-mediated endocytosis, which were contributed by MNP and folic acid, respectively. Furthermore, site-specific uptake of the developed carrier was confirmed by incubating with several other cell lines. Based on synergistically enhanced intracellular uptake, efficient cytotoxicity and apoptotic activity of HeLa cells incubated with anticancer drug loaded hybrid hydrogels were successfully achieved. The developed dual-targeted hybrid hydrogels are expected to provide a platform for the next generation intelligent drug delivery systems.

  18. Comparative Genomics of Smut Pathogens: Insights From Orphans and Positively Selected Genes Into Host Specialization

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    Juliana Benevenuto

    2018-04-01

    Full Text Available Host specialization is a key evolutionary process for the diversification and emergence of new pathogens. However, the molecular determinants of host range are poorly understood. Smut fungi are biotrophic pathogens that have distinct and narrow host ranges based on largely unknown genetic determinants. Hence, we aimed to expand comparative genomics analyses of smut fungi by including more species infecting different hosts and to define orphans and positively selected genes to gain further insights into the genetics basis of host specialization. We analyzed nine lineages of smut fungi isolated from eight crop and non-crop hosts: maize, barley, sugarcane, wheat, oats, Zizania latifolia (Manchurian rice, Echinochloa colona (a wild grass, and Persicaria sp. (a wild dicot plant. We assembled two new genomes: Ustilago hordei (strain Uhor01 isolated from oats and U. tritici (strain CBS 119.19 isolated from wheat. The smut genomes were of small sizes, ranging from 18.38 to 24.63 Mb. U. hordei species experienced genome expansions due to the proliferation of transposable elements and the amount of these elements varied among the two strains. Phylogenetic analysis confirmed that Ustilago is not a monophyletic genus and, furthermore, detected misclassification of the U. tritici specimen. The comparison between smut pathogens of crop and non-crop hosts did not reveal distinct signatures, suggesting that host domestication did not play a dominant role in shaping the evolution of smuts. We found that host specialization in smut fungi likely has a complex genetic basis: different functional categories were enriched in orphans and lineage-specific selected genes. The diversification and gain/loss of effector genes are probably the most important determinants of host specificity.

  19. Antibacterial effects of hydrogen peroxide and silver composition on selected pathogenic enterobacteriaceae

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    Mojtaba Davoudi

    2012-01-01

    Full Text Available Aims: Antibacterial effects of hydrogen peroxide and silver composition on selected pathogenic enterobacteriaceae was investigated in this study. Materials and Methods: The efficacy of 30 ppb silver in 0.3% hydrogen peroxide solution for inactivation of selected Enterobacteriaceae, including Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae was assessed for 72 hours in a designated nutrient broth medium and steel surface. The bactericidal growth ability was determined for each bacterium genus by the conventional colony count method and turbidimetry via an optical density (OD assay at 450 nm in a time interval of 24 hours. Results: Suspensions of K.pneumoniae, and P.mirabilis showed a significant OD reduction at three 24-hour intervals (CI = 0.95; P < 0.05, for both, along with blocked growth in a designated broth medium during 24 to 48 hours of exposure. The disinfectant was also significantly efficient for inactivating of the mentioned bacteria on steel surfaces after a 15-minute time exposure (CI = 0.95; P < 0.05. For E.coli, the OD decreased slightly during the initial exposure time, but increased after 24 hours. Viable E.coli cells were proved by colonies grown on the plate. A qualitative surface decontamination test showed that three pathogenic bacteria were inactivated significantly after disinfectant exposure (CI = 0.95, P < 0.05. Conclusions: In conclusion, a combination of hydrogen peroxide and silver ions was proposed as a strong disinfecting agent both in suspensions and on the surfaces against these three important human pathogens.

  20. In vitro antimicrobial activity of essential oils from aromatic plants against selected foodborne pathogens.

    Science.gov (United States)

    Rota, C; Carramiñana, J J; Burillo, J; Herrera, A

    2004-06-01

    The purpose of this study was to examine the effectiveness of selected essential oils for the control of growth and survival of pathogenic microorganisms of significant importance in food hygiene and to determine whether the antimicrobial effect was due to the major compounds of the oils. MIC and MBC were determined by the tube dilution method. Essential oils from Thymus vulgaris from Spain and France, Salvia sclarea, Salvia officinalis, Salvia lavandulifolia, Lavandula latifolia, Lavandula angustifolia, three hybrids of Lavandula latifolia x Lavandula angustifolia (Lavandin 'Super', Lavandin 'Abrialis', and Lavandin 'Grosso'), Rosmarinus officinalis, Hissopus officinalis, and Satureja montana were evaluated. Inhibition ranged from the strong activity of Satureja montana and Thymus vulgaris (France) to no inhibition with Salvia sclarea and Hissopus officinalis for each of the test strains: Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli O157:H7, Yersinia enterocolitica, Shigella flexneri, Listeria monocytogenes serovar 4b, and Staphylococcus aureus. Because some of the essential oils were highly inhibitory in small quantities to selected pathogenic microorganisms, they may provide alternatives to conventional antimicrobial additives in foods.

  1. High throughput phenotypic selection of Mycobacterium tuberculosis mutants with impaired resistance to reactive oxygen species identifies genes important for intracellular growth.

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    Olga Mestre

    Full Text Available Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS. Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

  2. Intrarenal transfer of an intracellular fluorescent fusion of angiotensin II selectively in proximal tubules increases blood pressure in rats and mice

    Science.gov (United States)

    Li, Xiao C.; Cook, Julia L.; Rubera, Isabelle; Tauc, Michel; Zhang, Fan

    2011-01-01

    The present study tested the hypothesis that intrarenal adenoviral transfer of an intracellular cyan fluorescent fusion of angiotensin II (ECFP/ANG II) selectively in proximal tubules of the kidney increases blood pressure by activating AT1 (AT1a) receptors. Intrarenal transfer of ECFP/ANG II was induced in the superficial cortex of rat and mouse kidneys, and the sodium and glucose cotransporter 2 (sglt2) promoter was used to drive ECFP/ANG II expression selectively in proximal tubules. Intrarenal transfer of ECFP/ANG II induced a time-dependent, proximal tubule-selective expression of ECFP/ANG II in the cortex, which peaked at 2 wk and was sustained for 4 wk. ECFP/ANG II expression was low in the glomeruli and the entire medulla and was absent in the contralateral kidney or extrarenal tissues. At its peak of expression in proximal tubules at day 14, ANG II was increased by twofold in the kidney (P tubules (P kidney, and proximal tubule ANG II, or sodium excretion. These results provide evidence that proximal tubule-selective transfer of an intracellular ANG II fusion protein increases blood pressure by activating AT1a receptors and increasing sodium reabsorption in proximal tubules. PMID:21307128

  3. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

    NARCIS (Netherlands)

    Ortega, Alvaro D.; Quereda, Juan J; Pucciarelli, M Graciela; García-del Portillo, Francisco

    2014-01-01

    Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles

  4. Pathogenic and Saprophytic Leptospira Species in Water and Soils from Selected Urban Sites in Peninsular Malaysia

    Science.gov (United States)

    Benacer, Douadi; Woh, Pei Yee; Mohd Zain, Siti Nursheena; Amran, Fairuz; Thong, Kwai Lin

    2013-01-01

    Leptospira species were studied in water and soils from selected urban sites in Malaysia. A total of 151 water (n=121) and soil (n=30) samples were collected from 12 recreational lakes and wet markets. All samples were filtered and inoculated into semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media supplemented with additional 5-fluorouracil. The cultures were then incubated at 30°C and observed under a dark field microscope with intervals of 10 days. A PCR assay targeting the rrs gene was used to confirm the genus Leptospira among the isolates. Subsequently, the pathogenic status of the isolates was determined using primer sets G1/G2 and Sapro1/Sapro2, which target the secY and rrs genes, respectively. The isolates were identified at serogroup level using the microscopic agglutination test (MAT) while their genetic diversity was assessed by pulsed field gel electrophoresis (PFGE). Based on dark field microscopy, 23.1% (28/121) water and 23.3% (7/30) soil cultures were positive for Leptospira spp. Of the 35 positive cultures, only 8 were pure and confirmed as Leptospira genus by PCR assay. Two out of 8 isolates were confirmed as pathogenic, 5 were saprophytic and one was intermediate. These 8 isolates were negative for the 25 reference hyperimmune rabbit sera tested in the MAT. PFGE showed that all 8 of these environmental Leptospira spp. were genetically diverse. In conclusion, the presence of pathogenic Leptospira spp. in the urban Malaysian environment may indicate and highlight the importance of water screening, especially in recreational lakes, in order to minimize any chance of Leptospira infection. PMID:23363618

  5. Natural selection on coding and noncoding DNA sequences is associated with virulence genes in a plant pathogenic fungus.

    Science.gov (United States)

    Rech, Gabriel E; Sanz-Martín, José M; Anisimova, Maria; Sukno, Serenella A; Thon, Michael R

    2014-09-04

    Natural selection leaves imprints on DNA, offering the opportunity to identify functionally important regions of the genome. Identifying the genomic regions affected by natural selection within pathogens can aid in the pursuit of effective strategies to control diseases. In this study, we analyzed genome-wide patterns of selection acting on different classes of sequences in a worldwide sample of eight strains of the model plant-pathogenic fungus Colletotrichum graminicola. We found evidence of selective sweeps, balancing selection, and positive selection affecting both protein-coding and noncoding DNA of pathogenicity-related sequences. Genes encoding putative effector proteins and secondary metabolite biosynthetic enzymes show evidence of positive selection acting on the coding sequence, consistent with an Arms Race model of evolution. The 5' untranslated regions (UTRs) of genes coding for effector proteins and genes upregulated during infection show an excess of high-frequency polymorphisms likely the consequence of balancing selection and consistent with the Red Queen hypothesis of evolution acting on these putative regulatory sequences. Based on the findings of this work, we propose that even though adaptive substitutions on coding sequences are important for proteins that interact directly with the host, polymorphisms in the regulatory sequences may confer flexibility of gene expression in the virulence processes of this important plant pathogen. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  6. Contamination of herbal medicinal products marketed in Kaduna metropolis with selected pathogenic bacteria.

    Science.gov (United States)

    Abba, Danladi; Inabo, Helen I; Yakubu, Sabo E; Olonitola, Olayeni S

    2008-10-25

    The study aimed to evaluate the bacterial contamination of powdered herbal medicinal preparations sourced from identified herbal retail outlets in different parts of Kaduna metropolis. The assessments of the contamination of the herbal products were carried out using standard procedures: total aerobic bacterial plate count, measurement of some physical parameters, isolation and characterization of selected bacterial pathogens etc. The results showed that out of a total of 150, 70 (46.67%) herbal remedies were contaminated with Salmonella typhi, twenty nine (19.33%) with Shigella spp. Eighty eight (58.67%) and 98 (65.33%) were contaminated with Escherichia coli and Staphylococcus aureus, respectively. The total aerobic plate count results showed that the highest average count of > 5x10(7) cfu/g was found in 89 (59.33%) of the preparations, while average plate count of < or = 5x10(7) cfu/g was found in 42 (28%) and no bacterial count was obtained in 19 (12.67%) of the preparations. Correlation was positive (P = 0.01; r = +0.109) between the physical parameters tested and the bacterial load. Antibacterial activities result of some common antibiotics showed that all the antibiotics had activities on the test bacterial isolates at various minimum inhibitory concentrations. Most traditionally prepared herbal medications in Kaduna state are likely to be contaminated with a wide variety of potentially pathogenic bacteria. The quality assurance of these products should be thoroughly enforced and monitored in the production and distribution of herbal preparations.

  7. Balancing selection at the tomato RCR3 Guardee gene family maintains variation in strength of pathogen defense.

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    Anja C Hörger

    Full Text Available Coevolution between hosts and pathogens is thought to occur between interacting molecules of both species. This results in the maintenance of genetic diversity at pathogen antigens (or so-called effectors and host resistance genes such as the major histocompatibility complex (MHC in mammals or resistance (R genes in plants. In plant-pathogen interactions, the current paradigm posits that a specific defense response is activated upon recognition of pathogen effectors via interaction with their corresponding R proteins. According to the "Guard-Hypothesis," R proteins (the "guards" can sense modification of target molecules in the host (the "guardees" by pathogen effectors and subsequently trigger the defense response. Multiple studies have reported high genetic diversity at R genes maintained by balancing selection. In contrast, little is known about the evolutionary mechanisms shaping the guardee, which may be subject to contrasting evolutionary forces. Here we show that the evolution of the guardee RCR3 is characterized by gene duplication, frequent gene conversion, and balancing selection in the wild tomato species Solanum peruvianum. Investigating the functional characteristics of 54 natural variants through in vitro and in planta assays, we detected differences in recognition of the pathogen effector through interaction with the guardee, as well as substantial variation in the strength of the defense response. This variation is maintained by balancing selection at each copy of the RCR3 gene. Our analyses pinpoint three amino acid polymorphisms with key functional consequences for the coevolution between the guardee (RCR3 and its guard (Cf-2. We conclude that, in addition to coevolution at the "guardee-effector" interface for pathogen recognition, natural selection acts on the "guard-guardee" interface. Guardee evolution may be governed by a counterbalance between improved activation in the presence and prevention of auto-immune responses in

  8. Selective extraction of intracellular components from the microalga Chlorella vulgaris by combined pulsed electric field-temperature treatment.

    Science.gov (United States)

    Postma, P R; Pataro, G; Capitoli, M; Barbosa, M J; Wijffels, R H; Eppink, M H M; Olivieri, G; Ferrari, G

    2016-03-01

    The synergistic effect of temperature (25-65 °C) and total specific energy input (0.55-1.11 kWh kgDW(-1)) by pulsed electric field (PEF) on the release of intracellular components from the microalgae Chlorella vulgaris was studied. The combination of PEF with temperatures from 25 to 55 °C resulted in a conductivity increase of 75% as a result of cell membrane permeabilization. In this range of temperatures, 25-39% carbohydrates and 3-5% proteins release occurred and only for carbohydrate release a synergistic effect was observed at 55 °C. Above 55 °C spontaneous cell lysis occurred without PEF. Combined PEF-temperature treatment does not sufficiently disintegrate the algal cells to release both carbohydrates and proteins at yields comparable to the benchmark bead milling (40-45% protein, 48-58% carbohydrates). Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Antimicrobial properties of commercial annatto extracts against selected pathogenic, lactic acid, and spoilage microorganisms.

    Science.gov (United States)

    Galindo-Cuspinera, Veronica; Westhoff, Dennis C; Rankin, Scott A

    2003-06-01

    Annatto preparations are used to impart distinctive flavor and color to foods and are a primary colorant in dairy foods such as cheese and butter. There are several reports indicating that certain fractions of the annatto plant have biological activities against microorganisms of significance in food fermentation, food preservation, and human health. However, little is reported describing the nature of the antimicrobial compound(s) or their potential presence in commercial annatto colorant preparations. This study was conducted to determine whether commonly available annatto extracts are capable of influencing the outgrowth of selected lactic acid, spoilage, and pathogenic microorganisms. Disk diffusion and tube macrodilution techniques were used to determine the MICs and MBCs of double-strength water-soluble annatto extracts. Standard antibiotic disks were used as controls for the disk diffusion assay. The results demonstrate that annatto has an inhibitory effect on Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus, with MICs of 0.08, 0.31, and 0.16% (vol/vol) and diameters of inhibition of 9 to 10, 12 to 13, and 15 to 16 mm, respectively. A concentration of 0.63% (vol/vol) inhibited the growth of Streptococcus thermophilus, Lactobacillus casei subsp. casei, Lactococcus lactis, and Paenibacillus polymyxa. The MICs for Listeria monocytogenes and Enterococcus durans were 1.25 and 2.5% (vol/vol), respectively. No activity was detected against Lactobacillus plantarum, Bifidobacterium bifidum, yeasts, or selected gram-negative bacteria.

  10. Prevalence Survey of Selected Bovine Pathogens in Water Buffaloes in the North Region of Brazil

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    Jenevaldo Barbosa da Silva

    2014-01-01

    Full Text Available Although the largest buffalo herd in the occident is in the north region of Brazil, few studies have been conducted to assess the prevalence of selected parasitic diseases in buffalo herd. The present study was therefore conducted to investigate the epidemiological of Toxoplasma gondii, Neospora caninum, Anaplasma marginale, Babesia bigemina, and Babesia bovis in water buffaloes in the north region of Brazil. A total of 4796 buffalo blood samples were randomly collected from five provinces and simultaneously analyzed by the IFAT and ELISA. The serological prevalence of T. gondii and N. caninum was 41.3% and 55.5% in ELISA and 35.7% and 48.8% in IFAT, respectively. The overall prevalence of A. marginale, B. bovis, and B. bigemina was 63%, 25%, and 21% by ELISA and 50.0%, 22.5%, and 18.8% by IFAT, respectively. This study shows valuable information regarding the serological survey of selected bovine pathogens in water buffaloes in the north region of Brazil which will likely be very beneficial for the management and control programs of this disease.

  11. Serum antibody levels against select bacterial pathogens in Atlantic bottlenose dolphins, Tursiops truncatus, from Beaufort NC USA and Charleston Harbor, Charleston, SC, USA.

    Science.gov (United States)

    Beck, B M; Rice, C D

    2003-03-01

    Concern over the emergence of zoonotic diseases in marine organisms is growing. In response to this concern, this study set out to measure antibody activities against bacterial pathogens in Atlantic bottlenose dolphins, Tursiops truncatus, from the coastal estuaries of NC and SC, USA. Individuals from Charleston SC harbor, a heavily industrialized shipping harbor estuary, and from Beaufort NC, a non-shipping estuary, were examined. Purified IgG was obtained from pooled sera using ammonium sulfate precipitation steps and protein-G procedures, which was then used to generate a panel of IgG-specific monoclonal antibodies. Two of these antibodies, mAbs BB-10-2 (IgG1) and BB-32-2 (IgG2b), were then used to determine total serum IgG concentrations using a sandwich capture ELISA. Circulating IgG levels were variable between individuals and between the two pods. MAb BB-10-2 was then used in an indirect ELISA to determine serum antibody activities against several common marine bacteria as well as the human pathogens E. coli and E. coli strain 0157:H7, Vibrio parahemolyticus, V. vulnificus, V. cholerae, Mycobacteria marinum, M. fortuitum, and M. chelonae. The highest antibody activities were against mycobacteria, two of which are zoonotic pathogens. Males had the highest antibody activities, thus suggesting low cell-mediated immunity against intracellular pathogens in these individuals. T-cell proliferation in response to Con-A, an indicator of cell-mediated immune function, was then measured in the Beaufort population. Males had the lowest proliferation responses, however a negative correlation between antibody activities and T-cell proliferation in individuals could not be established for either of the Mycobacteria species. Overall, antibody activities against all bacteria, including innocuous species such as V. anguillarum, V. natrigens, and M. xenopi were highly variable between individual dolphins and the two pods, with some animals exhibiting very high activities

  12. Functional genomics of intracellular bacteria.

    Science.gov (United States)

    de Barsy, Marie; Greub, Gilbert

    2013-07-01

    During the genomic era, a large amount of whole-genome sequences accumulated, which identified many hypothetical proteins of unknown function. Rapidly, functional genomics, which is the research domain that assign a function to a given gene product, has thus been developed. Functional genomics of intracellular pathogenic bacteria exhibit specific peculiarities due to the fastidious growth of most of these intracellular micro-organisms, due to the close interaction with the host cell, due to the risk of contamination of experiments with host cell proteins and, for some strict intracellular bacteria such as Chlamydia, due to the absence of simple genetic system to manipulate the bacterial genome. To identify virulence factors of intracellular pathogenic bacteria, functional genomics often rely on bioinformatic analyses compared with model organisms such as Escherichia coli and Bacillus subtilis. The use of heterologous expression is another common approach. Given the intracellular lifestyle and the many effectors that are used by the intracellular bacteria to corrupt host cell functions, functional genomics is also often targeting the identification of new effectors such as those of the T4SS of Brucella and Legionella.

  13. The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.

    Directory of Open Access Journals (Sweden)

    Rongman Cai

    2011-08-01

    Full Text Available Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

  14. High Yield of Pathogenic Germline Mutations Causative or Likely Causative of the Cancer Phenotype in Selected Children with Cancer

    NARCIS (Netherlands)

    Diets, Illja J.; Waanders, Esme; Ligtenberg, Marjolijn J.; van Bladel, Diede A. G.; Kamping, Eveline J.; Hoogerbrugge, Peter M.; Hopman, Saskia; Olderode-Berends, Maran J.; Gerkes, Erica H.; Koolen, David A.; Marcelis, Carlo; Santen, Gijs W.; van Belzen, Martine J.; Mordaunt, Dylan; McGregor, Lesley; Thompson, Elizabeth; Kattamis, Antonis; Pastorczak, Agata; Mlynarski, Wojciech; Ilencikova, Denisa; Vulto-van Silfhout, Anneke; Gardeitchik, Thatjana; de Bont, Eveline S.; Loeffen, Jan; Wagner, Anja; Mensenkamp, Arjen R.; Kuiper, Roland P.; Hoogerbrugge, Nicoline; Jongmans, Marjolijn C.

    2018-01-01

    Purpose: In many children with cancer and characteristics suggestive of a genetic predisposition syndrome, the genetic cause is still unknown. We studied the yield of pathogenic mutations by applying whole-exome sequencing on a selected cohort of children with cancer. Experimental Design: To

  15. High Yield of Pathogenic Germline Mutations Causative or Likely Causative of the Cancer Phenotype in Selected Children with Cancer

    NARCIS (Netherlands)

    Diets, I.J.; Waanders, E.; Ligtenberg, M.J.L.; Bladel, D.A.G. van; Kamping, E.J.; Hoogerbrugge, P.M.; Hopman, S.; Olderode-Berends, M.J.; Gerkes, E.H.; Koolen, D.A.; Marcelis, C.L.; Santen, G.W.E.; Belzen, M.J. van; Mordaunt, D.; McGregor, L.; Thompson, E.; Kattamis, A.; Pastorczak, A.; Mlynarski, W.; Ilencikova, D.; Vulto-van Silfhout, A.T.; Gardeitchik, T.; Bont, E.S. de; Loeffen, J.; Wagner, A.; Mensenkamp, A.R.; Kuiper, R.P.; Hoogerbrugge, N.; Jongmans, M.C.

    2018-01-01

    Purpose: In many children with cancer and characteristics suggestive of a genetic predisposition syndrome, the genetic cause is still unknown. We studied the yield of pathogenic mutations by applying whole-exome sequencing on a selected cohort of children with cancer.Experimental Design: To identify

  16. Differences in pathogen colonization and mortality of genetically selected Japanese quail lines subjected to heat stress and Escherichia coli challenge

    Science.gov (United States)

    Japanese quail selected for divergent corticosterone response to restraint stress were evaluated for their resistance to heat stress and aerosol challenge with avian pathogenic Escherichia coli (APEC) to determine the impact of stress response on APEC pathogenesis and colonization with food-borne pa...

  17. Serologic survey for selected arboviruses and other potential pathogens in wildlife from Mexico.

    Science.gov (United States)

    Aguirre, A A; McLean, R G; Cook, R S; Quan, T J

    1992-07-01

    During 1988 and 1989, a serologic survey of wildlife was conducted in northeastern Mexico to determine the presence, prevalence, and distribution of arboviruses and other selected disease agents. Eighty mammal specimens were tested. Antibodies to vesicular stomatitis-Indiana, Venezuelan equine encephalitis-Mena II, Rio Grande virus, and vesicular stomatitis-New Jersey were detected predominantly in small mammals. Deer and mouflon (Ovis musimon) had antibodies to bluetongue and epizootic hemorrhagic disease. Two species had serologic evidence of recent exposure to Francisella tularensis. A white-tailed deer (Odocoileus virginianus) had antibodies to Anaplasma marginale. All specimens tested for antibodies against Yersinia pestis and Brucella abortus were negative. Sera from 315 birds were tested for antibody against five equine encephalitis viruses and six avian pathogens. During 1988, antibodies to Venezuelan equine encephalitis-Mena II, Venezuelan equine encephalitis-TC83, St. Louis encephalitis, eastern equine encephalitis, and western equine encephalitis were detected in birds of several species. Antibodies to Pasteurella multocida and Newcastle disease virus were also detected. Birds from five species presented antibodies to Mycoplasma meleagridis. Specimens tested for M. gallisepticum, M. synoviae, and Chlamydia psittaci were negative. To the best of our knowledge, this survey represents the first serologic evidence of bluetongue, Cache Valley virus, epizootic hemorrhagic disease, Jamestown Canyon virus, vesicular stomatitis-Indiana, vesicular stomatitis-New Jersey, Rio Grande virus, and tularemia reported among wildlife in Mexico.

  18. A field survey on parasites and antibodies against selected pathogens in owned dogs in Lilongwe, Malawi

    Directory of Open Access Journals (Sweden)

    Karin Alvåsen

    2016-07-01

    Full Text Available The aim of this study was to screen for selected parasites and antibody levels against vectorborne pathogens in owned dogs in Lilongwe, Malawi. The study population consisted of 100 dogs; 80 participating in vaccination–spaying campaigns and 20 visiting a veterinary clinic as paying clients. All dogs went through a general physical examination including visual examination for signs of ectoparasites. A total of 100 blood samples were analysed using commercial snap tests and 40 faecal samples by egg flotation in saturated sodium chloride. The sampled dogs had a seroprevalence of 12% for Anaplasma spp., 22% for Ehrlichia spp., 4% for Dirofilaria immitis and 1% for Leishmania spp. Eggs from Ancylostoma spp. were found in 80% of the faecal samples, whereas eggs of Trichuris vulpis, Toxocara canis and Toxascaris leonina were only present in 3%, 8% and 13% of the samples, respectively. Ectoparasites such as Ctenocephalides sp., Trichodectes sp. and ticks were present on 98%, 25% and 11%, respectively, of the campaign dogs. Among client dogs, 35% had Ctenocephalides fleas, 10% had Trichodectes lice and none had ticks. Public education and prophylactic treatment could be used to improve the animal welfare of dogs; this would most likely also have positive impact on public health.

  19. Selected medicinal plants used in herbal industries; their toxicity against pathogenic microorganisms

    International Nuclear Information System (INIS)

    Fazal, H.; Ahmad, M.; Abbasi, B.H.

    2012-01-01

    Multi-drug resistant strains of fungi and bacteria are imposing the need for new drugs. Reliable natural sources with minor side effects are needed to control anti-human pathogenic invaders specially bacteria. Given the demands for natural products that are inherently safe and environmentally compatible, the advancement in antimicrobial potential has provided a better alternative to synthetic resistance antibiotics. In the present investigation such types of medicinal plants were selected for analyses that are used by local herbal practioners for multiple diseases. Thirty three extracts of Achillea millefolium, Acorus calamus, Arnebia nobilis, Fumaria indica, Gymnema sylvestre, Origanum vulgare, Paeonia emodi, Peganum harmala, Psoralea corylifolia, Rauwolfia serpentina and Vetiveria zizanioides in chloroform, ethanol and hexane were investigated for their antimicrobial potential. These extracts were tested against eight microorganisms including four gram negative bacterial strains viz., Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi, three gram positive bacterial strains Staphylococcus aureus, Bacillus subtilis and Bacillus cereus and a fungal strain viz., Candida albicans. Majority of the extracts showed marked antimicrobial potential against the tested microorganisms. (author)

  20. A field survey on parasites and antibodies against selected pathogens in owned dogs in Lilongwe, Malawi.

    Science.gov (United States)

    Alvåsen, Karin; Johansson, Sandra M; Höglund, Johan; Ssuna, Richard; Emanuelson, Ulf

    2016-07-29

    The aim of this study was to screen for selected parasites and antibody levels against vectorborne pathogens in owned dogs in Lilongwe, Malawi. The study population consisted of 100 dogs; 80 participating in vaccination-spaying campaigns and 20 visiting a veterinary clinic as paying clients. All dogs went through a general physical examination including visual examination for signs of ectoparasites. A total of 100 blood samples were analysed using commercial snap tests and 40 faecal samples by egg flotation in saturated sodium chloride. The sampled dogs had a seroprevalence of 12% for Anaplasma spp., 22% for Ehrlichia spp., 4% for Dirofilaria immitis and 1% for Leishmania spp. Eggs from Ancylostoma spp. were found in 80% of the faecal samples, whereas eggs of Trichuris vulpis, Toxocara canis and Toxascaris leonina were only present in 3%, 8% and 13% of the samples, respectively. Ectoparasites such as Ctenocephalides sp., Trichodectes sp. and ticks were present on 98%, 25% and 11%, respectively, of the campaign dogs. Among client dogs, 35% had Ctenocephalides fleas, 10% had Trichodectes lice and none had ticks. Public education and prophylactic treatment could be used to improve the animal welfare of dogs; this would most likely also have positive impact on public health.

  1. INFLUENCE OF INCUBATION TIME, GAMMA RAYS AND ELECTRON BEAM ON RADIATION RESISTANCE OF SOME SELECTED PATHOGENS

    International Nuclear Information System (INIS)

    EL-HIFNAWI, H.N.; EL-TABLAWY, S.Y.

    2009-01-01

    The effect of different growth phases on the radiation resistance, antibiotic susceptibility and pathogenicity of certain selected pathogens (Escherichia coli, Candida albicans and Staphylococcus aureus) was studied in mice. The obtained results showed that Escherichia coli was slightly more resistant to gamma radiation in 18 h than 24 h or 48 h but it was relatively more resistant to electron beam in 24 h and 48 h than 18 h. Candida albicans showed radiation resistance nearly the same in all incubation times in the case of gamma radiation while for electron beam, its radiation resistance was slightly more in 24 h and 48 h than in 18 h. On the other hand, Staphylococcus aureus recorded much more resistance to gamma radiation in the 48 h than in 24 h or 18 h whereas in the case of electron beam, it was slightly more resistant in 18 h than in 24 h and 48 h.The antibiotic susceptibility of Escherichia coli reported that the exposure to gamma radiation at 3 kGy and electron beam at 6 kGy increase the susceptibility to the nalidixic acid and nitrofurantoin. When Candida albicans was exposed to 3 kGy gamma radiation and 6 kGy electron beam, the same sensitivity to nystatin was observed in comparison with the unexposed one while the sensitivity of Staphylococcus aureus to some antibiotics (amoxicillin, nitrofurantoin and tetracycline) was decreased after exposure to gamma radiation at 0.75 and 2 kGy and electron beam at 6 kGy, but for other antibiotics (trimethoprim/ sulfamethoxazole), the sensitivity was increased at 6 kGy electron beam.The lethality percent recorded after the oral ingestion of the mice with the unexposed Escherichia coli and Candida albicans were 25% and 100%, respectively, and for 6 kGy exposure to electron beam was 0% . The cotaneous disease and abscesses caused by the intradermal injection of the mice with unexposed Staphylococcus aureus was 75% and for 6 kGy exposure to electron beam was 25%.

  2. Soil feedback of exotic savanna grass relates to pathogen absence and mycorrhizal selectivity

    NARCIS (Netherlands)

    Putten, van der W.H.; Kowalchuk, G.A.; Brinkman, E.P.; Doodeman, G.T.A.; Kraaij, van der R.M.; Kamp, A.F.D.; Menting, F.B.J.; Veenendaal, E.M.

    2007-01-01

    Enemy release of exotic plants from soil pathogens has been tested by examining plant¿soil feedback effects in repetitive growth cycles. However, positive soil feedback may also be due to enhanced benefit from the local arbuscular mycorrhizal fungi (AMF). Few studies actually have tested pathogen

  3. Soil feedback of exotic savanna grass relates to pathogen absence and mycorrhizal selectivity.

    NARCIS (Netherlands)

    van der Putten, W.H.; Kowalchuk, G.A.; Doodeman, G.T.A.; van der Kaaij, R.M.; Kamp, A.F.D.; Menting, F.B.J.; Veenendaal, E.M.

    2007-01-01

    Enemy release of exotic plants from soil pathogens has been tested by examining plant-soil feedback effects in repetitive growth cycles. However, positive soil feedback may also be due to enhanced benefit from the local arbuscular mycorrhizal fungi (AMF). Few studies actually have tested pathogen

  4. [A multi-pathogen selective enrichment broth for simultaneous growth of Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes].

    Science.gov (United States)

    Liu, Yuanyuan; Xiao, Xinglong; Yu, Yigang; Chen, Gu; Li, Xiaofeng; Tang, Yuqian; Wu, Hui

    2009-10-01

    A selective enrichment broth (SSL) was formulated to allow simultaneous growth of Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogens. Suitable additive agents were selected by single factor experiment, the enrichment effect of the broth for the three pathogens were evaluated by conventional detection method and real-time PCR. A selective enrichment broth, SSL, was obtained by adding the selective agents, including nalidixic acid, lithium chloride, and potassium tellurite, in the basic broth, and sodium pyruvate and mannitol as the supplemented elements. Recovery of three target pathogens in SSL was obtained within 24 h of incubation at 37 degrees C, yielding cell dnesities of 10(7) - 10(8) CFU/mL. Meanwhile, SSL broth effectively inhibited the growth of non-target organisms. 710 samples were detected by SSL with real-time PCR, and there is no error report. SSL is demonstrated to be a promising new multiplex selective enrichment broth for simultaneous detection of the three most prominent foodborn pathogens by multipathogen detection on a single assay platform.

  5. Antimicrobial susceptibility profile of selected bacteraemic pathogens from private institutions in South Africa.

    Science.gov (United States)

    Brink, Adrian; Moolman, Johan; da Silva, Mark Cruz; Botha, Maria

    2007-04-01

    The National Antimicrobial Surveillance Forum is a continuous surveillance organisation comprising all academic/public and private sector laboratories in South Africa. The antibiotic susceptibility of blood culture isolates of Escherichia coli, Klebsiella pneumoniae, Enterobacter species, Pseudomonas aeruginosa, Acinetobacter baumannii and Staphylococcus aureus from patients in private hospitals in five major centres were investigated. Antimicrobial susceptibility tests were performed by 12 participating laboratories according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Extended-spectrum b-lactamase (ESBL) production was determined in selected species of Enterobacteriaceae irrespective of source. The overall prevalence of ampicillin resistance in blood culture isolates of E. coli (N = 471) was 84%, and 20% were resistant to the fluoroquinolones. Considerable geographical differences were noted between the centres with regard to the K. pneumoniae (N = 636) resistance rates for ceftriaxone and/or cefotaxime (39 - 87%). The most active agents in the Enterobacter spp. (N = 244) were imipenem/meropenem, ertapenem, ciprofloxacin, levofloxacin and cefepime, with 100%, 94%, 88%, 87% and 80% susceptibility, respectively. Carbapenem resistance in P. aeruginosa (N = 382) varied between 42% and 45%, and in the case of A. baumannii (N = 190) resistance varied between 32% and 33% for meropenem and imipenem respectively. The nationwide incidence of oxacillin resistance in S. aureus (N = 629) was 36%. Overall, the prevalence of ESBL production among all isolates of K. pneumoniae was 26% (N = 7 514), while in Enterobacter spp. it was 12% (N = 4 031) and in E. coli 5% (N = 28 412). The data highlight the widespread problem of antibiotic resistance among important bacteraemic pathogens in private institutions in South Africa. Continued surveillance is vital to guide appropriate empirical therapy for invasive infections.

  6. The antimicrobial spectra of selected Penicillia against some pathogenic bacteria and yeasts

    Directory of Open Access Journals (Sweden)

    Shashi Chauhan

    2014-08-01

    Full Text Available The antagonista activity of eight isolaies of penicillia bas been studied against 13 pathogenic organisms, which included 6 Gram-positive bacteria, 4 Gram-negative bacteria and 3 yeasts.

  7. Antibacterial activity of some medicinal plants against selected human pathogenic bacteria

    OpenAIRE

    Khan, Usman Ali; Rahman, Hazir; Niaz, Zeeshan; Qasim, Muhammad; Khan, Jafar; Tayyaba,; Rehman, Bushra

    2013-01-01

    Medicinal plants are traditionally used for the treatment of human infections. The present study was undertaken to investigate Bergenia ciliata, Jasminum officinale, and Santalum album for their potential activity against human bacterial pathogens.

  8. Selection of media for antimicrobial susceptibility testing of fish pathogenic bacteria

    DEFF Research Database (Denmark)

    Dalsgaard, Inger

    2001-01-01

    The available data concerning antimicrobial susceptibility testing of fish pathogens showed that there is no consensus to the basal medium currently being employed. Different media recommended for susceptibility testing of human pathogens (Mueller-Hinton Agar, Tryptone Soya Agar, Antibiotic Medium...... normally used for cultivation of pathogens with specific growth requirements like Flavobacterium species and Renibacterium salmoninarum have been used for susceptibility testing. The Mueller-Hinton Agar and different modifications of this medium was used most frequently in published studies on resistant...... 3, Diagnostic Sensitivity Test Agar) have been used in addition to media (Brain Heart Infusion Agar, Heart Infusion Agar, Columbia Blood Agar) normally utilized for cultivating fastidious bacteria. When testing marine pathogens, sodium chloride or seawater has been included in the media. Media...

  9. Selection of media for antimicrobial susceptibility testing of fish pathogenic bacteria

    DEFF Research Database (Denmark)

    Dalsgaard, Inger

    2001-01-01

    normally used for cultivation of pathogens with specific growth requirements like Flavobacterium species and Renibacterium salmoninarum have been used for susceptibility testing. The Mueller-Hinton Agar and different modifications of this medium was used most frequently in published studies on resistant......The available data concerning antimicrobial susceptibility testing of fish pathogens showed that there is no consensus to the basal medium currently being employed. Different media recommended for susceptibility testing of human pathogens (Mueller-Hinton Agar, Tryptone Soya Agar, Antibiotic Medium...... 3, Diagnostic Sensitivity Test Agar) have been used in addition to media (Brain Heart Infusion Agar, Heart Infusion Agar, Columbia Blood Agar) normally utilized for cultivating fastidious bacteria. When testing marine pathogens, sodium chloride or seawater has been included in the media. Media...

  10. Selection of media for antimicrobial susceptibility testing of fish pathogenic bacteria

    DEFF Research Database (Denmark)

    Dalsgaard, Inger

    2001-01-01

    The available data concerning antimicrobial susceptibility testing of fish pathogens showed that there is no consensus to the basal medium currently being employed. Different media recommended for susceptibility testing of human pathogens (Mueller-Hinton Agar, Tryptone Soya Agar, Antibiotic Medium...... 3, Diagnostic Sensitivity Test Agar) have been used in addition to media (Brain Heart Infusion Agar, Heart Infusion Agar, Columbia Blood Agar) normally utilized for cultivating fastidious bacteria. When testing marine pathogens, sodium chloride or seawater has been included in the media. Media...... normally used for cultivation of pathogens with specific growth requirements like Flavobacterium species and Renibacterium salmoninarum have been used for susceptibility testing. The Mueller-Hinton Agar and different modifications of this medium was used most frequently in published studies on resistant...

  11. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Directory of Open Access Journals (Sweden)

    Suh JS

    2014-03-01

    alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue-engineering purposes. Keywords: protein transduction, low-molecular-weight protamine (LMWP, transcriptional coactivator with PDZ-binding motif (TAZ, selective osteogenesis, bone-tissue engineering

  12. Species of pathogens and drug selection in liver cirrhosis patients with sepsis

    Directory of Open Access Journals (Sweden)

    ZHANG Lan

    2013-11-01

    Full Text Available ObjectiveTo identify the species of pathogens in liver cirrhosis patients with sepsis and screen out the effective antibiotics, and to provide a basis for effective clinical antimicrobial therapy. MethodsBlood samples were collected from liver cirrhosis patients with suspected bloodstream infections and were then injected into the blood culture bottles at the bedside. Blood was cultured using the BacT/Alert 3D system. The isolated strains were identified by the Vitec II or API system (BioMerieux, France. Drug susceptibility tests were performed by Kirby-Bauer method. The pathogens and effective drugs in recent 10 years were analyzed. ResultsA total of 8543 strains of pathogens were isolated from various samples in recent 10 years, and 2065 (24.2% of them were detected from cultured blood, including 1233 (59.7% Gram-negative bacillus strains, 787(38.1% Gram-positive coccus strains, 28(1.4% fungal strains, and 17(08% other pathogenic strains. The drug sensitivity tests showed that Gram-negative bacteria had relatively low resistance to meropenem (746%, imipenem (6.49%, and amikacin (5.27%, extended spectrum beta-lactamase (ESBL-producing strains were more resistant to beta-lactam antibiotics except carbapenems and cefmetazole than ESBL-negative strains, and Gram-positive bacteria were 100% sensitive to vancomycin and teicoplanin. ConclusionGram-negative bacteria are the main pathogens of bloodstream infections in liver cirrhosis patients in our hospital. The pathogens are characterized by diversity and high resistance to many antibiotics. In clinical practice, antimicrobial drugs should be properly used according to the species and drug sensitivity of pathogens, so as to improve the cure rate, and the changing trends in the species and drug sensitivity of pathogens need to be continuously monitored.

  13. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Science.gov (United States)

    Suh, Jin Sook; Lee, Jue Yeon; Choi, Yoon Jung; You, Hyung Keun; Hong, Seong-Doo; Chung, Chong Pyoung; Park, Yoon Jeong

    2014-01-01

    Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue

  14. Does heterogeneity of intracellular Ca[Formula: see text] dynamics underlie speed tuning of direction-selective responses in starburst amacrine cells?

    Science.gov (United States)

    Koizumi, Amane; Poznanski, Roman R

    2016-01-14

    The starburst amacrine cell (SAC) plays a fundamental role in retinal motion perception. In the vertebrate retina, SAC dendrites have been shown to be directionally selective in terms of their Ca[Formula: see text] responses for stimuli that move centrifugally from the soma. The mechanism by which SACs show Ca[Formula: see text] bias for centrifugal motion is yet to be determined with precision. Recent morphological studies support a presynaptic delay in glutamate receptor activation induced Ca[Formula: see text] release from bipolar cells preferentially contacting SACs. However, bipolar cells are known to be electrotonically coupled so time delays between the bipolar cells that provide input to SACs seem unlikely. Using fluorescent microscopy and imunnostaining, we found that the endoplasmic reticulum (ER) is omnipresent in the soma extending to the distal processes of SACs. Consequently, a working hypothesis on heterogeneity of intracellular Ca[Formula: see text] dynamics from ER is proposed as a possible explanation for the cause of speed tuning of direction-selective Ca[Formula: see text] responses in dendrites of SACs.

  15. Prevalence of foodborne pathogens in food from selected African countries – a meta-analysis

    DEFF Research Database (Denmark)

    Paudyal, Narayan; Anihouvi, Victor; Hounhouigan, Joseph

    2017-01-01

    , Staphylococcus aureus and Listeria monocytogenes were the most frequently reported organisms in those studies. Although the data were highly heterogeneous, a striking feature is high prevalence of the major pathogens in RTE foods, almost as high as in raw foods. E. coli averaged at 37.6% in raw foods and 31...... for general analysis, while 66 papers on contamination of pathogenic bacteria were used for meta-analysis of prevalence. The food items were split into two categories: raw foods and ready-to-eat (RTE) foods (including street food and beverages) for meta-analysis. Majority of the reviewed studies (67.2%, 78...... the prevalence of foodborne pathogens in seven African countries (Benin, Botswana, Ghana, Kenya, Nigeria, Sudan and Uganda) from papers in regional or international journals published between January 2000 and December 2015. One hundred and sixteen publications that dealt with food microbiology were reviewed...

  16. Antibacterial activity of essential oils extracted from Satureja hortensis against selected clinical pathogens

    Science.gov (United States)

    Görmez, Arzu; Yanmiş, Derya; Bozari, Sedat; Gürkök, Sumeyra

    2017-04-01

    The antibiotic resistance of pathogenic microorganisms has become a worldwide concern to public health. To overcome the current resistance problem, new antimicrobial agents are extremely needed. The aim of the present study was to evaluate the antibacterial activity of Satureja hortensis essential oils against seven clinical pathogens. Chemical compositions of hydro distillated essential oils from S. hortensis were analyzed by GS-MS. The antibacterial activity was investigated against Corynebacterium diphtheria, Salmonella typhimurium, Serratia plymuthica Yersinia enterocolitica, Y. frederiksenii, Y. pseudotuberculosis and Vibrio cholerae by the use of disc diffusion method and broth micro dilution method. The minimum inhibitory concentration (MIC) values of essential oils were found as low as 7.81 µg/mL. Notably, essential oils of S. hortensis exhibited remarkable antimicrobial activities against the tested clinical pathogens. The results indicate that these essential oils can be used in treatment of different infectious diseases.

  17. Serosurvey for selected pathogens in free-ranging American black bears (Ursus americanus) in Maryland, USA.

    Science.gov (United States)

    Bronson, Ellen; Spiker, Harry; Driscoll, Cindy P

    2014-10-01

    American black bears (Ursus americanus) in Maryland, USA, live in forested areas in close proximity to humans and their domestic pets. From 1999 to 2011, we collected 84 serum samples from 63 black bears (18 males; 45 females) in five Maryland counties and tested them for exposure to infectious, including zoonotic, pathogens. A large portion of the bears had antibody to canine distemper virus and Toxoplasma gondii, many at high titers. Prevalences of antibodies to zoonotic agents such as rabies virus and to infectious agents of carnivores including canine adenovirus and canine parvovirus were lower. Bears also had antibodies to vector-borne pathogens common to bears and humans such as West Nile virus, Borrelia burgdorferi, Rickettsia rickettsii, and Anaplasma phagocytophilum. Antibodies were detected to Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Canicola, Grippotyphosa, and Bratislava. We did not detect antibodies to Brucella canis or Ehrlichia canis. Although this population of Maryland black bears demonstrated exposure to multiple pathogens of concern for humans and domesticated animals, the low levels of clinical disease in this and other free-ranging black bear populations indicate the black bear is likely a spillover host for the majority of pathogens studied. Nevertheless, bear populations living at the human-domestic-wildlife interface with increasing human and domestic animal exposure should continue to be monitored because this population likely serves as a useful sentinel of ecosystem health.

  18. From select agent to established pathogen: The response to Phakopsora pachyrhizi (soybean rust) in North America

    Science.gov (United States)

    The pathogen causing soybean rust, Phakopsora pachyrhizi Syd., was first described in Japan in 1902. The disease was important in the Eastern Hemisphere for many decades before the fungus was reported in Hawaii in 1994, which was followed by reports from countries in Africa and South America. In 200...

  19. Prioritizing risks and uncertainties from intentional release of selected Category A pathogens.

    Directory of Open Access Journals (Sweden)

    Tao Hong

    Full Text Available This paper synthesizes available information on five Category A pathogens (Bacillus anthracis, Yersinia pestis, Francisella tularensis, Variola major and Lassa to develop quantitative guidelines for how environmental pathogen concentrations may be related to human health risk in an indoor environment. An integrated model of environmental transport and human health exposure to biological pathogens is constructed which 1 includes the effects of environmental attenuation, 2 considers fomite contact exposure as well as inhalational exposure, and 3 includes an uncertainty analysis to identify key input uncertainties, which may inform future research directions. The findings provide a framework for developing the many different environmental standards that are needed for making risk-informed response decisions, such as when prophylactic antibiotics should be distributed, and whether or not a contaminated area should be cleaned up. The approach is based on the assumption of uniform mixing in environmental compartments and is thus applicable to areas sufficiently removed in time and space from the initial release that mixing has produced relatively uniform concentrations. Results indicate that when pathogens are released into the air, risk from inhalation is the main component of the overall risk, while risk from ingestion (dermal contact for B. anthracis is the main component of the overall risk when pathogens are present on surfaces. Concentrations sampled from untracked floor, walls and the filter of heating ventilation and air conditioning (HVAC system are proposed as indicators of previous exposure risk, while samples taken from touched surfaces are proposed as indicators of future risk if the building is reoccupied. A Monte Carlo uncertainty analysis is conducted and input-output correlations used to identify important parameter uncertainties. An approach is proposed for integrating these quantitative assessments of parameter uncertainty with

  20. Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics.

    Science.gov (United States)

    Vanhove, Audrey S; Rubio, Tristan P; Nguyen, An N; Lemire, Astrid; Roche, David; Nicod, Julie; Vergnes, Agnès; Poirier, Aurore C; Disconzi, Elena; Bachère, Evelyne; Le Roux, Frédérique; Jacq, Annick; Charrière, Guillaume M; Destoumieux-Garzón, Delphine

    2016-03-01

    Recent studies revealed that several vibrio species have evolved the capacity to survive inside host cells. However, it is still often ignored if intracellular stages are required for pathogenicity. Virulence of Vibrio tasmaniensis LGP32, a strain pathogenic for Crassostrea gigas oysters, depends on entry into hemocytes, the oyster immune cells. We investigated here the mechanisms of LGP32 intracellular survival and their consequences on the host-pathogen interaction. Entry and survival inside hemocytes were required for LGP32-driven cytolysis of hemocytes, both in vivo and in vitro. LGP32 intracellular stages showed a profound boost in metabolic activity and a major transcription of antioxidant and copper detoxification genes, as revealed by RNA sequencing. LGP32 isogenic mutants showed that resistance to oxidative stress and copper efflux are two main functions required for vibrio intracellular stages and cytotoxicity to hemocytes. Copper efflux was also essential for host colonization and virulence in vivo. Altogether, our results identify copper resistance as a major mechanism to resist killing by phagocytes, induce cytolysis of immune cells and colonize oysters. Selection of such resistance traits could arise from vibrio interactions with copper-rich environmental niches including marine invertebrates, which favour the emergence of pathogenic vibrios resistant to intraphagosomal killing across animal species. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. Survey for selected pathogens in wild pigs (Sus scrofa) from Guam, Marianna Islands, USA.

    Science.gov (United States)

    Cleveland, Christopher A; DeNicola, Anthony; Dubey, J P; Hill, Dolores E; Berghaus, Roy D; Yabsley, Michael J

    2017-06-01

    Pigs (Sus scrofa) were introduced to Guam in the 1600's and are now present in high densities throughout the island. Wild pigs are reservoirs for pathogens of concern to domestic animals and humans. Exposure to porcine parvovirus, transmissible gastroenteritis, and Leptospira interrogans has been documented in domestic swine but data from wild pigs are lacking. The close proximity of humans, domestic animals, and wild pigs, combined with the liberal hunting of wild pigs, results in frequent opportunities for pathogen transmission. From February-March 2015, blood, tissue and ectoparasite samples were collected from 47 wild pigs. Serologic testing found exposure to Brucella spp. (2%), Toxoplasma gondii (11%), porcine reproductive and respiratory syndrome (PRRS) virus (13%), porcine circovirus type 2 (36%), pseudorabies virus (64%), Actinobacillus pleuropneumoniae (93%), Lawsonia intracellularis (93%), and porcine parvovirus (94%). Eleven (24%) samples had low titers (1:100) to Leptospira interrogans serovars Bratislava (n=6), Icterohaemorrhagiae (n=6), Pomona (n=2), and Hardjo (n=1). Kidney samples from nine pigs with Leptospira antibodies were negative for Leptospira antigens. Numerous pigs had Metastrongylus lungworms and three had Stephanurus dentatus. Lice (Hematopinus suis) and ticks (Amblyomma breviscutatum) were also detected. No antibodies to Influenza A viruses were detected. In contrast to the previous domestic swine survey, we found evidence of numerous pathogens in wild pigs including new reports of pseudorabies virus, PRRS virus, Brucella, and Leptospira in pigs on Guam. These findings highlight that domestic swine-wild pig interactions should be prevented and precautions are needed when handling wild pigs to minimize the risk of pathogen transmission. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. The Use of Predatory Bacteria to Control Select Pathogens and Treat Respiratory Infections

    Science.gov (United States)

    2015-04-13

    detectable levels of predators using RT- qPCR (Fig. 7). As previously stated, for the one hour time point experiment, an inoculation dose of 4 x 109 PFU... detect 1010 gene copies of Bdellovibrio in three of the mice. qPCR has been found to slightly overestimate quantities of bacteria compared to...smaller inductions of TNF and IL-6 as compared to that induced by pathogenic E. coli. As TLR-4 is responsible for detecting LPS expressed on or released

  3. Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids.

    Directory of Open Access Journals (Sweden)

    Laura M Sanchez

    Full Text Available Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1 was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.

  4. Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries.

    Science.gov (United States)

    Abdul Majid, Mohamad Azlan; Mahboob, Tooba; Mong, Brandon G J; Jaturas, Narong; Richard, Reena Leeba; Tian-Chye, Tan; Phimphila, Anusorn; Mahaphonh, Panomphanh; Aye, Kyaw Nyein; Aung, Wai Lynn; Chuah, Joon; Ziegler, Alan D; Yasiri, Atipat; Sawangjaroen, Nongyao; Lim, Yvonne A L; Nissapatorn, Veeranoot

    2017-01-01

    Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public.

  5. Selection of aggressive pathogenic and solopathogenic strains of Ustilago maydis to improve Huitlacoche production

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    Porfirio Raúl Galicia-García

    Full Text Available ABSTRACT Ustilago maydis is a basidiomycete known as the causative agent of 'common smut', worldwide disease of maize that is recognized by the galls it forms, which have considerable potential as a gourmet food. Results of infection are quite variable, even under optimal greenhouse conditions. In order to find pathogenic strains able to be used as a highly infective and stable inoculum for the successful production of galls either in greenhouses or in the field, ears with gall symptoms containing teliospores were recovered from maize plants. The teliospores were suspended in water and plated on nutrient-rich medium. Twenty-six colonies developed, containing three types of yeast-like colonies: saprotrophic, pathogenic, and solopathogenic. DAPI staining confirmed the presence of solopathogenic strains with diploid sporidia. Groups of different mating types were found when pairs of the 26 strains were arranged resembling partial-diallel combinations. Amplification of the partial b locus revealed that the strains found harbor the alleles b3 and b4, allowing the formation in dikaryotic strains of heterodimeric regulatory proteins associated with fungal development and pathogenicity. In this study, we isolated compatible haploid and solopathogenic diploid strains for their high capacity for inducing smut.

  6. Inactivation of selected bacterial pathogens in dairy cattle manure by mesophilic anaerobic digestion (balloon type digester).

    Science.gov (United States)

    Manyi-Loh, Christy E; Mamphweli, Sampson N; Meyer, Edson L; Okoh, Anthony I; Makaka, Golden; Simon, Michael

    2014-07-14

    Anaerobic digestion of animal manure in biogas digesters has shown promise as a technology in reducing the microbial load to safe and recommended levels. We sought to treat dairy manure obtained from the Fort Hare Dairy Farm by investigating the survival rates of bacterial pathogens, through a total viable plate count method, before, during and after mesophilic anaerobic digestion. Different microbiological media were inoculated with different serial dilutions of manure samples that were withdrawn from the biogas digester at 3, 7 and 14 day intervals to determine the viable cells. Data obtained indicated that the pathogens of public health importance were 90%-99% reduced in the order: Campylobacter sp. (18 days) anaerobic digestion process. In addition, the highest p-value i.e., 0.957 for E. coli showed the statistical significance of its model and the strongest correlation between its reductions with days of digestion. In conclusion, the results demonstrated that the specific bacterial pathogens in manure can be considerably reduced through anaerobic digestion after 133 days.

  7. Comparison of the 'Ca Liberibacter asiaticus' genome adapted for an intracellular lifestyle with other members of the rhizobiales

    Science.gov (United States)

    An intracellular plant pathogen ‘Ca. Liberibacter asiaticus,’ a member of the Rhizobiales, is related to Sinorhizobium meliloti, Bradyrhizobium japonicum, Agrobacterium tumefaciens and Bartonella henselae, an intracellular mammalian pathogen. Whole chromosome comparisons identified at least 52 clust...

  8. The Role of Pathogenic Autoantibodies in Autoimmunity

    Directory of Open Access Journals (Sweden)

    Merrill J. Rowley

    2015-11-01

    Full Text Available The serological presence of autoantibodies is diagnostic of autoimmunity, and these autoantibodies may be present for many years before the presentation of autoimmune disease (AID. Although a pathogenic role has been demonstrated for various autoantibodies reactive with cell surface and extracellular autoantigens, studies using monoclonal antibodies (mAb show not all antibodies in the polyclonal response are pathogenic. Differences depend on Fab-mediated diversity in epitope specificity, Fc-mediated effects based on immunoglobulin (Ig class and subclass, activation of complement, and the milieu in which the reaction occurs. These autoantibodies often occur in organ-specific AID and this review illustrates their pathogenic and highly specific effects. The role of autoantibodies associated with intracellular antigens is less clear. In vitro they may inhibit or adversely affect well-defined intracellular biochemical pathways, yet, in vivo they are separated from their autoantigens by multiple cellular barriers. Recent evidence that Ig can traverse cell membranes, interact with intracellular proteins, and induce apoptosis has provided new evidence for a pathogenic role for such autoantibodies. An understanding of how autoantibodies behave in the polyclonal response and their role in pathogenesis of AID may help identify populations of culprit B-cells and selection of treatments that suppress or eliminate them.

  9. [Comparison of antimicrobial resistance pattern of selected respiratory tract pathogens isolated from different animal species].

    Science.gov (United States)

    Wettstein, K; Frey, J

    2004-09-01

    The antibiotic resistance pattern of respiratory tract pathogens isolated of different animal species suffering from respiratory tract diseases has been investigated by antibiograms performed by agar diffusion test. The results show that the resistance situation in Switzerland is favourable compared with studies from other countries. However, high resistance rates were found in certain species: 61% of Streptococcus spp. were resistant to erythromycin and 44% to tetracycline, 59% of Bordetella bronchiseptica were resistant to ampicillin and 50% of Mannheimia (Pasteurella) haemolytica were multiresistant to tetracycline, ampicillin and streptomycine. The gram negative isolates were widely resistant to streptomycine.

  10. Prevalence of foodborne pathogens in food from selected African countries - A meta-analysis.

    Science.gov (United States)

    Paudyal, Narayan; Anihouvi, Victor; Hounhouigan, Joseph; Matsheka, Maitshwarelo Ignatius; Sekwati-Monang, Bonno; Amoa-Awua, Wisdom; Atter, Amy; Ackah, Nina Bernice; Mbugua, Samuel; Asagbra, Agnes; Abdelgadir, Warda; Nakavuma, Jesca; Jakobsen, Mogens; Fang, Weihuan

    2017-05-16

    Food safety information in the African region is insufficient and fragmented due to lack of surveillance, documentation and reporting, thereby resulting in inefficient utilization of resources, duplication of activities, and lack of synergy among the countries of the region. This paper reviews the prevalence of foodborne pathogens in seven African countries (Benin, Botswana, Ghana, Kenya, Nigeria, Sudan and Uganda) from papers in regional or international journals published between January 2000 and December 2015. One hundred and sixteen publications that dealt with food microbiology were reviewed for general analysis, while 66 papers on contamination of pathogenic bacteria were used for meta-analysis of prevalence. The food items were split into two categories: raw foods and ready-to-eat (RTE) foods (including street food and beverages) for meta-analysis. Majority of the reviewed studies (67.2%, 78/116) dealt with food of animal origin: 38.8% for meat and eggs, 17.2% for dairy products and 11.2% for aquatic products. Only 8.6% examined foods of plant origin (fruits and vegetables). The remaining 24.1% was the composite RTE food and beverages. Enterobacteriaceae, Escherichia coli, Salmonella, Staphylococcus aureus and Listeria monocytogenes were the most frequently reported organisms in those studies. Although the data were highly heterogeneous, a striking feature is high prevalence of the major pathogens in RTE foods, almost as high as in raw foods. E. coli averaged at 37.6% in raw foods and 31.6% in RTE foods. The corresponding prevalence for Salmonella was 19.9% vs 21.7%; S. aureus, 27.8% vs 25.1% and L. monocytogenes, 19.5% vs 6.7%. The average prevalence of foodborne pathogens in these countries was 34.2% (29.0-39.3%). Differences in food types as well as non-uniform protocols for sampling and identification might have contributed to high heterogeneity (I 2 >97%) although some high prevalence data could be factual with extensive varieties of raw and RTE foods

  11. The occurrence and pathogenicity of Phoma exigua Desm. var. exigua for selected species of herbs

    Directory of Open Access Journals (Sweden)

    Zofia Machowicz-Stefaniak

    2012-12-01

    Full Text Available P. exigua var. exigua was isolated from underground and aboveground organs of different herb plant species in the years 1998-2006. Pathogenicity tests of three randomly chosen isolates of the fungus T 299, T 261 and T 277 for thyme and of three isolates M 1657, M 1193 and M 1635 for lemon balm were carried out. The effect of water suspension of fungus spores on the germination ability of schizocarps and of infected soil on sprouting and seedling health was studied. The study of microscopic structures of the fungus developing on plants, isolation of the fungus on artificial media and macro- and microscopic analysis on standard media are essential for the correct identification of P. exigua var. exigua. The polyphagous nature of the fungus is confirmed by the obtained results. The tested isolates were found to be occasional pathogens of thyme and lemon balm in the studied conditions. The harmfulness of the tested isolates pointed to inhibition of schizocarp germination, sprouting of plants and the ability to cause necrosis of germs, roots and epicotyl.

  12. Comparison of the in vitro activities of several new fluoroquinolones against respiratory pathogens and their abilities to select fluoroquinolone resistance.

    Science.gov (United States)

    Boswell, F J; Andrews, J M; Jevons, G; Wise, R

    2002-10-01

    In this study the in vitro activities and pharmacodynamic properties of moxifloxacin, levofloxacin, gatifloxacin and gemifloxacin were compared on recently isolated respiratory pathogens and strains of Streptococcus pneumoniae with known mechanisms of fluoroquinolone resistance. In addition, the resistance selection frequencies of moxifloxacin and levofloxacin on three recently isolated respiratory pathogens and four strains of S. pneumoniae with known mechanisms of fluoroquinolone resistance were investigated. The four fluoroquinolones had similar activities against both Moraxella catarrhalis (MIC(90)s 0.015-0.06 mg/L) and Haemophilus influenzae (MIC(90)s 0.008-0.03 mg/L). More marked differences in activity were noted with S. pneumoniae, with MIC(90)s of 0.25, 1, 0.5 and 0.03 mg/L for moxifloxacin, levofloxacin, gatifloxacin and gemifloxacin, respectively. With the S. pneumoniae strains, the four fluoroquinolones exhibited similar concentration-dependent time-kill kinetics. The resistance selection frequencies of levofloxacin were higher than those of moxifloxacin at concentrations equivalent to those at the end of the dosing interval. Therefore moxifloxacin may have less of an impact on the development of resistance than levofloxacin.

  13. Antibacterial Effect of Carvacrol and Coconut Oil on Selected Pathogenic Bacteria

    Directory of Open Access Journals (Sweden)

    Božik M.

    2018-03-01

    Full Text Available Essential oils play a prominent role as flavouring agents and fragrances in the food and perfume industries. Carvacrol is a major component of various essential oils, such as oregano and thyme oils, and is responsible for their antimicrobial activity. Lauric acid is a medium-chain fatty acid (MCFA with a high antibacterial potential. Both carvacrol and MCFAs have been used empirically as antimicrobial agents. Here, we tested the inhibitory properties of carvacrol and coconut (Cocos nucifera L. oil containing a high percentage of MCFAs against 5 harmful bacterial pathogens: Escherichia coli, Salmonella Enteritidis, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus cecorum. Gas chromatography (GC-FID analysis of coconut oil showed a high concentration of lauric acid (41%. Microdilution antimicrobial assays showed that the combination of carvacrol and coconut oil had a stronger antibacterial effect against all tested bacteria than both agents separately. We conclude that carvacrol could significantly improve the antibacterial effect of coconut oil.

  14. Effectiveness of sanitizing products on controlling selected pathogen surrogates on retail deli slicers.

    Science.gov (United States)

    Yeater, Michael C; Kirsch, Katie R; Taylor, T Matthew; Mitchell, Jeff; Osburn, Wesley N

    2015-04-01

    The objectives of this study were (i) to assess the efficacy of quaternary ammonium chloride-based wet foam (WF) and dry foam (DF) sanitizer systems (600 ppm) for reducing Listeria innocua (a nonpathogenic surrogate of Listeria monocytogenes) or a 100.0 μg/ml rifampin-resistant Salmonella Typhimurium LT2 (a nonpathogenic surrogate of Salmonella enterica serovar Typhimurium) on niche and transfer point areas of an unwashed retail deli slicer as compared with traditional chlorine (Cl(-)) treatment (200 ppm) and (ii) to compare sanitizer surface contact times (10 and 15 min) for pathogen surrogate control. Turkey frankfurter slurries inoculated with L. innocua or Salmonella Typhimurium were used to inoculate seven high-risk sites on a commercial slicer. After 30 min of bacterial attachment, slicers were dry wiped to remove excess food matter, followed by a randomly assigned sanitizer treatment. Surviving pathogen surrogate cells were enumerated on modified Oxford's agar not containing antimicrobic supplement (L. innocua) or on tryptic soy agar supplemented with 100 μg/ml rifampin (Salmonella Typhimurium LT2). Replicate-specific L. innocua and Salmonella Typhimurium reductions were calculated as log CFU per square centimeter of control minus log CFU per square centimeter of enumerated survivors for each site. For both organisms, all sanitizer treatments differed from each other, with Cl(-) producing the least reduction and WF the greatest reduction. A significant (P < 0.05) site-by-treatment interaction was observed. The results of the study indicate that quaternary ammonium chloride sanitizers (600 ppm) applied by both WF and DF were more effective at reducing L. innocua and Salmonella Typhimurium than a traditional Cl sanitizer (200 ppm) on unwashed slicer surfaces.

  15. Surveillance for zoonotic and selected pathogens in harbor seals Phoca vitulina from central California

    Science.gov (United States)

    Greig, Denise J.; Ip, Hon S.; Gulland, Frances M. D.; Miller, Woutrina A.; Conrad, Patricia A.; Field, Cara L.; Fleetwood, Michelle; Harvey, James T.; Jang, Spencer; Packham, Andrea; Wheeler, Elizabeth; Hall, Ailsa J.

    2014-01-01

    The infection status of harbor seals Phoca vitulina in central California, USA, was evaluated through broad surveillance for pathogens in stranded and wild-caught animals from 2001 to 2008, with most samples collected in 2007 and 2008. Stranded animals from Mendocino County to San Luis Obispo County were sampled at a rehabilitation facility: The Marine Mammal Center (TMMC, n = 175); wild-caught animals were sampled at 2 locations: San Francisco Bay (SF, n = 78) and Tomales Bay (TB, n = 97), that differed in degree of urbanization. Low prevalences of Salmonella, Campylobacter, Giardia, and Cryptosporidium were detected in the feces of stranded and wild-caught seals. Clostridium perfringens and Escherichia coli were more prevalent in the feces of stranded (58% [78 out of 135] and 76% [102 out of 135]) than wild-caught (42% [45 out of 106] and 66% [68 out of 106]) seals, whereas Vibrio spp. were 16 times more likely to be cultured from the feces of seals from SF than TB or TMMC (p < 0.005). Brucella DNA was detected in 3.4% of dead stranded harbor seals (2 out of 58). Type A influenza was isolated from feces of 1 out of 96 wild-caught seals. Exposure to Toxoplasma gondii, Sarcocystis neurona, and type A influenza was only detected in the wild-caught harbor seals (post-weaning age classes), whereas antibody titers to Leptospira spp. were detected in stranded and wild-caught seals. No stranded (n = 109) or wild-caught (n = 217) harbor seals had antibodies to phocine distemper virus, although a single low titer to canine distemper virus was detected. These results highlight the role of harbor seals as sentinel species for zoonotic and terrestrial pathogens in the marine environment.

  16. Rapid evolution of introduced tree pathogens via episodic selection and horizontal gene transfer

    Science.gov (United States)

    Clive Brasier

    2012-01-01

    Routine selection is simply defined as “the ecological constraints experienced by an endemic organism that favor a relatively stable but fluctuating population structure over time.” Its antithesis is episodic selection, defined as “any sudden ecological disturbance likely to lead to a significant alteration in a species’ population structure” (Brasier 1986, 1995). In...

  17. Genetic versus antigenic differences among highly pathogenic H5N1 avian influenza A viruses: Consequences for vaccine strain selection.

    Science.gov (United States)

    Peeters, Ben; Reemers, Sylvia; Dortmans, Jos; de Vries, Erik; de Jong, Mart; van de Zande, Saskia; Rottier, Peter J M; de Haan, Cornelis A M

    2017-03-01

    Highly pathogenic H5N1 avian influenza A viruses display a remarkable genetic and antigenic diversity. We examined to what extent genetic distances between several H5N1 viruses from different clades correlate with antigenic differences and vaccine performance. H5-specific antisera were generated, and cross-reactivity and antigenic distances between 12 different viruses were determined. In general, antigenic distances increased proportional to genetic distances although notable exceptions were observed. Antigenic distances correlated better with genetic variation in 27 selected, antigenically-relevant H5 residues, than in the complete HA1 domain. Variation in these selected residues could accurately predict the antigenic distances for a novel H5N8 virus. Protection provided by vaccines against heterologous H5N1 challenge viruses indicated that cross-protection also correlates better with genetic variation in the selected antigenically-relevant residues than in complete HA1. When time is limited, variation at these selected residues may be used to accurately predict antigenic distance and vaccine performance. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. PREVALENCE OF ANTIBODIES FOR SELECTED CANINE PATHOGENS AMONG WOLVES (CANIS LUPUS) FROM THE ALASKA PENINSULA, USA.

    Science.gov (United States)

    Watts, Dominique E; Benson, Anna-Marie

    2016-07-01

    We collected blood samples from wolves ( Canis lupus ) on the Alaska Peninsula, southwest Alaska, US, 2006-11 and tested sera for antibodies to canine adenovirus (CAV), canine coronavirus (CCV), canine distemper virus (CDV), canine herpesvirus (CHV), canine parainfluenza (CPI), canine parvovirus (CPV), Neospora caninum , and Toxoplasma gondii . Detected antibody prevalence was 90% for CAV, 28% for CCV, 12% for CDV, 93% for CHV, 0% for CPI, 20% for CPV, 0% for N. caninum, and 86% for T. gondii . Prevalence of CCV antibodies suggested a seasonal pattern with higher prevalence during spring (43%) than in fall (11%). Prevalence of CCV antibodies also declined during the 6-yr study with high prevalence during spring 2006-08 (80%, n=24) and low prevalence during spring 2009-11 (4%, n=24). Prevalence of N. caninum and T. gondii antibodies were highly variable in the study area during 2006-11. Results suggested that some pathogens might be enzootic on the Alaska Peninsula (e.g., CAV and CHV) while others may be epizootic (e.g., CCV, N. caninum , T. gondii ).

  19. Antifungal Activities of Extracts from Selected Lebanese Wild Plants against Plant Pathogenic Fungi

    Directory of Open Access Journals (Sweden)

    Y. Abou-Jawdah

    2004-12-01

    Full Text Available Extracts of nine plant species growing wild in Lebanon were tested for their efficacy against seven plant pathogenic fungi: Botrytis cinerea, Alternaria solani, Penicillium sp., Cladosporium sp., Fusarium oxysporum f. sp. melonis, Rhizoctonia solani and Sphaerotheca cucurbitae. Extracts of three of the plants, Origanum syriacum, Micromeria nervosa and Plumbago maritima, showed the highest levels of in vitro activity against spore germination and mycelial growth of the fungi tested. Inula viscosa showed high activity against spore germination but only moderate activity against mycelial growth. The other five plant species tested Calamintha origanifolia, Micromeria juliana, Ruta sp., Sideritis pullulans and Urginea maritima showed only moderate to low activity against these fungi. Preventive sprays with extracts of O. syriacum, M. nervosa, P. maritima and I. viscosa, applied at concentrations ranging between 4 and 8% to squash and cucumber seedlings, gave efficient protection against gray mold caused by B. cinerea and powdery mildew caused by S. cucurbitae. However, these extracts did not control green mold of citrus fruits caused by Penicillium sp. Thin layer chromatography revealed three inhibitory bands in extracts of O. syriacum, two in I. viscosa and only one in each of the other plants tested: M. nervosa, P. maritima, C. origanifolia and Ruta sp.

  20. New perspective of dendrobium crumenatum orchid for antimicrobial activity against selected pathogenic bacteria

    International Nuclear Information System (INIS)

    Sandrasagaran, U.M.; Murugaiyah, V.

    2014-01-01

    The present study was undertaken to investigate the potential anti-microbial activity from different parts of Dendrobium crumenatum (leaf, stem, root and pseudo-bulb) against 8 pathogenic bacteria. The antimicrobial activities were determined by using disc diffusion assay, microdilution test for determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The methanolic extracts of stem, root and pseudo-bulb displayed antimicrobial activity comparable to that of the standard antibiotics. Stem extract of D. crumenatum had the most potent antimicrobial activity against Staphylococcus aureus, Klebsiella pneumoniae and Enterobacter aerogenes with MIC values of 0.39, 0.195 and 0.195 mg/mL, respectively. Root and stem extracts were found to be active against Streptococcus pneumoniae, Shigella dysentriae and Saccharomyces cerevisiae with MIC values of 0.78 mg/ml compared to 0.00312 mg/mL, 0.025 mg/mL and 0.0125 mg/mL of standard antibiotics of amoxcillin, chloramphenicol and kanamycin. Stem and root extracts yield MBC values in the range of 0.78 mg/mL to 6.25 mg/mL against Staphylococcus aureus, Enterobacter aerogenes, Klebsiella pneumoniae and Saccharomyces cerevisiae. The present study showed that D. crumenatum exhibited potential antimicrobial activity which could be due to the presence of alkaloid and flavonoid compounds and this is a first report on South East Asia region's wild orchid. (author)

  1. Immunological and pathogenic properties of poliovirus variants selected for resistance to antiviral drug V-073.

    Science.gov (United States)

    Kouiavskaia, Diana V; Dragunsky, Eugenia M; Liu, Hong-Mei; Oberste, M Steven; Collett, Marc S; Chumakov, Konstantin M

    2011-01-01

    The National Research Council has recommended development of polio antiviral drugs to assist in management of outbreaks and to mitigate adverse consequences of vaccination. V-073 is a small molecule poliovirus capsid inhibitor that is being developed for these purposes. Antiviral use raises the potential of treatment-emergent resistance. Understanding virological consequences of resistance is important. Six independent laboratory-derived V-073-resistant poliovirus variants were characterized for their ability to be neutralized by conventional vaccine-induced immune sera, to elicit serum neutralizing antibodies upon CD-1 mouse immunization, and to replicate in and to cause paralysis of TgPVR21 mice. V-073-resistant variants were effectively neutralized by oral poliovirus vaccine and inactivated poliovirus vaccine human immune sera. All variants elicited virus neutralizing antibody titres in CD-1 mice that were comparable to drug-susceptible parental and Sabin vaccine strain viruses. Infection efficiency of TgPVR21 mice by variants was comparable to (1 of 6 variants) or considerably lower than (5 of 6 variants) parental viruses. Drug-resistant variants replicated to levels comparable to (1 of 6 variants) or substantially less than (5 of 6 variants) their drug-susceptible parental viruses and were on average 1.4 log(10) (range 0.3 to >2.8 log₁₀) less neurovirulent. Laboratory-derived V-073-resistant variants exhibit clear attenuation of pathogenic properties while maintaining immunological features of drug-susceptible viruses.

  2. Clinical significance and epidemiologic analyses of Mycobacterium avium and Mycobacterium intracellulare lung disease from post-marketing surveillance.

    Science.gov (United States)

    Suzuki, Katsuhiro; Kurashima, Atsuyuki; Tatsuno, Kinji; Kadota, Jun-Ichi

    2018-01-01

    In Japan, nontuberculous mycobacterial lung disease is mostly attributable to Mycobacterium avium complex (MAC), i.e., M. avium or M. intracellulare. However, clinical features of the disease caused by these two pathogens have not been studied sufficiently yet. A post-marketing survey of clarithromycin was performed at 130 facilities across Japan. The data on patients with M. avium infection and patients with M. intracellulare infection were selected from this survey for comparison of background variables and clinical features of the two pathogens. Among the patients analyzed (n = 368), 67.4% had M. avium infection and 32.6% had M. intracellulare infection. Stratified analysis revealed no significant differences between the ratio of the two pathogens based on gender, disease type, complication, past medical history, or smoking history. However, the percentage of patients with M. intracellulare infection was significantly higher among those with underlying lung disease than among those without lung disease (p = 0.0217). The percentage of patients with M. intracellulare infection rose significantly with age (p = 0.0296). This age-related change was more significant in women (p = 0.0018). When district-wise analysis was performed for Japan, the percentage of M. intracellulare infection was higher in the Chugoku/Shikoku and Kyushu districts whereas the percentage of M. avium infection was higher in the other districts. This survey revealed some differences in the clinical and epidemiologic features of M. avium and M. intracellulare infection. The significant predominance of M. avium infection among relatively young women is suggestive of an increase in the M. avium/M. intracellulare infection ratio among women in the future. Copyright © 2017 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

  3. In vitro growth control of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei-fermented milk.

    Science.gov (United States)

    Millette, M; Luquet, F M; Lacroix, M

    2007-03-01

    Food-borne pathogen inhibition was tested in the presence of a mixture of Lactobacillus acidophilus and Lactobacillus casei during fermentation under controlled pH conditions. The growth of Escherichia coli O157:H7, Salmonella serotype Typhimurium, Staphylococcus aureus, Listeria innocua, Enterococcus faecium and Enterococcus faecalis was evaluated for 48 h at 37 degrees C. In the presence of the lactic acid bacteria (LAB), an increase of the generation time was observed for all the gram-positive bacteria evaluated. Staphylococcus aureus was the most sensitive strain showing an increase of the generation time by 210%. However, for all the gram-negative bacteria evaluated, no inhibition occurred after 8 h of fermentation. The soluble portion of Lact. acidophilus- and Lact. casei-fermented milk was recuperated and tested for its antimicrobial activity. Listeria innocua and Staph. aureus were the most sensitive to the presence of fermented milk supernatant showing an inhibition of 85.9% and 84.7%, respectively. This soluble fraction was neutralized to eliminate the antimicrobial effect of the organic acids produced; the most sensitive strains were L. innocua and E. coli O157:H7 showing an inhibition of 65.9% and 61.9%, respectively. Finally, the soluble fraction was neutralized and irradiated at 45 kGy using a (60)Co source to eliminate the possible antimicrobial effect of both organic acids and bacteriocin-like substances. Enterococcus faecalis, E. coli O157:H7 and Staph. aureus were the most affected bacteria by this fraction, showing 39.1, 32 and 31.2% inhibition, respectively. The results obtained in this study suggest the implication of both organic acids and bacteriocin-like inhibitory substances in the antimicrobial activity observed in the soluble fraction of the probiotic preparation. This study revealed the antimicrobial mechanisms of action of Lact. acidophilus- and Lact. casei-fermented milk used to prevent antibiotic-associated diarrhoea.

  4. Molecular marker-assisted selection for resistance to pathogens in tomato

    International Nuclear Information System (INIS)

    Barone, A.; Frusciante, L.

    2007-01-01

    Since the 1980s, the use of molecular markers has been suggested to improve the efficiency of releasing resistant varieties, thus overcoming difficulties met with classical breeding. For tomato, a high-density molecular map is available in which more than 40 resistance genes are localized. Markers linked to these genes can be used to speed up gene transfer and pyramiding. Suitable PCR markers targeting resistance genes were constructed directly on the sequences of resistance genes or on restriction fragment length polymorphisms (RFLPs) tightly linked to them, and used to select resistant genotypes in backcross schemes. In some cases, the BC 5 generation was reached, and genotypes that cumulated two homozygous resistant genes were also obtained. These results supported the feasibility of using marker-assisted selection (MAS) in tomato and reinforcing the potential of this approach for other genes, which is today also driven by the development of new techniques and increasing knowledge about the tomato genome. (author)

  5. Antioxidant, antibacterial, and antibiofilm properties of polyphenols from muscadine grape (Vitis rotundifolia Michx.) pomace against selected foodborne pathogens.

    Science.gov (United States)

    Xu, Changmou; Yagiz, Yavuz; Hsu, Wei-Yea; Simonne, Amarat; Lu, Jiang; Marshall, Maurice R

    2014-07-16

    Polyphenols are predominantly secondary metabolites in muscadine grapes, playing an important role in the species' strong resistance to pests and diseases. This study examined the above property by evaluating the antioxidant, antibacterial, and antibiofilm activities of muscadine polyphenols against selected foodborne pathogens. Results showed that antioxidant activity for different polyphenols varied greatly, ranging from 5 to 11.1 mmol Trolox/g. Antioxidant and antibacterial activities for polyphenols showed a positive correlation. Muscadine polyphenols exhibited a broad spectrum of antibacterial activity against tested foodborne pathogens, especially Staphylococcus aureus (MIC = 67-152 mg/L). Muscadine polyphenols at 4 × MIC caused nearly a 5 log10 CFU/mL drop in cell viability for S. aureus in 6 h with lysis, whereas at 0.5 × MIC they inhibited its biofilm formation and at 16 × MIC they eradicated biofilms. Muscadine polyphenols showed synergy with antibiotics and maximally caused a 6.2 log10 CFU/mL drop in cell viability at subinhibitory concentration.

  6. Somatic mitochondrial DNA mutations in cancer escape purifying selection and high pathogenicity mutations lead to the oncocytic phenotype: pathogenicity analysis of reported somatic mtDNA mutations in tumors

    International Nuclear Information System (INIS)

    Pereira, Luísa; Soares, Pedro; Máximo, Valdemar; Samuels, David C

    2012-01-01

    The presence of somatic mitochondrial DNA (mtDNA) mutations in cancer cells has been interpreted in controversial ways, ranging from random neutral accumulation of mutations, to positive selection for high pathogenicity, or conversely to purifying selection against high pathogenicity variants as occurs at the population level. Here we evaluated the predicted pathogenicity of somatic mtDNA mutations described in cancer and compare these to the distribution of variations observed in the global human population and all possible protein variations that could occur in human mtDNA. We focus on oncocytic tumors, which are clearly associated with mitochondrial dysfunction. The protein variant pathogenicity was predicted using two computational methods, MutPred and SNPs&GO. The pathogenicity score of the somatic mtDNA variants were significantly higher in oncocytic tumors compared to non-oncocytic tumors. Variations in subunits of Complex I of the electron transfer chain were significantly more common in tumors with the oncocytic phenotype, while variations in Complex V subunits were significantly more common in non-oncocytic tumors. Our results show that the somatic mtDNA mutations reported over all tumors are indistinguishable from a random selection from the set of all possible amino acid variations, and have therefore escaped the effects of purifying selection that act strongly at the population level. We show that the pathogenicity of somatic mtDNA mutations is a determining factor for the oncocytic phenotype. The opposite associations of the Complex I and Complex V variants with the oncocytic and non-oncocytic tumors implies that low mitochondrial membrane potential may play an important role in determining the oncocytic phenotype

  7. Antimicrobial susceptibility survey of pathogens isolated from selected patients in Northern Italy

    Directory of Open Access Journals (Sweden)

    Elisabetta Maioli

    2005-03-01

    Full Text Available The Clinical Microbiology Laboratory of the University of Genoa participated, during the year 2003, in an international antimicrobial surveillance program.The collection of isolates was done according to the site of infection and/or type of patient. Four hundred twenty (420 clinical isolates were analyzed during this year and the frequencies of the different pathogens were investigated. A reference centre carried out susceptibility tests. Oxacillin-resistant Staphylococcus aureus represented 47.6% of all S. aureus isolates from blood stream infections and 33.3% of all S. aureus isolated from skin and soft tissue infections in hospitalised patients.These strains showed resistance to most of the antimicrobial agents evaluated, except vancomycin, teicoplanin, quinupristin/dalfopristin and linezolid which registered 100% of susceptibility. Some isolates from blood stream infections such as E. coli demonstrated resistance to ciprofloxacin (23.3%, levofloxacin (20%, and gatifloxacin (16.6%, and Klebsiella pneumoniae was resistant (18% to all fluoroquinolones tested. Pseudomonas aeruginosa manifested resistance to ciprofloxacin (16.6%, while 27.7% of these strains were resistant both to levofloxacin and gatifloxacin. All the Enterobacter cloacae isolated from blood were susceptible to ciprofloxacin, levofloxacin and gatifloxacin. Haemophilus influenzae and Moraxella catarrhalis collected from community-acquired respiratory tract infections were all inhibited by ciprofloxacin, levofloxacin and gatifloxacin. E. coli isolated from urinary tract infections in hospitalised patients were resistant to ciprofloxacin, levofloxacin and gatifloxacin (2.7%. All Salmonella spp. collected from samples of patients affected by infections of the gastro-intestinal tract were susceptible to all fluoroquinolones. Penicillin resistance in Streptococcus pneumoniae was found in 21.4% of isolates from patients with respiratory tract infections. Fluoroquinolone resistance was

  8. Selectable genes for transformation of the fungal plant pathogen Glomerella cingulata f. sp. phaseoli (Colletotrichum lindemuthianum).

    Science.gov (United States)

    Rodriguez, R J; Yoder, O C

    1987-01-01

    Glomerella cingulata f. sp. phaseoli (Gcp) was transformed using either of two selectable markers: the amdS + gene of Aspergillus nidulans, which encodes acetamidase and permits growth on acetamide as the sole nitrogen source and the hygBR gene of Escherichia coli which encodes hygromycin B (Hy) phosphotransferase and permits growth in the presence of the antibiotic Hy. The amdS+ gene functioned in Gcp under control of A. nidulans regulatory signals and hygBR was expressed after fusion to a promoter from Cochliobolus heterostrophus, another filamentous ascomycete. Protoplasts to be transformed were generated with the digestive enzyme complex Novozym 234 and then were exposed to plasmid DNA in the presence of 10 mM CaCl2 and polyethylene glycol. Transformation occurred by integration of single or multiple copies of either the amdS+ or hygBR plasmid into the fungal genome. There was no evidence of autonomous plasmid replication. Transformants were mitotically stable on selective and nonselective media. However, transforming DNA in hygBR transformants was observed to occasionally rearrange during nonselective growth, resulting in fewer copies of the plasmid per genome. These transformants were capable of infecting bean (Phaseolus vulgaris), the Gcp host plant, and after recovery from infected tissue were found to have retained both the transforming DNA unrearranged in their genomes and the Hy resistance phenotype. All single-conidial cultures derived from both amdS+ and hygBR transformants had the transplanted phenotype, suggesting that transformants were homokaryons.

  9. In-vitro selection of plants resistant or tolerant to pathogenic fungi

    International Nuclear Information System (INIS)

    Dutrecq, A.J.E.

    1977-01-01

    A technique was developed to test the inhibition of germination of barley seeds by culture filtrates of Helminthosporium sativum P., K and B. Seeds were soaked for 24h in toxin preparations, and then placed for 48h in wet sand. Using a log-probit scale, a quasilinear relationship was found between seed germination and toxin concentration. The toxic effect was markedly affected by the pH of culture filtrates, being maximum at pH 2.0. Control pH 2.0 filtrates of uninoculated medium did not affect the germination of barley seeds under similar condition. Culture filtrates of H. sativum were also tested as inhibitors of multiplication of barley cells in suspensions. The purified toxin helminthosporal isolated from culture filtrates of H. sativum inhibited rhizogenesis in wheat calluses obtained from tissue culture. The techniques described are being used for the selection of cereals tolerant to H. sativum toxin. (author)

  10. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    Science.gov (United States)

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  11. Phytochemical analysis and antimicrobial activity of some medicinal plants against selected common human pathogenic microorganisms

    Directory of Open Access Journals (Sweden)

    Divya Gupta

    2016-01-01

    Full Text Available Objective: To study the antimicrobial activity and phytochemicals of extracts from 5 different medicinal plants, as well as to evaluate the synergistic activity of potent plant extracts with suitable antibiotic discs and antibiotics susceptibility of tested microorganisms. Methods: The antimicrobial activities of different extracts were evaluated by using agar well diffusion method and antibiotics susceptibility of five selected microorganisms was tested by using disc diffusion method. For determination of synergistic activities of the potent plant extracts along with antibiotic discs, agar well diffusion and disc diffusion methods were combinedly used. Results: In the present investigation, the maximum in vitro inhibition of tested microorganisms, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Staphylococcus aureus was recorded in chloroform and methanol extracts of Terminalia arjuna, ethanol extract of Camellia sinensis, and petroleum ether extract of Polyalthia longifolia which offered inhibition zone ranged from 11 to 18 mm. The maximum antibacterial efficacy was exhibited by levofloxacin with an inhibition zone of 35 mm against Escherichia coli. The potent plant extracts showed positive synergistic effects against Staphylococcus aureus with lincomycin. The phytochemical analysis of the potent plant extracts revealed the presence of saponin, tannin, protein, carbohydrate, flavonoid, terpenoids and glycosides. Conclusions: According to the present study, Camellia sinensis, Terminalia arjuna and Polyalthia longifolia can be used as a potent source of natural antimicrobial agents by replacing commercially available synthetic drug that may have a large number of side effects.

  12. Prevalence of select vector-borne pathogens in stray and client-owned dogs from Algiers.

    Science.gov (United States)

    Azzag, Naouelle; Petit, Elisabeth; Gandoin, Christelle; Bouillin, Corinne; Ghalmi, Farida; Haddad, Nadia; Boulouis, Henri-Jean

    2015-02-01

    Data on the prevalence of vector-borne diseases agents infecting canines in Algeria is currently lacking. The purpose of this study is to assess by serological and molecular methods the prevalence of select arthropod borne-bacterial infections in client-owned and stray dogs. Antibodies to Anaplasma phagocytophilum were the most prevalent at 47.7%, followed by Borrelia burgdorferi s.l. at 37.6%, Ehrlichia canis at 30.0%, Bartonella henselae at 32.4% and Bartonella vinsonii subsp. berkhoffii at 27%. Seroprevalence was statistically significantly higher in stray dogs than those owned by clients. Seropositivity was not associated with health status, except for E. canis. Molecular evaluation indicates that 17.8% of the 213 analyzed dogs were positive for Ehrlichia and Anaplasma with a prevalence of 4.2% for E. canis, 14.1% for Anaplama platys and 0% for A. phagocytophilum. Seven (7.1%) of the tested dogs were positive for Bartonella spp. with two characterized as Bartonella rochalimae, four as B. henselae and one as B.v. subsp. berkhoffii. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Fate of selected pathogens in spiked «SALAME NOSTRANO» produced without added nitrates following the application of NONIT™ technology.

    Science.gov (United States)

    Cenci-Goga, B T; Karama, M; Sechi, P; Iulietto, M F; Grispoldi, L; Selvaggini, R; Ceccarelli, M; Barbera, S

    2018-02-14

    This study evaluated the effect of a novel formulation for starter culture associated with specific ripening conditions (NoNit™ technology) vs. a commercial» starter on the fate of selected pathogens and hygiene indicators during the fermentation and ripening of experimentally spiked salame nostrano (Italian dry sausage). Selected strains of Staphylococcus aureus 27R, Escherichia coli CSH26 K 12, Listeria innocua ATCC 33090 and Salmonella Derby 27 were inoculated into salami batter and challenged with two formulations of starter cultures (a commercial formulation and the NoNit™ formulation, consisting of Lactococcus lactis ssp. lactis, strain 340; L. lactis ssp. lactis, strain 16; Lactobacillus casei ssp. casei, strain 208 and Enterococcus faecium strain 614) with ripening at a low temperature. The proposed technology (NoNit™) performed better than the commercial formulation and limited the growth of spiked Escherichia coli, Staphylococcus aureus (including the production of enterotoxin), Salmonella Derby and Listeria innocua, yet maintained the basic product appearance and texture. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Gene flow, recombination, and positive selection in Stenotrophomonas maltophilia: mechanisms underlying the diversity of the widespread opportunistic pathogen.

    Science.gov (United States)

    Yu, Dong; Yin, Zhiqiu; Li, Beiping; Jin, Yuan; Ren, Hongguang; Zhou, Jing; Zhou, Wei; Liang, Long; Yue, Junjie

    2016-12-01

    Stenotrophomonas maltophilia is a global multidrug-resistant human opportunistic pathogen in clinical environments. Stenotrophomonas maltophilia is also ubiquitous in aqueous environments, soil, and plants. Various molecular typing methods have revealed that S. maltophilia exhibits high levels of phenotypic and genotypic diversity. However, information regarding the genomic diversity within S. maltophilia and the corresponding genetic mechanisms resulting in said diversity remain scarce. The genome sequences of 17 S. maltophilia strains were selected to investigate the mechanisms contributing to genetic diversity at the genome level. The core and large pan-genomes of the species were first estimated, resulting in a large, open pan-genome. A species phylogeny was also reconstructed based on 344 orthologous genes with one copy per genome, and the contribution of four evolutionary mechanisms to the species genome diversity was quantified: 15%-35% of the genes showed evidence for recombination, 0%-25% of the genes in one genome were likely gained, 0%-44% of the genes in some genomes were likely lost, and less than 0.3% of the genes in a genome were under positive selection pressures. We observed that, among the four main mechanisms, homologous recombination plays a key role in maintaining diversity in S. maltophilia. In this study, we provide an overview of evolution in S. maltophilia to provide a better understanding of its evolutionary dynamics and its relationship with genome diversity.

  15. Soil eukaryotic microorganism succession as affected by continuous cropping of peanut--pathogenic and beneficial fungi were selected.

    Directory of Open Access Journals (Sweden)

    Mingna Chen

    Full Text Available Peanut is an important oil crop worldwide and shows considerable adaptability but growth and yield are negatively affected by continuous cropping. Soil micro-organisms are efficient bio-indicators of soil quality and plant health and are critical to the sustainability of soil-based ecosystem function and to successful plant growth. In this study, 18S rRNA gene clone library analyses were employed to study the succession progress of soil eukaryotic micro-organisms under continuous peanut cultivation. Eight libraries were constructed for peanut over three continuous cropping cycles and its representative growth stages. Cluster analyses indicated that soil micro-eukaryotic assemblages obtained from the same peanut cropping cycle were similar, regardless of growth period. Six eukaryotic groups were found and fungi predominated in all libraries. The fungal populations showed significant dynamic change and overall diversity increased over time under continuous peanut cropping. The abundance and/or diversity of clones affiliated with Eurotiales, Hypocreales, Glomerales, Orbiliales, Mucorales and Tremellales showed an increasing trend with continuous cropping but clones affiliated with Agaricales, Cantharellales, Pezizales and Pyxidiophorales decreased in abundance and/or diversity over time. The current data, along with data from previous studies, demonstrated that the soil microbial community was affected by continuous cropping, in particular, the pathogenic and beneficial fungi that were positively selected over time, which is commonplace in agro-ecosystems. The trend towards an increase in fungal pathogens and simplification of the beneficial fungal community could be important factors contributing to the decline in peanut growth and yield over many years of continuous cropping.

  16. Survey of selected pathogens and blood parameters of northern yellowstone elk: Wolf sanitation effect implications

    Science.gov (United States)

    Barber-Meyer, S. M.; White, P.J.; Mech, L.D.

    2007-01-01

    The restoration or conservation of predators could reduce seroprevalences of certain diseases in prey if predation selectively removes animals exhibiting clinical signs. We assessed disease seroprevalences and blood parameters of 115 adult female elk (Cervus elaphus) wintering on the northern range of Yellowstone National Park [YNP] during 2000-2005 and compared them to data collected prior to wolf (Canis lupus) restoration (WR) in 1995 and to two other herds in Montana to assess this prediction. Blood parameters were generally within two standard deviations of the means observed in other Montana herds (Gravelly-Snowcrest [GS] and Garnet Mountain [GM]), but Yellowstone elk had higher seroprevalences of parainfluenza-3 virus (95% CI YNP = 61.1-78.6, GS = 30.3-46.5) and bovine-virus-diarrhea virus type 1 (95% CI YNP = 15.9-31.9, GM = 0). In comparisons between pre-wolf restoration [pre-WR] (i.e., prior to 1995) seroprevalences with those post-wolf restoration [post-WR] in Yellowstone, we found lower seroprevalences for some disease-causing agents post-wolf restoration (e.g., bovine-virus-diarrhea virus type-1 [95% CI pre-WR = 73.1-86.3, post-WR = 15.9-31.9] and bovine-respiratory syncytial virus [95% CI pre-WR = 70.0-83.8, post-WR = 0]), but similar (e.g., Brucella abortus [95% CI pre-WR = 0-4.45, post-WR = 0-4.74] and epizootic hemorrhagic disease virus [95% CI pre-WR = 0, post-WR = 0]) or higher for others (e.g., Anaplasma marginale [95% CI pre-WR = 0, post-WR = 18.5-38.7] and Leptospira spp. [95% CI pre-WR = 0.5-6.5, post-WR = 9.5-23.5]). Though we did not detect an overall strong predation effect through reduced disease seroprevalence using retrospective comparisons with sparse data, our reference values will facilitate future assessments of this issue.

  17. Positive directional selection in the proline-rich antigen (PRA) gene among the human pathogenic fungi Coccidioides immitis, C. posadasii and their closest relatives.

    Science.gov (United States)

    Johannesson, Hanna; Vidal, Pilar; Guarro, Josep; Herr, Roger A; Cole, Garry T; Taylor, John W

    2004-06-01

    In this study, we investigate the possibility of selection acting on the proline-rich antigen (PRA) gene in natural populations of the two human pathogens, Coccidioides immitis and Coccidioides posadasii, and three of their close relatives, Chrysosporium lucknowense, Chrysosporium queenslandicum, and Uncinocarpus reesii. We addressed the following questions: Is diversifying selection acting on PRA in the pathogenic species as a result of avoidance of the host's immune system, and has adaptation to a pathogenic life style lead to positive directional selection and increased rate of evolution in PRA between the species? For these purposes, we amplified and sequenced from 40 individuals belonging to the five species, the entire coding region of the PRA gene, as well as partial sequences from the coding region of each of the three housekeeping genes glyderaldehyde-3-phosphate dehydrogenase, glutamine synthetase A, and hexokinase A. We used likelihood-based methods to compare models of different types of selective pressure among codons to analyze the mode of evolution of the genes and found that the PRA gene evolves under positive selection, but the investigated parts of the housekeeping genes evolve primarily under purifying selection. We found a very low level of intraspecific variability and no evidence of diversifying selection, suggesting that the increased rate of evolution in the PRA gene is not a result of avoidance of the host's immune system. Neither did likelihood-based analyses suggest that selection was stronger on the branch separating pathogenic and nonpathogenic species. Instead, we suggest that positive selection act on PRA as a consequence of spore cell-wall morphogenesis unique to each species.

  18. Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate effectors

    OpenAIRE

    Sperschneider, Jana; Ying, Hua; Dodds, Peter N.; Gardiner, Donald M.; Upadhyaya, Narayana M.; Singh, Karam B.; Manners, John M.; Taylor, Jennifer M.

    2014-01-01

    Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with targe...

  19. A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo.

    Science.gov (United States)

    Naudin, Clément; Schumski, Ariane; Salo-Ahen, Outi M H; Herwald, Heiko; Smeds, Emanuel

    2017-05-01

    Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well-characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA-based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost-effective method that can be used to prevent unnecessary animal experiments. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  20. Enhanced photoresponsive polyethyleneimine/citric acid co-carbonized dots for facile and selective sensing and intracellular imaging of cobalt ions at physiologic pH

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Wen-Sheng, E-mail: wszou@ahjzu.edu.cn; Zhao, Qing-Chun; Zhang, Jun; Chen, Xiao-Ming; Wang, Xiu-Fang; Zhao, Lin; Chen, Shao-Hua; Wang, Ya-Qin, E-mail: yqwang@ahjzu.edu.cn

    2017-06-01

    Whether as an important biological element or as a radioactive source/medicine, the monitoring of trace levels of cobalt ions (Co) has become a non-negligible factor for human health and green environment. Current technologies for the detection of Co are cost-expensive and time-consuming, and require cumbersome sample pretreatment process. Herein a novel sensing platform has been developed for Co detection based on the quenching of the enhanced fluorescence signal of polyamine functionalized C-dots. Amine groups at the surface of the C-dots can capture Zn{sup 2+}/Cd{sup 2+} to form coordination compound, which can inhibit the photoinduced electron transfer pathways of C-dots and then induce the fluorescence enhancement of the C-dots by ∼80% margin. Also, Co interacts with these amine groups to form an absorbent complex, which can strongly quench the enhanced fluorescence of C-dots via an inner filter effect. This C-dots-based probe showed a wide linear response to Co with a concentration ranging from 0.012 to 12 μM, and a detection limit of 8.0 nM and RSD of 5.7% (n = 5). Significantly, the C-Dots exhibit excellent properties, such as negligible cytotoxicity, excellent biocompatibility, low-cost and high photostability, etc., which make C-dots favorable for label-free monitoring of Co and then successfully applied to the confocal imaging of intracellular Co. - Highlights: • Polyethyleneimine/citric acid co-carbonized dots were prepared. • Zn{sup 2+} can enhance fluorescence of C-dots by inhibiting PET pathways. • Co{sup 2+} can quench the enhanced fluorescence by an inner filter effect. • Bioprobe has been established for intracellular imaging Co.

  1. Antimicrobial activity of Piper nigrum L. and Cassia didymobotyra L. leaf extract on selected food borne pathogens

    Directory of Open Access Journals (Sweden)

    Mohd. Sayeed Akthar

    2014-09-01

    Full Text Available Objective: To investigate the antimicrobial activity of leaf extract of Piper nigrum (P. nigrum and Cassia didymobotyra (C. didymobotyra (aqueous, methanol, ethanol and petroleum ether against the food borne pathogenic bacteria [Staphylococcus aureus (S. aureus, Escherichia coli (E. coli, Salmonella typhimurium and Pseudomonas aeruginosa] and fungi [Aspergillus spp. and Candida albicans (C. albicans] and also to investigate the presence of various phytochemicals in the leaf extracts of tested plants. Methods: The antimicrobial activity was determined by disc diffusion method. Minimum inhibitory concentration (MIC, minimum bactericidal and fungicidal concentration were determined by serial dilution method. Results: Methanol leaf extract of test plants exhibited greater antimicrobial activity against the selected bacterial and fungal strains. The MIC results showed that ethanol, methanol and petroleum ether leaf extract of P. nigrum inhibited the growth of S. aureus and E. coli at concentration of 12.5 mg/mL. While, ethanol and methanol leaf extracts of C. didymobotyra inhibited the growth of S. aureus at concentration of 6.25 mg/mL. The MIC values for ethanol, methanol and petroleum ether leaf extract of P. nigrum inhibited the growth of C. albicans at concentration of 25.0 mg/mL. While, it was reported that at concentration of 12.5 mg/mL methanol leaf extract of P. nigrum was against the Aspergillus spp. The MIC values of methanol leaf extract of C. didymobotyra inhibited the growth of C. albicans and Aspergillus spp. at concentration of 12.5 mg/mL and 6.25 mg/mL, respectively. The minimum bactericidal concentration of ethanol, methanol leaf extract of P. nigrum for E. coli and ethanol, methanol leaf extract of C. didymobotyra for S. aureus was recorded at concentration 12.5 mg/mL. The minimum fungicidal concentration of ethanol and methanol leaf extract of P. nigrum and C. didymobotyra on C. albicans was recorded at concentration of 25.0 mg

  2. Host-pathogen interactions and subversion of autophagy.

    Science.gov (United States)

    McEwan, David G

    2017-12-12

    Macroautophagy ('autophagy'), is the process by which cells can form a double-membraned vesicle that encapsulates material to be degraded by the lysosome. This can include complex structures such as damaged mitochondria, peroxisomes, protein aggregates and large swathes of cytoplasm that can not be processed efficiently by other means of degradation. Recycling of amino acids and lipids through autophagy allows the cell to form intracellular pools that aid survival during periods of stress, including growth factor deprivation, amino acid starvation or a depleted oxygen supply. One of the major functions of autophagy that has emerged over the last decade is its importance as a safeguard against infection. The ability of autophagy to selectively target intracellular pathogens for destruction is now regarded as a key aspect of the innate immune response. However, pathogens have evolved mechanisms to either evade or reconfigure the autophagy pathway for their own survival. Understanding how pathogens interact with and manipulate the host autophagy pathway will hopefully provide a basis for combating infection and increase our understanding of the role and regulation of autophagy. Herein, we will discuss how the host cell can identify and target invading pathogens and how pathogens have adapted in order to evade destruction by the host cell. In particular, we will focus on interactions between the mammalian autophagy gene 8 (ATG8) proteins and the host and pathogen effector proteins. © 2017 The Author(s).

  3. Harmonizing methods for wildlife abundance estimation and pathogen detection in Europe-a questionnaire survey on three selected host-pathogen combinations

    DEFF Research Database (Denmark)

    Schulz, Jana; Ryser-Degiorgis, Marie-Pierre; Kuiken, Thijs

    2017-01-01

    Background: The need for wildlife health surveillance as part of disease control in wildlife, domestic animals and humans on the global level is widely recognized. However, the objectives, methods and intensity of existing wildlife health surveillance programs vary greatly among European countries......, resulting in a patchwork of data that are difficult to merge and compare. This survey aimed at evaluating the need and potential for data harmonization in wildlife health in Europe. The specific objective was to collect information on methods currently used to estimate host abundance and pathogen prevalence...... estimation, there is an urgent need to develop tools for the routine collection of host abundance data in a harmonized way. Wildlife health experts are encouraged to apply the harmonized APHAEA protocols in epidemiological studies in wildlife and to increase cooperation....

  4. Interferon-¿ production by human T cells and natural killer cells in vitro in response to antigens from the two intracellular pathogens Mycobacterium tuberculosis and Leishmania major

    DEFF Research Database (Denmark)

    Kemp, K; Hviid, L; Kharazmi, A

    1997-01-01

    Acquired resistance to both mycobacteria and Leishmania is primarily mediated by interferon-gamma (IFN-gamma), which triggers mechanisms leading to the death of the microorganism in macrophages. In this study, cell activation and IFN-gamma production was investigated in human peripheral blood...... mononuclear cells (PBMC) from individuals previously sensitized to tuberculin and without known exposure to Leishmania parasites. Immune staining for intracellular IFN-gamma and surface markers allowed flow cytometric identification of the cellular sources of IFN-gamma in cell cultures incubated with purified...

  5. In-Vitro Activity of Saponins of Bauhinia Purpurea Madhuca Longifolia Celastrus Paniculatus and Semecarpus Anacardium on Selected Oral Pathogens

    Directory of Open Access Journals (Sweden)

    K. S. Jyothi

    2012-01-01

    Full Text Available Objective: Dental caries, periodontitis and other mucosal diseases are caused by a complex community of microorganisms. This study aimed to investigate the antimicrobial properties of saponins of four important oil yielding medicinal plant extracts on selected oral pathogens that are involved in such diseases.Materials and Methods: Saponins were extracted from Bauhinia purpurea, Madhuca longifolia, Celastrus paniculatus and Semecarpus anacardium and purified. Antimicrobial properties of these saponins against Streptococcus mutans, Streptococcus mitis, Streptococcus salivarius, Staphylococcus aureus and Lactobacillus acidophilus were determined using well diffusion method. The minimum inhibitory concentration (MIC was determined as the lowest concentration of saponins inhibiting bacterial growth after 14 h of incubation at 37°C. The bactericidal activity was evaluated using the viable cell count method.Results: The minimum inhibitory concentration (MIC and minimum bactericidal concentration (MBC of Madhuca longifolia saponin on Streptococcus mutans MTCC 890, Streptococcus mitis and Staphylococcus aureus was 18.3 ± 0.15/34.4 ± 0.24 µg/ml, 19.0 ± 0.05/32.2 ± 0.0 µg/ml and 21.2 ± 0.35/39.0 ± 0.30 µg/ml, respectively and Bauhinia purpurea saponin on Streptococcus mutans MTCC 890, Staphylococcus aureus and Lactobacillus acidophilus was 26.4 ± 0.20/43.0 ± 0.40 µg/ml, 29.0 ± 0.30/39.6 ± 0.12 µg/ml and 20.2 ± 0.05/36.8 ± 0.23 µg/ml, respectively.Conclusion: The strong antimicrobial activity of Madhuca longifolia and Bauhinia purpurea may be due to the presence of complex triterpenoid saponins, oleanane type triterpenoid glycosides or atypical pentacyclic triterpenoid saponin. Hence, these extracted saponins may be used in food and oral products to prevent and control oral diseases.

  6. Use of oligonucleotide array for identification of six foodborne pathogens and Pseudomonas aeruginosa grown on selective media.

    Science.gov (United States)

    Lin, Miao Chu; Huang, Ay Huey; Tsen, Hau Yang; Wong, Hin-Chung; Chang, Tsung Chain

    2005-11-01

    Identification of presumptive foodborne pathogens grown on selective media may take one to several days and requires a different battery of biochemical tests for each microorganism. A molecular identification method was developed in which universal primers were used to amplify the 16S to 23S rDNA intergenic spacer of target microorganisms, and PCR products were hybridized to a panel of species-specific oligonucleotides that were immobilized on a nylon membrane. The seven target microorganisms were Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and Vibrio parahaemolyticus. After testing a large collection of target bacteria (29 to 51 strains) and nontarget bacteria (> 500 strains), the performances (sensitivity and specificity) of the oligonucleotide array were as follows: B. cereus (100 and 77%), E. coli (100 and 100%), L. monocytogenes (100 and 90%), P. aeruginosa (100 and 100%), Salmonella (100 and 100%), S. aureus (100 and 100%), and V. parahaemolyticus (100 and 94.2%). Other species in the B. cereus group cross-hybridized to the probes used for identification of B. cereus, and positive results should be confirmed by additional morphological observation of colonies. Listeria innocua cross-reacted with probes used to identify L. monocytogenes, but a simple hemolysis test was used to differentiate the two species. Some strains of Vibrio harveyi and Vibrio mimicus cross-hybridized with probes used for identification of V. parahaemolyticus and caused false-positive reactions. The advantage of the array is that a common protocol was used to identify the seven target microorganisms and multiple different microorganisms could be simultaneously identified on a single array.

  7. Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

    Directory of Open Access Journals (Sweden)

    Ahmed R El-Awady

    2015-02-01

    Full Text Available Signaling via pattern recognition receptors (PRRs expressed on professional antigen presenting cells, such as dendritic cells (DCs, is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs. We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

  8. Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

    Science.gov (United States)

    El-Awady, Ahmed R; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B; Palani, Chithra D; Arce, Roger M; Waller, Jennifer L; Genco, Caroline A; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V; Cutler, Christopher W

    2015-02-01

    Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

  9. Macrophage defense mechanisms against intracellular bacteria.

    Science.gov (United States)

    Weiss, Günter; Schaible, Ulrich E

    2015-03-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. © 2015 The Authors

  10. Macrophage defense mechanisms against intracellular bacteria

    Science.gov (United States)

    Weiss, Günter; Schaible, Ulrich E

    2015-01-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. PMID:25703560

  11. Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate effectors.

    Science.gov (United States)

    Sperschneider, Jana; Ying, Hua; Dodds, Peter N; Gardiner, Donald M; Upadhyaya, Narayana M; Singh, Karam B; Manners, John M; Taylor, Jennifer M

    2014-01-01

    Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defense proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialized gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control.

  12. Diversifying Selection in the Wheat Stem Rust Fungus Acts Predominantly on Pathogen-Associated Gene Families and Reveals Candidate Effectors

    Directory of Open Access Journals (Sweden)

    Jana eSperschneider

    2014-09-01

    Full Text Available Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defence proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialised gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control.

  13. A method for functional trans-complementation of intracellular Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    Shaun Steele

    Full Text Available Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional

  14. The HLA-B landscape of Africa: Signatures of pathogen-driven selection and molecular identification of candidate alleles to malaria protection

    Czech Academy of Sciences Publication Activity Database

    Sanchez-Mazas, A.; Černý, V.; Di, D.; Buhler, S.; Podgorná, Eliška; Chevallier, E.; Brunet, L.; Weber, S.; Kervaire, B.; Testi, M.; Andreani, M.; Tiercy, J.-M.; Villard, J.; Nunes, J. M.

    2017-01-01

    Roč. 26, č. 22 (2017), s. 6238-6252 ISSN 0962-1083 R&D Projects: GA ČR GA13-37998S Institutional support: RVO:67985912 Keywords : African populations * geographic patterns * HLA polymorphism and disease associations * human population genetics * malaria protection * pathogen-driven selection Subject RIV: AC - Archeology, Anthropology, Ethnology OBOR OECD: Archaeology Impact factor: 6.086, year: 2016

  15. Cell-selective labeling of bacterial proteomes with an orthogonal phenylalanine amino acid reporter.

    Science.gov (United States)

    Grammel, Markus; Dossa, Paul D; Taylor-Salmon, Emma; Hang, Howard C

    2012-02-01

    Orthogonal amino acid reporters allow the selective labeling of different cell types in heterogeneous populations through the expression of engineered aminoacyl tRNA synthetases. Here, we demonstrate that para-ethynylphenylalanine (PEP) can be used as an orthogonal amino acid reporter for efficient selective labeling of an intracellular bacterial pathogen during infection. This journal is © The Royal Society of Chemistry 2012

  16. Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations.

    Science.gov (United States)

    Ul Ain, Qurrat; Lee, Jong Hwan; Woo, Young Sun; Kim, Yong-Hee

    2016-09-01

    Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein's nature, transduction efficiency and stability.

  17. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

    DEFF Research Database (Denmark)

    Gold, Marielle C.; McLaren, James E.; Reistetter, Joseph A.

    2015-01-01

    with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped...

  18. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

    DEFF Research Database (Denmark)

    Gold, Marielle C.; McLaren, James E.; Reistetter, Joseph A.

    2014-01-01

    with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped...

  19. Environmental E.coli isolate characterization and selection as improved indicators for pathogens associated with produce contamination

    Science.gov (United States)

    Contamination of food and water by pathogens is a substantial public health issue in the United States. According to the Centers for Disease Control and Prevention, 49% of foodborne illness is caused by produce, and despite efforts to reduce produce contamination, these issues persist. Escherichia c...

  20. Repression of fungal plant pathogens and fungal-related contaminants: Selected ecosystem services by soil fauna communities in agroecosystems

    Science.gov (United States)

    Meyer-Wolfarth, Friederike; Schrader, Stefan; Oldenburg, Elisabeth; Brunotte, Joachim; Weinert, Joachim

    2017-04-01

    In agroecosystems soil-borne fungal plant diseases are major yield-limiting factors which are difficult to control. Fungal plant pathogens, like Fusarium species, survive as a saprophyte in infected tissue like crop residues and endanger the health of the following crop by increasing the infection risk for specific plant diseases. In infected plant organs, these pathogens are able to produce mycotoxins. Mycotoxins like deoxynivalenol (DON) persist during storage, are heat resistant and of major concern for human and animal health after consumption of contaminated food and feed, respectively. Among fungivorous soil organisms, there are representatives of the soil fauna which are obviously antagonistic to a Fusarium infection and the contamination with mycotoxins. Specific members of the soil macro-, meso-, and microfauna provide a wide range of ecosystem services including the stimulation of decomposition processes which may result in the regulation of plant pathogens and the degradation of environmental contaminants. Investigations under laboratory conditions and in field were conducted to assess the functional linkage between soil faunal communities and plant pathogenic fungi (Fusarium culmorum). The aim was to examine if Fusarium biomass and the content of its mycotoxin DON decrease substantially in the presence of soil fauna (earthworms: Lumbricus terrestris, collembolans: Folsomia candida and nematodes: Aphelenchoides saprophilus) in a commercial cropping system managed with conservation tillage located in Northern Germany. The results of our investigations pointed out that the degradation performance of the introduced soil fauna must be considered as an important contribution to the biodegradation of fungal plant diseases and fungal-related contaminants. Different size classes within functional groups and the traits of keystone species appear to be significant for soil function and the provision of ecosystem services as in particular L. terrestris revealed to

  1. Salmonella Intracellular Lifestyles and Their Impact on Host-to-Host Transmission.

    Science.gov (United States)

    Pucciarelli, M Graciela; García-Del Portillo, Francisco

    2017-07-01

    More than a century ago, infections by Salmonella were already associated with foodborne enteric diseases with high morbidity in humans and cattle. Intestinal inflammation and diarrhea are hallmarks of infections caused by nontyphoidal Salmonella serovars, and these pathologies facilitate pathogen transmission to the environment. In those early times, physicians and microbiologists also realized that typhoid and paratyphoid fever caused by some Salmonella serovars could be transmitted by "carriers," individuals outwardly healthy or at most suffering from some minor chronic complaint. In his pioneering study of the nontyphoidal serovar Typhimurium in 1967, Takeuchi published the first images of intracellular bacteria enclosed by membrane-bound vacuoles in the initial stages of the intestinal epithelium penetration. These compartments, called Salmonella -containing vacuoles, are highly dynamic phagosomes with differing biogenesis depending on the host cell type. Single-cell studies involving real-time imaging and gene expression profiling, together with new approaches based on genetic reporters sensitive to growth rate, have uncovered unprecedented heterogeneous responses in intracellular bacteria. Subpopulations of intracellular bacteria displaying fast, reduced, or no growth, as well as cytosolic and intravacuolar bacteria, have been reported in both in vitro and in vivo infection models. Recent investigations, most of them focused on the serovar Typhimurium, point to the selection of persisting bacteria inside macrophages or following an autophagy attack in fibroblasts. Here, we discuss these heterogeneous intracellular lifestyles and speculate on how these disparate behaviors may impact host-to-host transmissibility of Salmonella serovars.

  2. The ability of selected plant essential oils to enhance the action of recommended antibiotics against pathogenic wound bacteria.

    Science.gov (United States)

    Sienkiewicz, Monika; Łysakowska, Monika; Kowalczyk, Edward; Szymańska, Grażyna; Kochan, Ewa; Krukowska, Jolanta; Olszewski, Jurek; Zielińska-Bliźniewska, Hanna

    2017-03-01

    The aim of this work was to characterize the ability of essential oils to support antibiotics against pathogenic bacteria in wounds. Gram-positive and Gram-negative bacteria obtained from wound infections were identified according to standard microbiological methods. Essential oils were analysed by GC-FID-MS. The susceptibility of bacteria to antibiotics, essential oils and their combination was assessed using the disc-diffusion method. The Minimal Inhibitory Concentration and Minimum Bactericidal Concentration of the essential oils were established by the micro-dilution broth method. Although cinnamon, clove, thyme and lavender essential oils were found to have the greatest antibacterial activity when used alone, the greatest additive and synergistic effects against pathogenic wound bacteria in combination with recommended antibiotics were demonstrated by basil, clary sage and rosemary oils. Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.

  3. Mycoplasmas and bovine respiratory disease: studies related to pathogenicity and the immune response--a selective review.

    Science.gov (United States)

    Howard, C J

    1983-01-01

    Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum. M. bovis is the most pathogenic of these species but the disease outbreaks with which it is associated are sporadic. M. dispar is regularly isolated from pneumonic calves but is also found causing mild superficial and asymptomatic infections of the respiratory mucosa. The bovine ureaplasmas are serologically complex. They are distinct from ureaplasmas isolated from other non-ruminants by PAGE analysis, G + C content of DNA, and serology. A second species within the genus ureaplasma has been proposed to accommodate the bovine ureaplasmas, U. diversum. Control of mycoplasma respiratory infections of cattle based on immunization might be possible. Calves have been immunized against M. bovis and immunity has been related to antibody in the lung. M. dispar appears less immunogenic in calves than M. bovis and this may contribute to its pathogenicity.

  4. Immune escape mutants of Highly Pathogenic Avian Influenza H5N1 selected using polyclonal sera: identification of key amino acids in the HA protein.

    Directory of Open Access Journals (Sweden)

    Ioannis Sitaras

    Full Text Available Evolution of Avian Influenza (AI viruses--especially of the Highly Pathogenic Avian Influenza (HPAI H5N1 subtype--is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1, using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.

  5. Subversion of the cytoskeleton by intracellular bacteria: lessons from Listeria, Salmonella, and Vibrio

    Science.gov (United States)

    de Souza Santos, Marcela; Orth, Kim

    2018-01-01

    Summary Entry into host cells and intracellular persistence by invasive bacteria are tightly coupled to the ability of the bacterium to disrupt the eukaryotic cytoskeletal machinery. Herein we review the main strategies used by three intracellular pathogens to harness key modulators of the cytoskeleton. Two of these bacteria, namely Listeria monocytogenes and Salmonella enterica serovar Typhimurium, exhibit quite distinct intracellular lifestyles, and therefore, provide a comprehensive panel for the understanding of the intricate bacteria-cytoskeleton interplay during infections. The emerging intracellular pathogen Vibrio parahaemolyticus is depicted as a developing model for the uncovering of novel mechanisms used to hijack the cytoskeleton. PMID:25440316

  6. Viruses of plant pathogenic fungi.

    Science.gov (United States)

    Ghabrial, Said A; Suzuki, Nobuhiro

    2009-01-01

    Mycoviruses are widespread in all major groups of plant pathogenic fungi. They are transmitted intracellularly during cell division, sporogenesis, and cell fusion, but apparently lack an extracellular route for infection. Their natural host ranges are limited to individuals within the same or closely related vegetative compatibility groups. Recent advances, however, allowed the establishment of experimental host ranges for a few mycoviruses. Although the majority of known mycoviruses have dsRNA genomes that are packaged in isometric particles, an increasing number of usually unencapsidated mycoviruses with positive-strand RNA genomes have been reported. We discuss selected mycoviruses that cause debilitating diseases and/or reduce the virulence of their phytopathogenic fungal hosts. Such fungal-virus systems are valuable for the development of novel biocontol strategies and for gaining an insight into the molecular basis of fungal virulence. The availability of viral and host genome sequences and of transformation and transfection protocols for some plant pathogenic fungi will contribute to progress in fungal virology.

  7. Serosurvey for Selected Viral Pathogens among Sympatric Species of the African Large Predator Guild in Northern Botswana.

    Science.gov (United States)

    Chaber, Anne-Lise; Cozzi, Gabriele; Broekhuis, Femke; Hartley, Robyn; W McNutt, John

    2017-01-01

    The recent increase in the creation of transboundary protected areas and wildlife corridors between them lends importance to information on pathogen prevalence and transmission among wildlife species that will become connected. One such initiative is the Kavango Zambezi Transfrontier Conservation Area of which Botswana's Okavango Delta constitutes a major contribution for wildlife and ecosystems. Between 2008 and 2011, we collected serum samples from 14 lions ( Panthera leo ), four leopards ( Panthera pardus ), 19 spotted hyenas ( Crocuta crocuta ), and six cheetahs ( Acinonyx jubatus ) in the Okavango. Samples were tested for antibodies against canine distemper virus (CDV), feline panleukopenia virus, enteric coronavirus, feline calicivirus, feline herpesvirus (FHV-1), and feline immunodeficiency virus (FIV). Evidence of exposure to all of these pathogens was found, to varying degrees, in at least one of the species sampled. High antibody prevalence (>90%) was only found to FHV-1 and FIV in lions. Only hyenas (26%, 5/19) were positive for CDV antibody. Except for one case, all individuals displayed physical conditions consistent with normal health for ≥12 mo following sampling. Our results emphasize the need for a comprehensive, multispecies approach to disease monitoring and the development of coordinated management strategies for subpopulations likely to be connected in transboundary initiatives.

  8. Pattern of Bacterial Pathogens and Their Susceptibility Isolated from Surgical Site Infections at Selected Referral Hospitals, Addis Ababa, Ethiopia

    Directory of Open Access Journals (Sweden)

    Walelign Dessie

    2016-01-01

    Full Text Available Background. The emergence of multidrug resistant bacterial pathogens in hospitals is becoming a challenge for surgeons to treat hospital acquired infections. Objective. To determine bacterial pathogens and drug susceptibility isolated from surgical site infections at St. Paul Specialized Hospital Millennium Medical College and Yekatit 12 Referral Hospital Medical College, Addis Ababa, Ethiopia. Methods. A cross-sectional study was conducted between October 2013 and March 2014 on 107 surgical site infected patients. Wound specimens were collected using sterile cotton swab and processed as per standard operative procedures in appropriate culture media; and susceptibility testing was done using Kirby-Bauer disc diffusion technique. The data were analyzed by using SPSS version 20. Result. From a total of 107 swabs collected, 90 (84.1% were culture positive and 104 organisms were isolated. E. coli (24 (23.1% was the most common organism isolated followed by multidrug resistant Acinetobacter species (23 (22.1%. More than 58 (75% of the Gram negative isolates showed multiple antibiotic resistance (resistance ≥ 5 drugs. Pan-antibiotic resistance was noted among 8 (34.8% Acinetobacter species and 3 (12.5% E. coli. This calls for abstinence from antibiotic abuse. Conclusion. Gram negative bacteria were the most important isolates accounting for 76 (73.1%. Ampicillin, amoxicillin, penicillin, cephazoline, and tetracycline showed resistance while gentamicin and ciprofloxacin were relatively effective antimicrobials.

  9. Detection of selected antibiotic resistance genes using multiplex PCR assay in mastitis pathogens in the Czech Republic

    Directory of Open Access Journals (Sweden)

    Vladimir Pyatov

    2017-01-01

    Full Text Available The aim of this research was to develop multiplex polymerase chain reaction assays for the detection of aminoglycoside (strA, strB, sulphonamide (sulI, sulII, tetracycline (tetA, tetB, tetK, tetM, tetO, macrolide and lincosamide (msrA, ermA, ermB, ermC, mefA/E genes of resistance in mastitis pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae. Applying the established assays, we investigated the distribution of antibiotic resistance genes in the above mentioned species isolated from milk samples in the Czech Republic. Each assay consisted of seven pairs of primers. Six of them amplified fragments of antibiotic resistance genes and one pair a fragment of a species specific gene. Polymerase chain reaction conditions were optimized to amplify seven gene fragments simultaneously in one reaction. In total, 249 isolates were used, among which 111 were positive for E. coli, 52 for S. aureus and 86 for Streptococcus spp. The majority (60.2% of bacteria carried at least one antibiotic resistance gene and 44.6% were multidrug-resistant. The designed multiplex polymerase chain reaction assays may be applied as diagnostic method to replace or complement standard techniques of antibiotic susceptibility testing in the mentioned pathogens.

  10. From Select Agent to an Established Pathogen: The Response to Phakopsora pachyrhizi (Soybean Rust) in North America.

    Science.gov (United States)

    Kelly, Heather Y; Dufault, Nicholas S; Walker, David R; Isard, Scott A; Schneider, Raymond W; Giesler, Loren J; Wright, David L; Marois, James J; Hartman, Glen L

    2015-07-01

    The pathogen causing soybean rust, Phakopsora pachyrhizi, was first described in Japan in 1902. The disease was important in the Eastern Hemisphere for many decades before the fungus was reported in Hawaii in 1994, which was followed by reports from countries in Africa and South America. In 2004, P. pachyrhizi was confirmed in Louisiana, making it the first report in the continental United States. Based on yield losses from countries in Asia, Africa, and South America, it was clear that this pathogen could have a major economic impact on the yield of 30 million ha of soybean in the United States. The response by agencies within the United States Department of Agriculture, industry, soybean check-off boards, and universities was immediate and complex. The impacts of some of these activities are detailed in this review. The net result has been that the once dreaded disease, which caused substantial losses in other parts of the world, is now better understood and effectively managed in the United States. The disease continues to be monitored yearly for changes in spatial and temporal distribution so that soybean growers can continue to benefit by knowing where soybean rust is occurring during the growing season.

  11. Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

    2008-12-09

    For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.

  12. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens

    DEFF Research Database (Denmark)

    Abdul Momin, Muhd Haziq F; Bean, David C; Hendriksen, Rene S.

    2017-01-01

    Purpose. A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture...... for the growth of COL-resistant bacterial pathogens. Due to the low limit of detection (101 c.f.u.), it may be useful as a primary isolation medium in the surveillance and recovery of COL-resistant bacteria from complex human, veterinary and environmental samples, especially those with plasmid-mediated MCR-1...... medium (SuperPolymyxin). Methodology. The medium was challenged with 84 isolates, including polymyxin B (POL B)-susceptible and -resistant type strains and colistin (COL)-resistant organisms recovered from human and animal samples. Susceptibility to COL and POL B was determined by agar dilution and broth...

  13. The Black Yeast Exophiala dermatitidis and Other Selected Opportunistic Human Fungal Pathogens Spread from Dishwashers to Kitchens.

    Directory of Open Access Journals (Sweden)

    Jerneja Zupančič

    Full Text Available We investigated the diversity and distribution of fungi in nine different sites inside 30 residential dishwashers. In total, 503 fungal strains were isolated, which belong to 10 genera and 84 species. Irrespective of the sampled site, 83% of the dishwashers were positive for fungi. The most frequent opportunistic pathogenic species were Exophiala dermatitidis, Candida parapsilosis sensu stricto, Exophiala phaeomuriformis, Fusarium dimerum, and the Saprochaete/Magnusiomyces clade. The black yeast E. dermatitidis was detected in 47% of the dishwashers, primarily at the dishwasher rubber seals, at up to 106 CFU/cm2; the other fungi detected were in the range of 102 to 105 CFU/cm2. The other most heavily contaminated dishwasher sites were side nozzles, doors and drains. Only F. dimerum was isolated from washed dishes, while dishwasher waste water contained E. dermatitidis, Exophiala oligosperma and Sarocladium killiense. Plumbing systems supplying water to household appliances represent the most probable route for contamination of dishwashers, as the fungi that represented the core dishwasher mycobiota were also detected in the tap water. Hot aerosols from dishwashers contained the human opportunistic yeast C. parapsilosis, Rhodotorula mucilaginosa and E. dermatitidis (as well as common air-borne genera such as Aspergillus, Penicillium, Trichoderma and Cladosporium. Comparison of fungal contamination of kitchens without and with dishwashers revealed that virtually all were contaminated with fungi. In both cases, the most contaminated sites were the kitchen drain and the dish drying rack. The most important difference was higher prevalence of black yeasts (E. dermatitidis in particular in kitchens with dishwashers. In kitchens without dishwashers, C. parapsilosis strongly prevailed with negligible occurrence of E. dermatitidis. F. dimerum was isolated only from kitchens with dishwashers, while Saprochaete/Magnusiomyces isolates were only found within

  14. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

    Science.gov (United States)

    Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E.; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R.; Ross, Ian; Parton, Robert G.; Böcking, Till; Gambin, Yann

    2016-01-01

    Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils. PMID:27892477

  15. Chemical Composition and Antibacterial Activity of Essential Oils of Tagetes minuta (Asteraceae) against Selected Plant Pathogenic Bacteria

    Science.gov (United States)

    Wagacha, John M.; Dossaji, Saifuddin F.

    2016-01-01

    The objective of this study was to determine the chemical composition and antibacterial activity of essential oils (EOs) of Tagetes minuta against three phytopathogenic bacteria Pseudomonas savastanoi pv. phaseolicola, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas axonopodis pv. manihotis. The essential oils were extracted using steam distillation method in a modified Clevenger-type apparatus while antibacterial activity of the EOs was evaluated by disc diffusion method. Gas chromatography coupled to mass spectrometry (GC/MS) was used for analysis of the chemical profile of the EOs. Twenty compounds corresponding to 96% of the total essential oils were identified with 70% and 30% of the identified components being monoterpenes and sesquiterpenes, respectively. The essential oils of T. minuta revealed promising antibacterial activities against the test pathogens with Pseudomonas savastanoi pv. phaseolicola being the most susceptible with mean inhibition zone diameters of 41.83 and 44.83 mm after 24 and 48 hours, respectively. The minimum inhibitory concentrations and minimum bactericidal concentrations of the EOs on the test bacteria were in the ranges of 24–48 mg/mL and 95–190 mg/mL, respectively. These findings provide a scientific basis for the use of T. minuta essential oils as a botanical pesticide for management of phytopathogenic bacteria. PMID:27721831

  16. Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations

    Science.gov (United States)

    Gammage, Payam A; Rorbach, Joanna; Vincent, Anna I; Rebar, Edward J; Minczuk, Michal

    2014-01-01

    We designed and engineered mitochondrially targeted obligate heterodimeric zinc finger nucleases (mtZFNs) for site-specific elimination of pathogenic human mitochondrial DNA (mtDNA). We used mtZFNs to target and cleave mtDNA harbouring the m.8993T>G point mutation associated with neuropathy, ataxia, retinitis pigmentosa (NARP) and the “common deletion” (CD), a 4977-bp repeat-flanked deletion associated with adult-onset chronic progressive external ophthalmoplegia and, less frequently, Kearns-Sayre and Pearson's marrow pancreas syndromes. Expression of mtZFNs led to a reduction in mutant mtDNA haplotype load, and subsequent repopulation of wild-type mtDNA restored mitochondrial respiratory function in a CD cybrid cell model. This study constitutes proof-of-principle that, through heteroplasmy manipulation, delivery of site-specific nuclease activity to mitochondria can alleviate a severe biochemical phenotype in primary mitochondrial disease arising from deleted mtDNA species. PMID:24567072

  17. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence.

    Science.gov (United States)

    Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R; Ross, Ian; Parton, Robert G; Böcking, Till; Gambin, Yann

    2016-11-28

    Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

  18. Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing

    NARCIS (Netherlands)

    Rienksma, R.A.; Suarez Diez, M.; Mollenkopf, H.J.; Dolganov, G.M.; Dorhoi, A.; Schoolnik, G.K.; Martins Dos Santos, V.A.P.; Kaufmann, S.; Schaap, P.J.; Gengenbacher, M.

    2015-01-01

    BackgroundThe human pathogen Mycobacterium tuberculosis has the capacity to escape eradication by professional phagocytes. During infection, M. tuberculosis resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular

  19. Foodborne pathogens

    Directory of Open Access Journals (Sweden)

    Thomas Bintsis

    2017-06-01

    Full Text Available Foodborne pathogens are causing a great number of diseases with significant effects on human health and economy. The characteristics of the most common pathogenic bacteria (Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cronobacter sakazakii, Esherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococccus aureus, Vibrio spp. and Yersinia enterocolitica, viruses (Hepatitis A and Noroviruses and parasites (Cyclospora cayetanensis, Toxoplasma gondii and Trichinella spiralis, together with some important outbreaks, are reviewed. Food safety management systems based on to classical hazard-based approach has been proved to be inefficient, and risk-based food safety approach is now suggested from leading researchers and organizations. In this context, a food safety management system should be designed in a way to estimate the risks to human health from food consumption and to identify, select and implement mitigation strategies in order to control and reduce these risks. In addition, the application of suitable food safety education programs for all involved people in the production and consumption of foods is suggested.

  20. Characterization of HIV-Specific CD4+T Cell Responses against Peptides Selected with Broad Population and Pathogen Coverage

    DEFF Research Database (Denmark)

    Buggert, Marcus; Norstrom, Melissa M.; Czarnecki, Chris

    2012-01-01

    this challenge, and identify a set of potential HLA class II-restricted HIV epitopes that in concert will provide optimal viral and host coverage. Using this selection strategy, we identified 64 putative epitopes (peptides) located in the Gag, Nef, Env, Pol and Tat protein regions of HIV. In total, 73......CD4+ T cells orchestrate immunity against viral infections, but their importance in HIV infection remains controversial. Nevertheless, comprehensive studies have associated increase in breadth and functional characteristics of HIV-specific CD4+ T cells with decreased viral load. A major challenge...... for the identification of HIV-specific CD4+ T cells targeting broadly reactive epitopes in populations with diverse ethnic background stems from the vast genomic variation of HIV and the diversity of the host cellular immune system. Here, we describe a novel epitope selection strategy, PopCover, that aims to resolve...

  1. Antimicrobial Effect of Filipendula ulmaria Plant Extract Against Selected Foodborne Pathogenic and Spoilage Bacteria in Laboratory Media, Fish Flesh and Fish Roe Product

    Directory of Open Access Journals (Sweden)

    Charalampos Proestos

    2011-01-01

    Full Text Available Water-methanol extract from Filipendula ulmaria contains a variety of phenolic compounds, such as caffeic, p-coumaric and vanillic acid, myricetin, etc, which demonstrate antibacterial activity. Monitoring this activity in the broth using absorbance measurements showed that species of the Enterobacteriaceae family were more resistant than other Gram-negative and Gram-positive bacteria tested. Acidic environment enhanced the antibacterial activity of Filipendula ulmaria extract when it was tested against Salmonella Enteritidis PT4 and Listeria monocytogenes Scott A. The efficacy of Filipendula ulmaria extract against selected foodborne psychrotrophic bacteria was also tested using solid laboratory media and low incubation temperatures for better simulation of food preservation conditions. Higher concentrations of the extract, compared to minimum inhibitory concentration determined in the broth, were needed for satisfactory inhibition of spoilage bacteria. Potential use of Filipendula ulmaria extract as natural food preservative was also examined against natural spoilage flora and inoculated pathogenic bacteria on fish flesh and fish roe product (tarama salad. No significant differences of viable populations of spoilage or pathogenic bacteria were found between the treated samples and controls. Further trials of Filipendula ulmaria extract should be carried out in acidic foods with low fat and protein content, supplemented with additional adjuncts, in order to explore its potential as effective natural food antimicrobial agent.

  2. Highly selective and sensitive fluorescence sensing of nanomolar Zn2+ions in aqueous medium using Calix[4]arene passivated Carbon Quantum Dots based on fluorescence enhancement: Real-time monitoring and intracellular investigation.

    Science.gov (United States)

    Kaur, Harpreet; Raj, Pushap; Sharma, Hemant; Verma, Meenakshi; Singh, Narinder; Kaur, Navneet

    2018-06-07

    Carbon Quantum Dots (CDs) owing to its unique photophysical properties have attracted immense consideration of the researchers in last decade to explore its applications in the fields of bio-imaging and sensing. In the present work, we have developed a novel CDs functionalized with Calix[4]arene derivative by hydrothermal route to detect biologically pertinent Zn 2+ ions. Surface modification of CDs enhances fluorescence efficiency apart from introducing active surface sites broadening its sensor capabilities. The obtained surface-functionalized CDs exhibits a strong blue fluorescence under UV light (365 nm) with a quantum yield (Φ) of 56% which is appreciably good. Zn 2+ ions get apprehended in the cavity formed due to passivation by Calix[4]arene moeity on the exterior of CDs having profuse -NH 2 groups. Addition of Zn 2+ ions into the solution of CDs results in remarkable enhancement in the fluorescence intensity attributed to Photo-induced Electron Transfer (PET) "OFF" phenomenon. The CDs based nanosensor exhibited a wide linear response with a detection limit of 7.34 nM. Significantly, CDs were shown to display insignificant cytotoxicity, decent biocompatibility, and virtuous resistance to photo-bleaching, good stability over a wide range of pH, insignificant variation in the emission intensity in the presence of high concentration of salt and excellent bio-labeling ability when tested on E. coli cell lines. Additionally, the detection results of Zn 2+ ions obtained in real samples were in complete agreement with that obtained by Atomic Absorption Spectroscopy (AAS), signifying the prospective application of the proposed probe as a selective and sensitive sensor for Zn 2+ ions. The proposed sensor was also applied for fluorescence imaging of intracellular Zn 2+ ions. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Facile preparation and characterization of new green emitting carbon dots for sensitive and selective off/on detection of Fe3+ion and ascorbic acid in water and urine samples and intracellular imaging in living cells.

    Science.gov (United States)

    Shamsipur, Mojtaba; Molaei, Karam; Molaabasi, Fatemeh; Alipour, Mohsen; Alizadeh, Naader; Hosseinkhani, Saman; Hosseini, Morteza

    2018-06-01

    Carbon dots (CDs) have gained great attention as multifunctional materials because of their interesting properties and general applicability. However, there are some reports for the preparation of highly luminescent green-emitting CDs (G-CDs), although these reports seem not to be extensible. Herein, new G-CDs (quantum yield: 27.2%) were synthesized from a facile hydrothermal treatment of p-aminosalicylic acid and ethylene glycol dimethacrylate as both carbon and nitrogen source and cross-linking agent, respectively. The chemical composition and optical properties of the as-prepared G-CDs were successfully investigated using transmission electron microscopy, atomic force microscopy, dynamic light scattering, X-ray diffraction, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and fluorescence and UV-vis spectroscopies. Interestingly, the fluorescence intensity of G-CDs was selectivity quenched by Fe 3+ in the range of 0.05-10.0 µmol L- 1 , with a detection limit of 13.7 nmol L- 1 . Meanwhile, ascorbic acid found to reduce Fe 3+ to Fe 2+ , thereby causing restoration of the fluorescence of G-CDs. The detection limit for ascorbic acid detection was estimated as 82.0 nmol L- 1 over a linear range from 0.2 to 11.0 µmol L- 1 . Furthermore, the designed sensing platform was successfully utilized to the detection of Fe 3+ and ascorbic acid in water and urine samples and to intracellular imaging without surface modification. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Albinism and disease causing pathogens in Tanzania: are alleles that are associated with OCA2 being maintained by balancing selection?

    Science.gov (United States)

    Tuli, Abbas M; Valenzuela, Robert K; Kamugisha, Erasmus; Brilliant, Murray H

    2012-12-01

    Oculocutaneous albinism type 2 (OCA2) is present at significantly higher frequencies in sub-Saharan African populations compared to populations in other regions of the world. In Tanzania and other sub-Saharan countries, most OCA2 is associated with a common 2.7kb deletion allele. Leprosy is also in high prevalence in sub-Saharan African populations. The infectious agent of leprosy, Mycobacterium leprae, contains a gene, 38L, that is similar to OCA2. Hypopigmented patches of skin are early symptoms that present with infection of leprosy. In consideration of both the genetic similarity of OCA2 and the 38L gene of M. leprae and the involvement of pigmentation in both disorders, we hypothesized that the high rates of OCA2 may be due to heterozygote advantage. Hence, we hypothesized that carriers of the 2.7kb deletion allele of OCA2 may provide a protective advantage from infection with leprosy. We tested this hypothesis by determining the carrier frequency of the 2.7kb deletion allele from a sample of 240 individuals with leprosy from Tanzania. The results were inconclusive due to the small sample size; however, they enabled us to rule out a large protective effect, but perhaps not a small advantage. Mycobacterium tuberculosis is another infectious organism prevalent in sub-Saharan Africa that contains a gene, arsenic-transport integral membrane protein that is also similar to OCA2. Interestingly, chromosomal region 15q11-13, which also contains OCA2, was reported to be linked to tuberculosis susceptibility. Although variants within OCA2 were tested for association, the 2.7kb deletion allele of OCA2 was not tested. This led us to hypothesize that the deletion allele may confer resistance to susceptibility. Confirmation of our hypothesis would enable development of novel pharmocogenetic therapies for the treatment of tuberculosis, which in turn, may enable development of drugs that target other pathogens that utilize a similar infection mechanism as M. tuberculosis

  5. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens.

    Science.gov (United States)

    Abdul Momin, Muhd Haziq F; Bean, David C; Hendriksen, Rene S; Haenni, Marisa; Phee, Lynette M; Wareham, David W

    2017-11-01

    A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture medium (SuperPolymyxin). The medium was challenged with 84 isolates, including polymyxin B (POL B)-susceptible and -resistant type strains and colistin (COL)-resistant organisms recovered from human and animal samples. Susceptibility to COL and POL B was determined by agar dilution and broth microtitre dilution. The lower limit for the detection of COL-resistant organisms was also calculated for both CHROMagar COL-APSE and SuperPolymyxin media. The ability to isolate and correctly differentiate COL-resistant organisms within mixed cultures was also assessed and compared using both media. Using CHROMagar COL-APSE, Gram-negative pathogens (n=71) with intrinsic (n=8) or acquired COL (n=63) resistance were recovered with 100 % specificity down to the lower limit of detection of 10 1 colony-forming units (c.f.u.). The growth on SuperPolymyxin was similar, but notably weaker for COL-resistant non-fermentative bacteria (Acinetobacter, Pseudomonas and Stenotrophomonas). CHROMagar COL-APSE was also more sensitive in supporting the growth of Enterobacteriaceae with COL resistance associated with the carriage of mcr-1. CHROMagar COL-APSE is a sensitive and specific medium for the growth of COL-resistant bacterial pathogens. Due to the low limit of detection (10 1  c.f.u.), it may be useful as a primary isolation medium in the surveillance and recovery of COL-resistant bacteria from complex human, veterinary and environmental samples, especially those with plasmid-mediated MCR-1 or novel mechanisms of polymyxin resistance.

  6. STRATEGY TO IDENTIFY POSSIBLE INTRACELLULAR SURVIVAL MECHANISMS ANO PATHOGENICITY

    OpenAIRE

    VERA SEPULVEDA, TAMARA SCARLETT

    2013-01-01

    P. sa/monis es una bacteria gram-negativa, intracelular facultativa, agente causal del Síndrome Riketsial Salmonídeo (SRS), que en los últimos treinta años ha causado grandes pérdidas en la industria chilena del salmón. Se describió por primera vez en salmón coho en el sur de Chile, en 1989 por Bravo y Campos y en un primer momento se calificó como intracelular obligada y hace ya unos años se logró cultivar en medios artificiales sólidos, libres de células. Sin embargo, debido ...

  7. Footprint of Positive Selection in Treponema pallidum subsp. pallidum Genome Sequences Suggests Adaptive Microevolution of the Syphilis Pathogen

    Science.gov (United States)

    Centurion-Lara, Arturo; Jeffrey, Brendan M.; Le, Hoavan T.; Molini, Barbara J.; Lukehart, Sheila A.; Sokurenko, Evgeni V.; Rockey, Daniel D.

    2012-01-01

    In the rabbit model of syphilis, infection phenotypes associated with the Nichols and Chicago strains of Treponema pallidum (T. pallidum), though similar, are not identical. Between these strains, significant differences are found in expression of, and antibody responses to some candidate virulence factors, suggesting the existence of functional genetic differences between isolates. The Chicago strain genome was therefore sequenced and compared to the Nichols genome, available since 1998. Initial comparative analysis suggested the presence of 44 single nucleotide polymorphisms (SNPs), 103 small (≤3 nucleotides) indels, and 1 large (1204 bp) insertion in the Chicago genome with respect to the Nichols genome. To confirm the above findings, Sanger sequencing was performed on most loci carrying differences using DNA from Chicago and the Nichols strain used in the original T. pallidum genome project. A majority of the previously identified differences were found to be due to errors in the published Nichols genome, while the accuracy of the Chicago genome was confirmed. However, 20 SNPs were confirmed between the two genomes, and 16 (80.0%) were found in coding regions, with all being of non-synonymous nature, strongly indicating action of positive selection. Sequencing of 16 genomic loci harboring SNPs in 12 additional T. pallidum strains, (SS14, Bal 3, Bal 7, Bal 9, Sea 81-3, Sea 81-8, Sea 86-1, Sea 87-1, Mexico A, UW231B, UW236B, and UW249C), was used to identify “Chicago-“ or “Nichols -specific” differences. All but one of the 16 SNPs were “Nichols-specific”, with Chicago having identical sequences at these positions to almost all of the additional strains examined. These mutations could reflect differential adaptation of the Nichols strain to the rabbit host or pathoadaptive mutations acquired during human infection. Our findings indicate that SNPs among T. pallidum strains emerge under positive selection and, therefore, are likely to be functional in

  8. Thermosensing coordinates a cis-regulatory module for transcriptional activation of the intracellular virulence system in Salmonella enterica serovar Typhimurium.

    Science.gov (United States)

    Duong, Nancy; Osborne, Suzanne; Bustamante, Víctor H; Tomljenovic, Ana M; Puente, José L; Coombes, Brian K

    2007-11-23

    The expression of bacterial virulence genes is tightly controlled by the convergence of multiple extracellular signals. As a zoonotic pathogen, virulence gene regulation in Salmonella enterica serovar Typhimurium must be responsive to multiple cues from the general environment as well as from multiple niches within animal and human hosts. Previous work has identified combined magnesium and phosphate limitation as an environmental cue that activates genes required for intracellular virulence. One unanswered question is how virulence genes that are expressed within the host are inhibited in non-host environments that satisfy the phosphate and magnesium limitation cues. We report here that thermosensing is the major mechanism controlling incongruous activation of the intracellular virulence phenotype. Bacteria grown at 30 degrees C or lower were unable to activate the intracellular type III secretion system even under strong inducing signals such as synthetic medium, contact with macrophages, and exposure to the murine gut. Thermoregulation was fully recapitulated in a Salmonella bongori strain engineered to contain the intracellular virulence genes of S. enterica sv. Typhimurium, suggesting that orthologous thermoregulators were available. Accordingly, virulence gene repression at the nonpermissive temperature required Hha and H-NS, two nucleoid-like proteins involved in virulence gene control. The use of combined environmental cues to control transcriptional "logic gates" allows for transcriptional selectivity of virulence genes that would otherwise be superfluous if activated in the non-host environment. Thus, thermosensing by Salmonella provides integrated control of host niche-specific virulence factors.

  9. Xylem defense wood of Norway spruce compromised by the pathogenic white-rot fungus Heterobasidion parviporum shows a prolonged period of selective decay.

    Science.gov (United States)

    Nagy, Nina Elisabeth; Ballance, Simon; Kvaalen, Harald; Fossdal, Carl Gunnar; Solheim, Halvor; Hietala, Ari M

    2012-10-01

    Heterobasidion parviporum, a common pathogenic white-rot fungus in managed Norway spruce forests in northern and central Europe, causes extensive decay columns within stem heartwood of the host tree. Infected trees combat the lateral spread of decay by bordering the heartwood with a fungistatic reaction zone characterized by elevated pH and phenol content. To examine the mode of fungal feeding in the reaction zone of mature Norway spruce trees naturally infected by H. parviporum, we conducted spatial profiling of pectin and hemicellulose composition, and established transcript levels of candidate fungal genes encoding enzymes involved in degradation of the different cell wall components of wood. Colonized inner heartwood showed pectin and hemicellulose concentrations similar to those of healthy heartwood, whereas the carbohydrate profiles of compromised reaction zone, irrespective of the age of fungal activity in the tissue, indicated selective fungal utilization of galacturonic acid, arabinose, xylose and mannose. These data show that the rate of wood decay in the reaction zone is slow. While the up-regulation of genes encoding pectinases and hemicellulases preceded that of the endoglucanase gene during an early phase of fungal interaction with xylem defense, the manganese peroxidase gene showed similar transcript levels during different phases of wood colonization. It seems plausible that the reaction zone components of Norway spruce interfere with both lignin degradation and the associated co-hydrolysis of hemicelluloses and pectin, resulting in a prolonged phase of selective decay.

  10. Evidence for Directional Selection at a Novel Major Histocompatibility Class I Marker in Wild Common Frogs (Rana temporaria) Exposed to a Viral Pathogen (Ranavirus)

    Science.gov (United States)

    Teacher, Amber G. F.; Garner, Trenton W. J.; Nichols, Richard A.

    2009-01-01

    Whilst the Major Histocompatibility Complex (MHC) is well characterized in the anuran Xenopus, this region has not previously been studied in another popular model species, the common frog (Rana temporaria). Nor, to date, have there been any studies of MHC in wild amphibian host-pathogen systems. We characterise an MHC class I locus in the common frog, and present primers to amplify both the whole region, and specifically the antigen binding region. As no more than two expressed haplotypes were found in over 400 clones from 66 individuals, it is likely that there is a single class I locus in this species. This finding is consistent with the single class I locus in Xenopus, but contrasts with the multiple loci identified in axolotls, providing evidence that the diversification of MHC class I into multiple loci likely occurred after the Caudata/Anura divergence (approximately 350 million years ago) but before the Ranidae/Pipidae divergence (approximately 230 mya). We use this locus to compare wild populations of common frogs that have been infected with a viral pathogen (Ranavirus) with those that have no history of infection. We demonstrate that certain MHC supertypes are associated with infection status (even after accounting for shared ancestry), and that the diseased populations have more similar supertype frequencies (lower FST) than the uninfected. These patterns were not seen in a suite of putatively neutral microsatellite loci. We interpret this pattern at the MHC locus to indicate that the disease has imposed selection for particular haplotypes, and hence that common frogs may be adapting to the presence of Ranavirus, which currently kills tens of thousands of amphibians in the UK each year. PMID:19240796

  11. Effects of flood irrigation on the risk of selected zoonotic pathogens in an arid and semi-arid area in the eastern Kenya.

    Science.gov (United States)

    Bett, Bernard; Said, Mohammed Y; Sang, Rosemary; Bukachi, Salome; Wanyoike, Salome; Kifugo, Shem C; Otieno, Fredrick; Ontiri, Enoch; Njeru, Ian; Lindahl, Johanna; Grace, Delia

    2017-01-01

    To investigate the effects of irrigation on land cover changes and the risk of selected zoonotic pathogens, we carried out a study in irrigated, pastoral and riverine areas in the eastern Kenya. Activities implemented included secondary data analyses to determine land use and land cover (LULC) changes as well as human, livestock and wildlife population trends; entomological surveys to characterize mosquitoes population densities and species distribution by habitat and season; and serological surveys in people to determine the risk of Rift Valley fever virus (RVFV), West Nile fever virus (WNV), dengue fever virus (DFV), Leptospira spp. and Brucella spp. Results demonstrate a drastic decline in vegetation cover over ≈25 years particularly in the irrigated areas where cropland increased by about 1,400% and non-farm land (under closed trees, open to closed herbaceous vegetation, bushlands and open trees) reduced by 30-100%. The irrigated areas had high densities of Aedes mcintoshi, Culex spp. and Mansonia spp. (important vectors for multiple arboviruses) during the wet and dry season while pastoral areas had high densities of Ae. tricholabis specifically in the wet season. The seroprevalences of RVFV, WNV and DFV were higher in the irrigated compared to the pastoral areas while those for Leptospira spp and Brucella spp. were higher in the pastoral compared to the irrigated areas. It is likely that people in the pastoral areas get exposed to Leptospira spp by using water fetched from reservoirs that are shared with livestock and wildlife, and to Brucella spp. by consuming raw or partially cooked animal-source foods such as milk and meat. This study suggests that irrigation increases the risk of mosquito-borne infections while at the same time providing a protective effect against zoonotic pathogens that thrive in areas with high livestock population densities.

  12. Therapeutic Antibodies against Intracellular Tumor Antigens

    Directory of Open Access Journals (Sweden)

    Iva Trenevska

    2017-08-01

    Full Text Available Monoclonal antibodies are among the most clinically effective drugs used to treat cancer. However, their target repertoire is limited as there are relatively few tumor-specific or tumor-associated cell surface or soluble antigens. Intracellular molecules represent nearly half of the human proteome and provide an untapped reservoir of potential therapeutic targets. Antibodies have been developed to target externalized antigens, have also been engineered to enter into cells or may be expressed intracellularly with the aim of binding intracellular antigens. Furthermore, intracellular proteins can be degraded by the proteasome into short, commonly 8–10 amino acid long, peptides that are presented on the cell surface in the context of major histocompatibility complex class I (MHC-I molecules. These tumor-associated peptide–MHC-I complexes can then be targeted by antibodies known as T-cell receptor mimic (TCRm or T-cell receptor (TCR-like antibodies, which recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells. Advances in the production of TCRm antibodies have enabled the generation of multiple TCRm antibodies, which have been tested in vitro and in vivo, expanding our understanding of their mechanisms of action and the importance of target epitope selection and expression. This review will summarize multiple approaches to targeting intracellular antigens with therapeutic antibodies, in particular describing the production and characterization of TCRm antibodies, the factors influencing their target identification, their advantages and disadvantages in the context of TCR therapies, and the potential to advance TCRm-based therapies into the clinic.

  13. Application of high-throughput sequencing to measure the performance of commonly used selective cultivation methods for the foodborne pathogen Campylobacter.

    Science.gov (United States)

    Oakley, Brian B; Morales, Cesar A; Line, J Eric; Seal, Bruce S; Hiett, Kelli L

    2012-02-01

    Campylobacter is an important foodborne human pathogen, which has traditionally been studied using a variety of selective cultivation methods. Here we use next-generation sequencing to ask the following: (i) how selective are commonly used Campylobacter cultivation methods relative to the initial sample and (ii) how do the specificity and sensitivity of these methods compare with one another? To answer these questions, we used 16S rRNA tagged-pyrosequencing to sequence directly from a pooled fecal sample representing a c. 16,000 bird poultry flock and compared these data to exhaustive sequencing of colonies formed after plating. We compared five commonly used media [Cefex, Cape Town, modified cefoperazone charcoal deoxycholate agar (mCCDA), Campy-Line agar (CLA), and Campy-CVA agar (CVA)], two incubation atmospheres (10% CO(2), 5% O(2), 85% N(2) and 10% CO(2), 10% H(2), 80% N(2)), and two incubation temperatures (37 and 42 °C). Analysis of 404,104 total sequence reads, including 19 472 total fecal reads, revealed Campylobacter represented only a small proportion (media type. Incubation atmosphere had little effect on recovery, but a significant difference in media specificity (more non-Campylobacter OTUs; P = 0.028) was found at 42 vs. 37 °C. The most common non-Campylobacter sequence type was Proteus, which ranged from 0.04% of sequences (mCCDA) to 10.8% (Cape Town). High-throughput sequencing provides a novel and powerful approach to measure the performance of selective media, which remain widely used for research and regulatory purposes. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.

  14. Avoidance and Subversion of Eukaryotic Homeostatic Autophagy Mechanisms by Bacterial Pathogens.

    Science.gov (United States)

    Miller, Cheryl; Celli, Jean

    2016-08-28

    Autophagy is a conserved lysosomal recycling process, which maintains cellular homeostasis during stress and starvation conditions by degrading and recycling proteins, lipids, and carbohydrates, ultimately increasing nutrient availability in eukaryotes. An additional function of autophagy, termed xenophagy, is to detect, capture, and destroy invading microorganisms, such as viruses, bacteria, and protozoa, providing autophagy with a role in innate immunity. Many intracellular pathogens have, however, developed mechanisms to avoid xenophagy and have evolved strategies to take advantage of select autophagic processes to undergo their intracellular life cycle. This review article will discuss the molecular mechanisms used by the intracellular bacterial pathogens Francisella spp. and Brucella spp. to manipulate components of the autophagic pathway, promoting cytosolic growth in the case of Francisella spp. and facilitating cellular egress and cell-to-cell spread in the case of Brucella spp. These examples highlight how successful, highly infectious bacterial pathogens avoid or subvert host autophagy mechanisms normally employed to maintain eukaryotic homeostasis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli

    Science.gov (United States)

    Shaffer, Carrie L.; Zhang, Ellisa W.; Dudley, Anne G.; Dixon, Beverly R. E. A.; Guckes, Kirsten R.; Breland, Erin J.; Floyd, Kyle A.; Casella, Daniel P.; Algood, Holly M. Scott; Clayton, Douglass B.

    2016-01-01

    ABSTRACT The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement. PMID:27795353

  16. The intracellular pharmacokinetics of terminally capped peptides.

    NARCIS (Netherlands)

    Ruttekolk, I.R.R.; Witsenburg, J.J.; Glauner, H.B.; Bovee-Geurts, P.H.M.; Ferro, E.S.; Verdurmen, W.P.R.; Brock, R.E.

    2012-01-01

    With significant progress in delivery technologies, peptides and peptidomimetics are receiving increasing attention as potential therapeutics also for intracellular applications. However, analyses of the intracellular behavior of peptides are a challenge; therefore, knowledge on the intracellular

  17. Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

    DEFF Research Database (Denmark)

    Johnson, Erin E; Srikanth, Chittur V; Sandgren, Andreas

    2010-01-01

    that siderocalin expression is upregulated following M.tb infection of mouse macrophage cell lines and primary murine alveolar macrophages. Furthermore, siderocalin added exogenously as a recombinant protein or overexpressed in the RAW264.7 macrophage cell line inhibited the intracellular growth of the pathogen......Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show....... A variant form of siderocalin, which is expressed only in the macrophage cytosol, inhibited intracellular M.tb growth as effectively as the normal, secreted form, an observation that provides mechanistic insight into how siderocalin might influence iron acquisition by the bacteria in the phagosome. Our...

  18. Nosocomial pathogens

    African Journals Online (AJOL)

    remains an important problem in intensive care units. Hospital wards had been shown to act as reservoirs of pathogenic microorganisms associated with infection. To assess the prevalence of pathogenic organisms in the environment of the neonatal unit, 92 swabs were randomly collected from cots, incubators and various ...

  19. Invitro Evaluation Of Antibacterial Activity Of Lactic Acid Bacteria Isolated From Ergo And Qotchqotcha Ethiopian Traditional Fermented Foods Against Some Selected Food Borne Pathogens

    Directory of Open Access Journals (Sweden)

    Lamenew Fenta

    2017-09-01

    Full Text Available The prevention of pathogenic bacteria by lactic acid bacteria LAB isolated directly from foods is an innovative approach. With the aim of determining the anti-bacterial activity of Lactic acid bacteria isolated from ergo and qotchqotcha Ethiopian fermented food 12 samples of each were taken from the 4 different kebeles of Assosa town. Isolation of LAB from the selected samples were carried out using MRS media. The different set of isolates were characterized using primary biochemical tests. Isolates which were gram positive catalase negative and KOH negative were considered to be presumptive LAB and further characterized by using different biochemical tests for further identification. 16 isolates from ergo samples were isolated. Based on Bergeys manual of determinative bacteriology the 16 isolates belonged to four 4 LAB species namely Lactobacillus acidophilus 18.75 Lactobacillus casei 31.25 Streptococcus thermophiles 25 and Lactobacillus bulgaricus 25. Likewise 5 isolates were isolated from Qotchqotcha and the five isolates were found to be Lactobacillus acidophilus 80 and Pediococcus acidilactici 20. Cell free solution from MRS broth culture of theses LAB was prepared and tested against Escherichia coli O157H7 and Staphylococcus aureus using agar-well diffusion method. Of the 16 isolates isolated from ergo 9 of them show antimicrobial activity against E. coli O157H7 with a largest inhibition zone measured about 7.331.20mm by EK0101 and 12 of them show antimicrobial activity against S. aureus with a largest inhibition zone measured about 11.66 0.88mm by EK0201. On the basis of morphological and biochemical test done EK0101 was found to be presumptive Lactobacillus acidophilus and EK0201 to be Streptococcus thermophiles. All of the isolates isolated from Qotchqotcha showed antimicrobial activity against the tested organisms though there was a significant difference in their activity P0.05. The isolate QK0201 showed an inhibition zone of about 6

  20. PDlim2 selectively interacts with the PDZ binding motif of highly pathogenic avian H5N1 influenza A virus NS1.

    Directory of Open Access Journals (Sweden)

    Jia Yu

    Full Text Available The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the C-terminus of NS1 constitute a type I PDZ domain binding motif (PBM. Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1 by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12-NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1. A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV, but not PR8-NS1 (RSEV, enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins.

  1. Evaluation of Low-Dose Ultraviolet Light C for Reduction of Select ESKAPE Pathogens in a Canine Skin and Muscle Model.

    Science.gov (United States)

    Dujowich, Mauricio; Case, J Brad; Ellison, Gary; Wellehan, James F X

    2016-08-01

    This study aimed at comparing the ability of low-dose UVC, 0.05% chlorhexidine, and combined UVC with 0.05% chlorhexidine to reduce colony-forming units (CFUs) on select ESKAPE pathogens (Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecium) in a canine skin and muscle model. Surgical site infections (SSIs) result in increased morbidity and cost. UVC damages DNA, rendering bacteria nonviable and does not discriminate between drug-sensitive and multi-drug-resistant organisms. Specimens were inoculated with one of three pathogens. Samples were treated with a 254 nm UVC mercury lamp or a 270 nm UVC LED light at 0.015, 0.03, or 0.04 J/cm(2) doses; 0.05% and 2% chlorhexidine were used as positive controls. To evaluate synergism, 0.05% chlorhexidine was used with 0.015 J/cm(2) of UVC. CFUs were counted and compared against the negative control. There were no significant differences in CFU counts between samples of the same tissue type treated with different light sources of the same UVC dose. UVC significantly decreased CFUs when compared against all negative controls in both skin and muscle. There was no consistently superior bactericidal UVC dose identified for individual bacteria or for tissue type. The bactericidal activity of UVC at 0.015 J/cm(2) versus 0.05% chlorhexidine was not different in muscle for any bacteria. The bactericidal activity of UVC at 0.015 J/cm(2) was superior to 0.05% chlorhexidine in skin for S. aureus and K. pneumonia, but not E. faecium. Combination of UVC and 0.05% chlorhexidine showed synergy against E. faecium when evaluated on skin. Low-dose UVC shows promise as a rapid, effective, and synergistic means of reducing bacterial burdens, which may decrease the incidence of SSIs. It should be further evaluated for use when 2% chlorhexidine would be contraindicated or impractical, such as open wounds or surgical sites.

  2. A first step toward liposome-mediated intracellular bacteriophage therapy.

    Science.gov (United States)

    Nieth, Anita; Verseux, Cyprien; Barnert, Sabine; Süss, Regine; Römer, Winfried

    2015-01-01

    The emergence of antibiotic-resistant bacteria presents a severe challenge to medicine and public health. While bacteriophage therapy is a promising alternative to traditional antibiotics, the general inability of bacteriophages to penetrate eukaryotic cells limits their use against resistant bacteria, causing intracellular diseases like tuberculosis. Bacterial vectors show some promise in carrying therapeutic bacteriophages into cells, but also bring a number of risks like an overload of bacterial antigens or the acquisition of virulence genes from the pathogen. As a first step in the development of a non-bacterial vector for bacteriophage delivery into pathogen-infected cells, we attempted to encapsulate bacteriophages into liposomes. Here we report effective encapsulation of the model bacteriophage λeyfp and the mycobacteriophage TM4 into giant liposomes. Furthermore, we show that liposome-associated bacteriophages are taken up into eukaryotic cells more efficiently than free bacteriophages. These are important milestones in the development of an intracellular bacteriophage therapy that might be useful in the fight against multi-drug-resistant intracellular pathogens like Mycobacterium tuberculosis.

  3. Human pathogen subversion of antigen presentation.

    Science.gov (United States)

    Brodsky, F M; Lem, L; Solache, A; Bennett, E M

    1999-04-01

    Many pathogens have co-evolved with their human hosts to develop strategies for immune evasion that involve disruption of the intracellular pathways by which antigens are bound by class I and class II molecules of the major histocompatibility complex (MHC) for presentation to T cells. Here the molecular events in these pathways are reviewed and pathogen interference is documented for viruses, extracellular and intracellular bacteria and intracellular parasites. In addition to a general review, data from our studies of adenovirus, Chlamydia trachomatis and Coxiella burnetii are summarized. Adenovirus E19 is the first viral gene product described that affects class I MHC molecule expression by two separate mechanisms, intracellular retention of the class I heavy chain by direct binding and by binding to the TAP transporter involved in class I peptide loading. Coxiella and Chlamydia both affect peptide presentation by class II MHC molecules as a result of their residence in endocytic compartments, although the properties of the parasitophorous vacuoles they form are quite different. These examples of active interference with antigen presentation by viral gene products and passive interference by rickettsiae and bacteria are typical of the strategies used by these different classes of pathogens, which need to evade different types of immune responses. Pathogen-host co-evolution is evident in these subversion tactics for which the pathogen crime seems tailored to fit the immune system punishment.

  4. Amphipathic β2,2-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae.

    Science.gov (United States)

    Hanski, Leena; Ausbacher, Dominik; Tiirola, Terttu M; Strøm, Morten B; Vuorela, Pia M

    2016-01-01

    We demonstrate in the current work that small cationic antimicrobial β2,2-amino acid derivatives (Mw molecules for development of antimicrobial agents against this hard-to-treat intracellular pathogen.

  5. Purification and proteomics of pathogen-modified vacuoles and membranes.

    Science.gov (United States)

    Herweg, Jo-Ana; Hansmeier, Nicole; Otto, Andreas; Geffken, Anna C; Subbarayal, Prema; Prusty, Bhupesh K; Becher, Dörte; Hensel, Michael; Schaible, Ulrich E; Rudel, Thomas; Hilbi, Hubert

    2015-01-01

    Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.

  6. Intracellular antibody-mediated immunity and the role of TRIM21.

    Science.gov (United States)

    McEwan, William A; Mallery, Donna L; Rhodes, David A; Trowsdale, John; James, Leo C

    2011-11-01

    Protection against bacterial and viral pathogens by antibodies has always been thought to end at the cell surface. Once inside the cell, a pathogen was understood to be safe from humoral immunity. However, it has now been found that antibodies can routinely enter cells attached to viral particles and mediate an intracellular immune response. Antibody-coated virions are detected inside the cell by means of an intracellular antibody receptor, TRIM21, which directs their degradation by recruitment of the ubiquitin-proteasome system. In this article we assess how this discovery alters our view of the way in which antibodies neutralise viral infection. We also consider the antiviral function of TRIM21 in the context of its other reported roles in immune signalling and autoimmunity. Finally, we discuss the conceptual implications of intracellular antibody immunity and how it alters our view of the discrete separation of extracellular and intracellular environments. Copyright © 2011 WILEY Periodicals, Inc.

  7. The effect of intracellular trafficking of CD1d on the formation of TCR repertoire of NKT cells.

    Science.gov (United States)

    Shin, Jung Hoon; Park, Se-Ho

    2014-05-01

    CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to αβ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of αβ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant Vα14-Jα18 TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire.

  8. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells.

    Science.gov (United States)

    Ortega, Alvaro D; Quereda, Juan J; Pucciarelli, M Graciela; García-del Portillo, Francisco

    2014-01-01

    Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of "intact" intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

  9. Modulation of host central carbon metabolism and in situ glucose uptake by intracellular Trypanosoma cruzi amastigotes.

    Science.gov (United States)

    Shah-Simpson, Sheena; Lentini, Gaelle; Dumoulin, Peter C; Burleigh, Barbara A

    2017-11-01

    Obligate intracellular pathogens satisfy their nutrient requirements by coupling to host metabolic processes, often modulating these pathways to facilitate access to key metabolites. Such metabolic dependencies represent potential targets for pathogen control, but remain largely uncharacterized for the intracellular protozoan parasite and causative agent of Chagas disease, Trypanosoma cruzi. Perturbations in host central carbon and energy metabolism have been reported in mammalian T. cruzi infection, with no information regarding the impact of host metabolic changes on the intracellular amastigote life stage. Here, we performed cell-based studies to elucidate the interplay between infection with intracellular T. cruzi amastigotes and host cellular energy metabolism. T. cruzi infection of non-phagocytic cells was characterized by increased glucose uptake into infected cells and increased mitochondrial respiration and mitochondrial biogenesis. While intracellular amastigote growth was unaffected by decreased host respiratory capacity, restriction of extracellular glucose impaired amastigote proliferation and sensitized parasites to further growth inhibition by 2-deoxyglucose. These observations led us to consider whether intracellular T. cruzi amastigotes utilize glucose directly as a substrate to fuel metabolism. Consistent with this prediction, isolated T. cruzi amastigotes transport extracellular glucose with kinetics similar to trypomastigotes, with subsequent metabolism as demonstrated in 13C-glucose labeling and substrate utilization assays. Metabolic labeling of T. cruzi-infected cells further demonstrated the ability of intracellular parasites to access host hexose pools in situ. These findings are consistent with a model in which intracellular T. cruzi amastigotes capitalize on the host metabolic response to parasite infection, including the increase in glucose uptake, to fuel their own metabolism and replication in the host cytosol. Our findings enrich

  10. Immune regulation of Rab proteins expression and intracellular transport.

    Science.gov (United States)

    Pei, Gang; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel

    2012-07-01

    Compartmentalization in cells of the immune system, the focus of this review, facilitates the spatiotemporal organization of cellular responses essential for specialized immune functions. In this process of compartment maintenance, Rab proteins are central regulators of protein-mediated transport and fusion of intracellular structures. It is widely believed that the intracellular concentration of proteins that regulate intracellular transport, including Rab proteins, is constitutively mantained. However, there is a growing body of evidence indicating that transcriptional rates of Rab proteins can be modified. This process is especially evident during immune activation and argues that after activation, these cells require higher levels of Rab proteins. The aim of this review is to discuss evidence showing the increasing links between Rab protein expression and intracellular transport, particularly in monocytes and macrophages. We highlight here biological processes in which the expression of Rab GTPases is selectively regulated, leading to the activation of specific intracellular routes. Further, we focus on the immune regulation of intracellular transport after cytokine activation and microbial infection, with an emphasis in mycobacterial infection.

  11. Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenzae

    Science.gov (United States)

    Moleres, Javier; Sinha, Sunita; Fernández-Calvet, Ariadna; Porsch, Eric A.; St. Geme, Joseph W.; Nislow, Corey; Redfield, Rosemary J.; Garmendia, Junkal

    2016-01-01

    Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe “transformed recombinant enrichment profiling” (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2A Hi375 by hmw1A 86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A 86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A 86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A 86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation. PMID:27124727

  12. Mechanisms of Borrelia burgdorferi internalization and intracellular innate immune signaling

    Directory of Open Access Journals (Sweden)

    Tanja ePetnicki-Ocwieja

    2014-12-01

    Full Text Available Lyme disease is a long-term infection whose most severe pathology is characterized by inflammatory arthritis of the lower bearing joints, carditis and neuropathy. The inflammatory cascades are initiated through the early recognition of invading Borrelia burgdorferi spirochetes by cells of the innate immune response, such as neutrophils and macrophage. B. burgdorferi does not have an intracellular niche and thus much research has focused on immune pathways activated by pathogen recognition molecules at the cell surface, such as the Toll-like receptors (TLRs. However, in recent years, studies have shown that internalization of the bacterium by host cells is an important component of the defense machinery in response to B. burgdorferi. Upon internalization, B. burgdorferi is trafficked through an endo/lysosomal pathway resulting in the activation of a number of intracellular pathogen recognition receptors including TLRs and Nod-like receptors (NLRs. Here we will review the innate immune molecules that participate in both cell surface and intracellular immune activation by B. burgdorferi.

  13. Acanthamoeba spp. as a universal host for pathogenic microorganisms: One bridge from environment to host virulence.

    Science.gov (United States)

    Guimaraes, Allan J; Gomes, Kamilla Xavier; Cortines, Juliana Reis; Peralta, José Mauro; Peralta, Regina H Saramago

    2016-12-01

    Free-living amoebas (FLA) are ubiquitous environmental protists that have enormously contributed to the microbiological contamination of water sources. FLAs have displayed resistance to environmental adversities and germicides and have played important roles in the population control of microbial communities due to its predatory behavior and microbicidal activity. However, some organisms have developed resistance to the intracellular milieu of amoebas, as in the case of Acanthamoebas, which in turn, have been functioning as excellent reservoirs for amoeba-resistant microorganisms (ARMs), such as bacteria, viruses and fungi. Little is known about these relationships and interaction mechanisms, but it is speculated that the FLAs need a very broad repertoire or universal class of receptors to bind and recognize these diverse species of microorganisms. By harboring these organisms as a "Trojan Horse", the Achantamoeba has been working as an excellent vector for pathogens. Moreover, studies have demonstrated that the interaction of pathogens with Acanthamoeba results in environmental selective pressure responsible for induction and maintenance of virulence factors and increase in microbial pathogenicity. This phenomenon is correlated to the observation of higher gene number and DNA content of ARMs, when compared to their relatives which are adapted to other hosts, due to allopatric or sympatric gene transfer and acquisition, contradicting the overall genome reduction theory for intracellularly adapted pathogens. Thus, adaptation to FLAs indirectly provided a "learning" environment for pathogens to resist later to macrophages; besides the evolutionary distance, these phagocytes share similar predatory mechanisms, such as phagocytosis and phagolysossomal degradation. In this mini-review, we cover the most important aspects of Acanthamoeba biology and their interactions with endemically important human pathogens. Copyright © 2016 Elsevier GmbH. All rights reserved.

  14. Deciphering the Intracellular Fate of Propionibacterium acnes in Macrophages

    Science.gov (United States)

    Fischer, Natalie; Mak, Tim N.; Shinohara, Debika Biswal; Sfanos, Karen S.; Meyer, Thomas F.

    2013-01-01

    Propionibacterium acnes is a Gram-positive bacterium that colonizes various niches of the human body, particularly the sebaceous follicles of the skin. Over the last years a role of this common skin bacterium as an opportunistic pathogen has been explored. Persistence of P. acnes in host tissue has been associated with chronic inflammation and disease development, for example, in prostate pathologies. This study investigated the intracellular fate of P. acnes in macrophages after phagocytosis. In a mouse model of P. acnes-induced chronic prostatic inflammation, the bacterium could be detected in prostate-infiltrating macrophages at 2 weeks postinfection. Further studies performed in the human macrophage cell line THP-1 revealed intracellular survival and persistence of P. acnes but no intracellular replication or escape from the host cell. Confocal analyses of phagosome acidification and maturation were performed. Acidification of P. acnes-containing phagosomes was observed at 6 h postinfection but then lost again, indicative of cytosolic escape of P. acnes or intraphagosomal pH neutralization. No colocalization with the lysosomal markers LAMP1 and cathepsin D was observed, implying that the P. acnes-containing phagosome does not fuse with lysosomes. Our findings give first insights into the intracellular fate of P. acnes; its persistency is likely to be important for the development of P. acnes-associated inflammatory diseases. PMID:23862148

  15. Association between VDAC1 mRNA expression and intracellular ...

    African Journals Online (AJOL)

    One way in which xenobiotics induce apoptotic cell death is to alter the selective permeability of the intracellular voltage-dependent anion channel (VDAC1) in the mitochondrial membrane. In this study, we explored the association between VDAC1 mRNA expression and mitochondrial function during hexavalent chromium ...

  16. Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections

    DEFF Research Database (Denmark)

    Bahnsen, Jesper S; Franzyk, Henrik; Sayers, Edward J

    2015-01-01

    . TPk showed the highest antibacterial activity. SA-3 exhibited selective disruption of liposomes mimicking Gram-positive and Gram-negative membranes. CONCLUSION: PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected...

  17. Subversion of inflammasome activation and pyroptosis by pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Larissa D Cunha

    2013-11-01

    Full Text Available Activation of the inflammasome occurs in response to a notably high number of pathogenic microbes and is a broad innate immune response that effectively contributes to restriction of pathogen replication and generation of adaptive immunity. Activation of these platforms leads to caspase-1- and/or caspase-11-dependent secretion of proteins, including cytokines, and induction of a specific form of cell death called pyroptosis, which directly or indirectly contribute for restriction of pathogen replication. Not surprisingly, bona fide intracellular pathogens developed strategies for manipulation of cell death to guarantee intracellular replication. In this sense, the remarkable advances in the knowledge of the inflammasome field have been accompanied by several reports characterizing the inhibition of this platform by several pathogenic bacteria. Herein, we review some processes used by pathogenic bacteria, including Yersinia spp., Pseudomonas aeruginosa, Vibrio parahaemolyticus, Chlamydia trachomatis, Francisella tularensis, Shigella flexneri, Legionella pneumophila and Coxiella burnetii to evade the activation of the inflammasome and the induction of pyroptosis.

  18. Subversion of inflammasome activation and pyroptosis by pathogenic bacteria.

    Science.gov (United States)

    Cunha, Larissa D; Zamboni, Dario S

    2013-01-01

    Activation of the inflammasome occurs in response to a notably high number of pathogenic microbes and is a broad innate immune response that effectively contributes to restriction of pathogen replication and generation of adaptive immunity. Activation of these platforms leads to caspase-1- and/or caspase-11-dependent secretion of proteins, including cytokines, and induction of a specific form of cell death called pyroptosis, which directly or indirectly contribute for restriction of pathogen replication. Not surprisingly, bona fide intracellular pathogens developed strategies for manipulation of cell death to guarantee intracellular replication. In this sense, the remarkable advances in the knowledge of the inflammasome field have been accompanied by several reports characterizing the inhibition of this platform by several pathogenic bacteria. Herein, we review some processes used by pathogenic bacteria, including Yersinia spp., Pseudomonas aeruginosa, Vibrio parahaemolyticus, Chlamydia trachomatis, Francisella tularensis, Shigella flexneri, Legionella pneumophila, and Coxiella burnetii to evade the activation of the inflammasome and the induction of pyroptosis.

  19. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.

    2013-01-09

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

  20. Evaluation of essential oils in beef cattle manure slurries and applications of select compounds to beef feedlot surfaces to control zoonotic pathogens

    Science.gov (United States)

    Aims: To evaluate natural terpene compounds for antimicrobial activities and determine if these compounds could be used to control microbial activities and pathogens in production animal facilities. Methods and Results: Thymol, geraniol, glydox, linalool, pine oil, plinol, and terpineol were teste...

  1. A new strategy for healthcare-associated pneumonia: a 2-year prospective multicenter cohort study using risk factors for multidrug-resistant pathogens to select initial empiric therapy.

    Science.gov (United States)

    Maruyama, Takaya; Fujisawa, Takao; Okuno, Masataka; Toyoshima, Hirokazu; Tsutsui, Kiyoyuki; Maeda, Hikaru; Yuda, Hisamichi; Yoshida, Masamichi; Kobayashi, Hiroyasu; Taguchi, Osamu; Gabazza, Esteban C; Takei, Yoshiyuki; Miyashita, Naoyuki; Ihara, Toshiaki; Brito, Veronica; Niederman, Michael S

    2013-11-01

    Optimal empiric therapy for hospitalized patients with healthcare-associated pneumonia (HCAP) is uncertain. We prospectively applied a therapeutic algorithm, based on the presence of risk factors for multidrug-resistant (MDR) pathogens in a multicenter cohort study of 445 pneumonia patients, including both community-acquired pneumonia (CAP; n = 124) and HCAP (n = 321). MDR pathogens were more common (15.3% vs 0.8%, P risk factors, one of which was severity of illness (n = 170), vs HCAP patients with 0-1 risk factor (n = 151) had a significantly higher frequency of MDR pathogens (27.1% vs 2%, P risk factor, mortality was lower than with ≥2 MDR risk factors (8.6% vs 18.2%, P = .012). In multivariate analysis, initial treatment failure, but not inappropriate empiric antibiotic therapy, was a mortality risk factor (odds ratio, 72.0). Basing empiric HCAP therapy on its severity and the presence of risk factors for MDR pathogens is a potentially useful approach that achieves good outcomes without excessive use of broad-spectrum antibiotic therapy. Japan Medical Association Center for Clinical Trials, JMA-IIA00054.

  2. Comparative Analyses of Nonpathogenic, Opportunistic, and Totally Pathogenic Mycobacteria Reveal Genomic and Biochemical Variabilities and Highlight the Survival Attributes of Mycobacterium tuberculosis

    Science.gov (United States)

    Singh, Yadvir; Kohli, Sakshi; Ahmad, Javeed; Ehtesham, Nasreen Z.; Tyagi, Anil K.

    2014-01-01

    ABSTRACT Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size—their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. PMID:25370496

  3. An intracellular replication niche for Vibrio cholerae in the amoeba Acanthamoeba castellanii.

    Science.gov (United States)

    Van der Henst, Charles; Scrignari, Tiziana; Maclachlan, Catherine; Blokesch, Melanie

    2016-04-01

    Vibrio cholerae is a human pathogen and the causative agent of cholera. The persistence of this bacterium in aquatic environments is a key epidemiological concern, as cholera is transmitted through contaminated water. Predatory protists, such as amoebae, are major regulators of bacterial populations in such environments. Therefore, we investigated the interaction between V. cholerae and the amoeba Acanthamoeba castellanii at the single-cell level. We observed that V. cholerae can resist intracellular killing. The non-digested bacteria were either released or, alternatively, established a replication niche within the contractile vacuole of A. castellanii. V. cholerae was maintained within this compartment even upon encystment. The pathogen ultimately returned to its aquatic habitat through lysis of A. castellanii, a process that was dependent on the production of extracellular polysaccharide by the pathogen. This study reinforces the concept that V. cholerae is a facultative intracellular bacterium and describes a new host-pathogen interaction.

  4. Transcriptome-based analyses of phosphite-mediated suppression of rust pathogens Puccinia emaculata and Phakopsora pachyrhizi and functional characterization of selected fungal target genes.

    Science.gov (United States)

    Gill, Upinder S; Sun, Liang; Rustgi, Sachin; Tang, Yuhong; von Wettstein, Diter; Mysore, Kirankumar S

    2018-03-01

    Phosphite (Phi) is used commercially to manage diseases mainly caused by oomycetes, primarily due to its low cost compared with other fungicides and its persistent control of oomycetous pathogens. We explored the use of Phi in controlling the fungal pathogens Puccinia emaculata and Phakopsora pachyrhizi, the causal agents of switchgrass rust and Asian soybean rust, respectively. Phi primes host defenses and efficiently inhibits the growth of P. emaculata, P. pachyrhizi and several other fungal pathogens tested. To understand these Phi-mediated effects, a detailed molecular analysis was undertaken in both the host and the pathogen. Transcriptomic studies in switchgrass revealed that Phi activates plant defense signaling as early as 1 h after application by increasing the expression of several cytoplasmic and membrane receptor-like kinases and defense-related genes within 24 h of application. Unlike in oomycetes, RNA sequencing of P. emaculata and P. pachyrhizi did not exhibit Phi-mediated retardation of cell wall biosynthesis. The genes with reduced expression in either or both rust fungi belonged to functional categories such as ribosomal protein, actin, RNA-dependent RNA polymerase, and aldehyde dehydrogenase. A few P. emaculata genes that had reduced expression upon Phi treatment were further characterized. Application of double-stranded RNAs specific to P. emaculata genes encoding glutamate N-acetyltransferase and cystathionine gamma-synthase to switchgrass leaves resulted in reduced disease severity upon P. emaculata inoculation, suggesting their role in pathogen survival and/or pathogenesis. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  5. High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

    Science.gov (United States)

    Greub, Gilbert; Kebbi-Beghdadi, Carole; Bertelli, Claire; Collyn, François; Riederer, Beat M; Yersin, Camille; Croxatto, Antony; Raoult, Didier

    2009-12-23

    With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

  6. Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy.

    Science.gov (United States)

    Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

    2017-08-01

    To target bacterial pathogens that invade and proliferate inside host cells, it is necessary to design intervention strategies directed against bacterial attachment, cellular invasion and intracellular proliferation. We present an automated microscopy-based, fast, high-throughput method for analyzing size and number of intracellular bacterial colonies in infected tissue culture cells. Cells are seeded in 48-well plates and infected with a GFP-expressing bacterial pathogen. Following gentamicin treatment to remove extracellular pathogens, cells are fixed and cell nuclei stained. This is followed by automated microscopy and subsequent semi-automated spot detection to determine the number of intracellular bacterial colonies, their size distribution, and the average number per host cell. Multiple 48-well plates can be processed sequentially and the procedure can be completed in one working day. As a model we quantified intracellular bacterial colonies formed by uropathogenic Escherichia coli (UPEC) during infection of human kidney cells (HKC-8). Urinary tract infections caused by UPEC are among the most common bacterial infectious diseases in humans. UPEC can colonize tissues of the urinary tract and is responsible for acute, chronic, and recurrent infections. In the bladder, UPEC can form intracellular quiescent reservoirs, thought to be responsible for recurrent infections. In the kidney, UPEC can colonize renal epithelial cells and pass to the blood stream, either via epithelial cell disruption or transcellular passage, to cause sepsis. Intracellular colonies are known to be clonal, originating from single invading UPEC. In our experimental setup, we found UPEC CFT073 intracellular bacterial colonies to be heterogeneous in size and present in nearly one third of the HKC-8 cells. This high-throughput experimental format substantially reduces experimental time and enables fast screening of the intracellular bacterial load and cellular distribution of multiple

  7. Autophagy Evasion and Endoplasmic Reticulum Subversion: The Yin and Yang of Legionella Intracellular Infection.

    Science.gov (United States)

    Sherwood, Racquel Kim; Roy, Craig R

    2016-09-08

    The gram-negative bacterial pathogen Legionella pneumophila creates a novel organelle inside of eukaryotic host cells that supports intracellular replication. The L. pneumophila-containing vacuole evades fusion with lysosomes and interacts intimately with the host endoplasmic reticulum (ER). Although the natural hosts for L. pneumophila are free-living protozoa that reside in freshwater environments, the mechanisms that enable this pathogen to replicate intracellularly also function when mammalian macrophages phagocytose aerosolized bacteria, and infection of humans by L. pneumophila can result in a severe pneumonia called Legionnaires' disease. A bacterial type IVB secretion system called Dot/Icm is essential for intracellular replication of L. pneumophila. The Dot/Icm apparatus delivers over 300 different bacterial proteins into host cells during infection. These bacterial proteins have biochemical activities that target evolutionarily conserved host factors that control membrane transport processes, which results in the formation of the ER-derived vacuole that supports L. pneumophila replication. This review highlights research discoveries that have defined interactions between vacuoles containing L. pneumophila and the host ER. These studies reveal how L. pneumophila creates a vacuole that supports intracellular replication by subverting host proteins that control biogenesis and fusion of early secretory vesicles that exit the ER and host proteins that regulate the shape and dynamics of the ER. In addition to recruiting ER-derived membranes for biogenesis of the vacuole in which L. pneumophila replicates, these studies have revealed that this pathogen has a remarkable ability to interfere with the host's cellular process of autophagy, which is an ancient cell autonomous defense pathway that utilizes ER-derived membranes to target intracellular pathogens for destruction. Thus, this intracellular pathogen has evolved multiple mechanisms to control membrane

  8. LEGER: knowledge database and visualization tool for comparative genomics of pathogenic and non-pathogenic Listeria species

    OpenAIRE

    Dieterich, Guido; Kärst, Uwe; Fischer, Elmar; Wehland, Jürgen; Jänsch, Lothar

    2005-01-01

    Listeria species are ubiquitous in the environment and often contaminate foods because they grow under conditions used for food preservation. Listeria monocytogenes, the human and animal pathogen, causes Listeriosis, an infection with a high mortality rate in risk groups such as immune-compromised individuals. Furthermore, L.monocytogenes is a model organism for the study of intracellular bacterial pathogens. The publication of its genome sequence and that of the non-pathogenic species Lister...

  9. Iron in intracellular infection: to provide or to deprive?

    Directory of Open Access Journals (Sweden)

    Sandro eSilva-Gomes

    2013-12-01

    Full Text Available Due to their chemical versatility, transition metals were incorporated as cofactors for several basic metabolic pathways in living organisms. This same characteristic makes them potentially harmful, since they can be engaged in deleterious reactions like Fenton chemistry. As such, organisms have evolved highly specialized mechanisms to supply their own metal needs while keeping their toxic potential in check.This dual character comes into play in host-pathogen interactions, given that the host can either deprive the pathogen of these key nutrients or exploit them to induce toxicity towards the invading agent. Iron stands as the prototypic example of how a metal can be used to limit the growth of pathogens by nutrient deprivation, a mechanism widely studied in Mycobacterium infections. However, the host can also take advantage of iron-induced toxicity to control pathogen proliferation, as observed in infections caused by Leishmania. Whether we may harness either of the two pathways for therapeutical purposes is still ill-defined.In this review, we discuss how modulation of the host iron availability impacts the course of infections, focusing on those caused by two relevant intracellular pathogens, Mycobacterium and Leishmania.

  10. An intracellular anion channel critical for pigmentation.

    Science.gov (United States)

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-12-16

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.

  11. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

    Science.gov (United States)

    Schumacher, Dominik; Helma, Jonas; Schneider, Anselm F. L.; Leonhardt, Heinrich

    2018-01-01

    Abstract Nanobodies can be seen as next‐generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site‐specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens. PMID:28913971

  12. Aqueous extract of tamarind seeds selectively increases glucose transporter-2, glucose transporter-4, and islets' intracellular calcium levels and stimulates β-cell proliferation resulting in improved glucose homeostasis in rats with streptozotocin-induced diabetes mellitus.

    Science.gov (United States)

    Sole, Sushant Shivdas; Srinivasan, B P

    2012-08-01

    Tamarindus indica Linn. has been in use for a long time in Asian food and traditional medicine for different diseases including diabetes and obesity. However, the molecular mechanisms of these effects have not been fully understood. In view of the multidimensional activity of tamarind seeds due to their having high levels of polyphenols and flavonoids, we hypothesized that the insulin mimetic effect of aqueous tamarind seed extract (TSE) might increase glucose uptake through improvement in the expression of genes of the glucose transporter (GLUT) family and sterol regulatory element-binding proteins (SREBP) 1c messenger RNA (mRNA) in the liver. Daily oral administration of TSE to streptozotocin (STZ)-induced (90 mg/kg intraperitoneally) type 2 diabetic male Wistar rats at different doses (120 and 240 mg/kg body weight) for 4 weeks showed positive correlation with intracellular calcium and insulin release in isolated islets of Langerhans. Tamarind seed extract supplementation significantly improved the GLUT-2 protein and SREBP-1c mRNA expression in the liver and GLUT-4 protein and mRNA expression in the skeletal muscles of diabetic rats. The elevated levels of serum nitric oxide (NO), glycosylated hemoglobin level (hemoglobin (A1c)) and tumor necrosis factor α (TNF-α) decreased after TSE administration. Immunohistochemical findings revealed that TSE abrogated STZ-induced apoptosis and increased β-cell neogenesis, indicating its effect on islets and β-cell mass. In conclusion, it was found that the antidiabetic effect of TSE on STZ-induced diabetes resulted from complex mechanisms of β-cell neogenesis, calcium handling, GLUT-2, GLUT-4, and SREBP-1c. These findings show the scope for formulating a new herbal drug for diabetes therapy. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens

    DEFF Research Database (Denmark)

    Abdul Momin, Muhd Haziq F; Bean, David C; Hendriksen, Rene S.

    2017-01-01

    Purpose. A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture...

  14. Intracellular ion channels and cancer

    Directory of Open Access Journals (Sweden)

    Luigi eLeanza

    2013-09-01

    Full Text Available Several types of channels play a role in the maintenance of ion homeostasis in subcellular organelles including endoplasmatic reticulum, nucleus, lysosome, endosome and mitochondria. Here we give a brief overview of the contribution of various mitochondrial and other organellar channels to cancer cell proliferation or death. Much attention is focused on channels involved in intracellular calcium signaling and on ion fluxes in the ATP-producing organelle mitochondria. Mitochondrial K+ channels (Ca2+-dependent BKCa and IKCa, ATP-dependent KATP, Kv1.3, two-pore TWIK-related Acid-Sensitive K+ channel-3 (TASK-3, Ca2+ uniporter MCU, Mg2+-permeable Mrs2, anion channels (voltage-dependent chloride channel VDAC, intracellular chloride channel CLIC and the Permeability Transition Pore (MPTP contribute importantly to the regulation of function in this organelle. Since mitochondria play a central role in apoptosis, modulation of their ion channels by pharmacological means may lead to death of cancer cells. The nuclear potassium channel Kv10.1 and the nuclear chloride channel CLIC4 as well as the endoplasmatic reticulum (ER-located inositol 1,4,5-trisphosphate (IP3 receptor, the ER-located Ca2+ depletion sensor STIM1 (stromal interaction molecule 1, a component of the store-operated Ca2+ channel and the ER-resident TRPM8 are also mentioned. Furthermore, pharmacological tools affecting organellar channels and modulating cancer cell survival are discussed. The channels described in this review are summarized on Figure 1. Overall, the view is emerging that intracellular ion channels may represent a promising target for cancer treatment.

  15. Linking the transcriptional profiles and the physiological states of Mycobacterium tuberculosis during an extended intracellular infection.

    Directory of Open Access Journals (Sweden)

    Kyle H Rohde

    Full Text Available Intracellular pathogens such as Mycobacterium tuberculosis have evolved strategies for coping with the pressures encountered inside host cells. The ability to coordinate global gene expression in response to environmental and internal cues is one key to their success. Prolonged survival and replication within macrophages, a key virulence trait of M. tuberculosis, requires dynamic adaptation to diverse and changing conditions within its phagosomal niche. However, the physiological adaptations during the different phases of this infection process remain poorly understood. To address this knowledge gap, we have developed a multi-tiered approach to define the temporal patterns of gene expression in M. tuberculosis in a macrophage infection model that extends from infection, through intracellular adaptation, to the establishment of a productive infection. Using a clock plasmid to measure intracellular replication and death rates over a 14-day infection and electron microscopy to define bacterial integrity, we observed an initial period of rapid replication coupled with a high death rate. This was followed by period of slowed growth and enhanced intracellular survival, leading finally to an extended period of net growth. The transcriptional profiles of M. tuberculosis reflect these physiological transitions as the bacterium adapts to conditions within its host cell. Finally, analysis with a Transcriptional Regulatory Network model revealed linked genetic networks whereby M. tuberculosis coordinates global gene expression during intracellular survival. The integration of molecular and cellular biology together with transcriptional profiling and systems analysis offers unique insights into the host-driven responses of intracellular pathogens such as M. tuberculosis.

  16. Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

    Science.gov (United States)

    García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

    2013-01-01

    Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

  17. Intracellular Staphylococcus aureus: Live-in and let die

    Directory of Open Access Journals (Sweden)

    Martin eFraunholz

    2012-04-01

    Full Text Available Staphylococcus aureus uses a plethora of virulence factors to accomodate a diversity of niches in its human host. Aside from the classical manifestations of S. aureus-induced diseases, the pathogen also invades and survives within mammalian host cells. The survival strategies of the pathogen are as diverse as strains or host cell types used. S. aureus is able to replicate in the phagosome or freely in the cytoplasm of its host cells. It escapes the phagosome of professional and non-professional phagocytes, subverts autophagy, induces cell death mechanisms such as apoptosis and pyronecrosis, and even can induce anti-apoptotic programs in phagocytes. The focus of this review is to present a guide to recent research outlining the variety of intracellular fates of S. aureus.

  18. Economic Game Theory to Model the Attenuation of Virulence of an Obligate Intracellular Bacterium.

    Science.gov (United States)

    Tago, Damian; Meyer, Damien F

    2016-01-01

    Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host's defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g., with Ehrlichia ruminantium), there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria.

  19. Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.

    Science.gov (United States)

    Kang, Yoon-Suk; Kirby, James E

    2017-05-01

    We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus , B. melitensis , and B. suis , B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila , we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection. Copyright © 2017 American Society for Microbiology.

  20. Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli.

    Science.gov (United States)

    Shaffer, Carrie L; Zhang, Ellisa W; Dudley, Anne G; Dixon, Beverly R E A; Guckes, Kirsten R; Breland, Erin J; Floyd, Kyle A; Casella, Daniel P; Algood, Holly M Scott; Clayton, Douglass B; Hadjifrangiskou, Maria

    2017-01-01

    The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement. Copyright © 2016 American Society for Microbiology.

  1. Potent Antibacterial Nanoparticles against Biofilm and Intracellular Bacteria

    Science.gov (United States)

    Mu, Haibo; Tang, Jiangjiang; Liu, Qianjin; Sun, Chunli; Wang, Tingting; Duan, Jinyou

    2016-01-01

    The chronic infections related to biofilm and intracellular bacteria are always hard to be cured because of their inherent resistance to both antimicrobial agents and host defenses. Herein we develop a facile approach to overcome the above conundrum through phosphatidylcholine-decorated Au nanoparticles loaded with gentamicin (GPA NPs). The nanoparticles were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet-visible (UV-vis) absorption spectra which demonstrated that GPA NPs with a diameter of approximately 180 nm were uniform. The loading manner and release behaviors were also investigated. The generated GPA NPs maintained their antibiotic activities against planktonic bacteria, but more effective to damage established biofilms and inhibited biofilm formation of pathogens including Gram-positive and Gram-negative bacteria. In addition, GPA NPs were observed to be nontoxic to RAW 264.7 cells and readily engulfed by the macrophages, which facilitated the killing of intracellular bacteria in infected macrophages. These results suggested GPA NPs might be a promising antibacterial agent for effective treatment of chronic infections due to microbial biofilm and intracellular bacteria.

  2. Modulation of pathogen recognition by autophagy

    Directory of Open Access Journals (Sweden)

    Ji Eun eOh

    2012-03-01

    Full Text Available Autophagy is an ancient biological process for maintaining cellular homeostasis by degradation of long-lived cytosolic proteins and organelles. Recent studies demonstrated that autophagy is availed by immune cells to regulate innate immunity. On the one hand, cells exert direct effector function by degrading intracellular pathogens; on the other hand, autophagy modulates pathogen recognition and downstream signaling for innate immune responses. Pathogen recognition via pattern recognition receptors induces autophagy. The function of phagocytic cells is enhanced by recruitment of autophagy-related proteins. Moreover, autophagy acts as a delivery system for viral replication complexes to migrate to the endosomal compartments where virus sensing occurs. In another case, key molecules of the autophagic pathway have been found to negatively regulate immune signaling, thus preventing aberrant activation of cytokine production and consequent immune responses. In this review, we focus on the recent advances in the role of autophagy in pathogen recognition and modulation of innate immune responses.

  3. Prevalence study and risk factor analysis of selected bacterial, protozoal and viral, including vector-borne, pathogens in cats from Cyprus.

    Science.gov (United States)

    Attipa, Charalampos; Papasouliotis, Kostas; Solano-Gallego, Laia; Baneth, Gad; Nachum-Biala, Yaarit; Sarvani, Elpida; Knowles, Toby G; Mengi, Sena; Morris, David; Helps, Chris; Tasker, Séverine

    2017-03-13

    Feline infectious agent studies are lacking in Cyprus. The aims of this study were to determine the prevalence and risk factors for various feline infectious agents, including feline vector-borne pathogens (FVBP), in cats from Cyprus. A cross-sectional, descriptive, multicentre study was performed on 174 feline samples [138 owned and 36 shelter-feral, including both healthy (43) and non-healthy (131), cats] from private veterinary clinics from all six districts of Cyprus. Real-time quantitative polymerase chain reaction (qPCR) assays were used to detect Mycoplasma haemofelis (Mhf), "Candidatus Mycoplasma haemominutum" (CMhm) and "Candidatus Mycoplasma turicensis" (CMt). The population was tested for four FVBP including Bartonella henselae and Leishmania spp. using qPCR, while conventional PCR assays were used to detect Ehrlichia/Anaplasma spp. and Hepatozoon spp. Serological assays were performed to detect Leishmania infantum antibodies, feline leukaemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies. Statistical analysis was performed to test associations and possible risk factors between variables and infectious agents. Ninety-six (55.2%) of the 174 cats were PCR-positive for at least one infectious agent. Forty-six cats (26.4%) were haemoplasma positive, including 13 (7.5%) for Mhf, 36 (20.7%) for CMhm and 12 (6.9%) for CMt. Sixty-six cats (37.9%) were positive for Hepatozoon spp., while 19 (10.9%) were positive for B. henselae, four (2.3%) for Leishmania spp. and one (0.6%) for Ehrlichia/Anaplasma spp. Sequencing revealed the presence of Hepatozoon felis, L. infantum and Anaplasma platys. Of the 164 cats that underwent retroviral serology, 10 (6.1%) were FeLV-positive and 31 (18.9%) were FIV-positive, while L. infantum serology was positive in 7 (4.4%) of the 160 cats tested. Multivariable logistic regression revealed significant associations for various infectious agents including L. infantum with each of Hepatozoon spp. and CMt

  4. Intracellular survival of Staphylococcus aureus during persistent infection in the insect Tenebrio molitor.

    Science.gov (United States)

    McGonigle, John E; Purves, Joanne; Rolff, Jens

    2016-06-01

    Survival of bacteria within host cells and tissues presents a challenge to the immune systems of higher organisms. Escape from phagocytic immune cells compounds this issue, as immune cells become potential vehicles for pathogen dissemination. However, the duration of persistence within phagocytes and its contribution to pathogen load has yet to be determined. We investigate the immunological significance of intracellular persistence within the insect model Tenebrio molitor, assessing the extent, duration and location of bacterial recovery during a persistent infection. Relative abundance of Staphylococcus aureus in both intracellular and extracellular fractions was determined over 21 days, and live S. aureus were successfully recovered from both the hemolymph and within phagocytic immune cells across the entire time course. The proportion of bacteria recovered from within phagocytes also increased over time. Our results show that to accurately estimate pathogen load it is vital to account for bacteria persisting within immune cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. The glycoprotein TRP36 of Ehrlichia sp. UFMG-EV and related cattle pathogen Ehrlichia sp. UFMT-BV evolved from a highly variable clade of E. canis under adaptive diversifying selection.

    Science.gov (United States)

    Cabezas-Cruz, Alejandro; Valdés, James J; de la Fuente, José

    2014-12-10

    A new species of Ehrlichia, phylogenetically distant from E. ruminantium, was found in 2010 infecting cattle in Canada. In 2012 and 2013, we reported the in vitro propagation, molecular and ultrastructural characterization of Ehrlichia sp. UFMG-EV (E. mineirensis), a new species of Ehrlichia isolated from the haemolymph of Brazilian Rhipicephalus (Boophilus) microplus ticks. A new organism, named Ehrlichia sp. UFMT-BV, closely related to Ehrlichia sp. UFMG-EV, was recently described in Brazil and after experimental infection it was shown to be pathogenic for cattle. This new emerging clade of cattle Ehrlichia pathogens is closely related to E. canis. The major immunogenic Tandem Repeat Protein (TRP36; also known as gp36) is extensively used to characterize the genetic diversity of E. canis. Homologs of TRP36 were found in both Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV. Herein, we characterized the evolution of this new Ehrlichia clade using TRP36 sequences. Our working hypothesis is that Ehrlichia sp. UFMG-EV and related microorganisms evolved from a highly variable E. canis clade. In support of our hypothesis we found that Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV TRP36 evolved from a highly divergent and variable clade within E. canis and this clade evolved under episodic diversifying selection with a high proportion of sites under positive selection. Our results suggest that Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV evolved from a variable clade within E. canis.

  6. Proanthocyanidin-rich extracts from cranberry fruit (Vaccinium macrocarpon Ait.) selectively inhibit the growth of human pathogenic fungi Candida spp. and Cryptococcus neoformans.

    Science.gov (United States)

    Patel, Kunal D; Scarano, Frank J; Kondo, Miwako; Hurta, Robert A R; Neto, Catherine C

    2011-12-28

    Cranberry ( Vaccinium macrocarpon ) has been shown in clinical studies to reduce infections caused by Escherichia coli and other bacteria, and proanthocyanidins are believed to play a role. The ability of cranberry to inhibit the growth of opportunistic human fungal pathogens that cause oral, skin, respiratory, and systemic infections has not been well-studied. Fractions from whole cranberry fruit were screened for inhibition of five Candida species and Cryptococcus neoformans , a causative agent of fungal meningitis. Candida glabrata , Candida lusitaniae , Candida krusei , and Cryptococcus neoformans showed significant susceptibility to treatment with cranberry proanthocyanidin fractions in a broth microdilution assay, with minimum inhibitory concentrations as low as 1 μg/mL. MALDI-TOF MS analysis of subfractions detected epicatechin oligomers of up to 12 degrees of polymerization. Those containing larger oligomers caused the strongest inhibition. This study suggests that cranberry has potential as an antifungal agent.

  7. Healthcare workers’ occupational exposure to bloodborne pathogens: A 5-year observation in selected hospitals of the Małopolska province

    Directory of Open Access Journals (Sweden)

    Anna Różańska

    2014-10-01

    Full Text Available Objectives: The study presents data concerning occupational exposures among the staff of 5 hospitals in the Małopolska province in 2008–2012, taking into account the frequency and circumstances of exposure formation, occupational groups of hospital workers, as well as diversification of the reported rates in subsequent years between the hospitals and in each of them. An additional objective of the analysis was to assess the practical usefulness of the reported data for planning and evaluation of the effectiveness of procedures serving to minimize the risk of healthcare workers’ exposure to pathogens transmitted through blood. Material and Methods: Data were derived from occupational exposure registries kept by 5 hospitals of varying sizes and operational profiles from the Małopolska province from the years 2008–2012. Results: Seven hundred and seventy-five cases of exposure were found in a group of 3165 potentially exposed workers in the analyzed period. Most cases were observed in nurses (68% and these were mainly various types of needlestick injuries (78%. Exposure rates with respect to all workers ranged from 2.6% to 8.3% in individual hospitals, but the differences in their values registered in the hospitals in subsequent years did not bear any statistical significance, in a way similar to the rates calculated separately for each occupational group. Conclusions: There was no upward or downward trend in the number of reported cases of exposure to bloodborne pathogens in the studied period in any of the hospitals. Statistically significant differences in the percentages of exposures were reported between individual hospitals in some years of the analyzed period, which confirms the need for registries in individual units in order to plan and evaluate the effectiveness of preventative measures.

  8. MR imaging of intracellular and extracellular deoxyhemoglobin

    International Nuclear Information System (INIS)

    Janick, P.A.; Grossman, R.I.; Asakura, T.

    1989-01-01

    MR imaging was performed on varying concentrations of intracellular and extracellular deoxyhemoglobin as well as varying proportions of deoxyhemoglobin and oxyhemoglobin in vitro at 1.5T with use of standard spin-echo and gradient-refocused spin sequences. This study indicates that susceptibility-induced T2 shortening occurs over a broad range of intracellular deoxyhemoglobin concentrations (maximal at hematocrits between 20% and 45%), reflecting diffusional effects at the cellular level. T2* gradient-echo imaging enhances the observed hypointensity in images of intracellular deoxyhemoglobin. The characteristic MR appearance of acute hemotomas can be modeled by the behavior of intracellular and extracellular deoxyhemoglobin and oxyhemoglobin

  9. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

    Directory of Open Access Journals (Sweden)

    Onkar Sharma

    2016-03-01

    Full Text Available A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase. When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO, and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells.

  10. Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids.

    Science.gov (United States)

    Chongsiriwatana, Nathaniel P; Lin, Jennifer S; Kapoor, Rinki; Wetzler, Modi; Rea, Jennifer A C; Didwania, Maruti K; Contag, Christopher H; Barron, Annelise E

    2017-12-01

    Many organisms rely on antimicrobial peptides (AMPs) as a first line of defense against pathogens. In general, most AMPs are thought to kill bacteria by binding to and disrupting cell membranes. However, certain AMPs instead appear to inhibit biomacromolecule synthesis, while causing less membrane damage. Despite an unclear understanding of mechanism(s), there is considerable interest in mimicking AMPs with stable, synthetic molecules. Antimicrobial N-substituted glycine (peptoid) oligomers ("ampetoids") are structural, functional and mechanistic analogs of helical, cationic AMPs, which offer broad-spectrum antibacterial activity and better therapeutic potential than peptides. Here, we show through quantitative studies of membrane permeabilization, electron microscopy, and soft X-ray tomography that both AMPs and ampetoids trigger extensive and rapid non-specific aggregation of intracellular biomacromolecules that correlates with microbial death. We present data demonstrating that ampetoids are "fast killers", which rapidly aggregate bacterial ribosomes in vitro and in vivo. We suggest intracellular biomass flocculation is a key mechanism of killing for cationic, amphipathic AMPs, which may explain why most AMPs require micromolar concentrations for activity, show significant selectivity for killing bacteria over mammalian cells, and finally, why development of resistance to AMPs is less prevalent than developed resistance to conventional antibiotics.

  11. Blood, Tissue, and Intracellular Concentrations of Azithromycin during and after End of Therapy

    Science.gov (United States)

    Matzneller, P.; Krasniqi, S.; Kinzig, M.; Sörgel, F.; Hüttner, S.; Lackner, E.; Müller, M.

    2013-01-01

    Although azithromycin is extensively used in the treatment of respiratory tract infections as well as skin and skin-related infections, pharmacokinetics of azithromycin in extracellular space fluid of soft tissues, i.e., one of its therapeutic target sites, are not yet fully elucidated. In this study, azithromycin concentration-time profiles in extracellular space of muscle and subcutaneous adipose tissue, but also in plasma and white blood cells, were determined at days 1 and 3 of treatment as well as 2 and 7 days after the end of treatment. Of all compartments, azithromycin concentrations were highest in white blood cells, attesting for intracellular accumulation. However, azithromycin concentrations in both soft tissues were markedly lower than in plasma both during and after treatment. Calculation of the area under the concentration-time curve from 0 to 24 h (AUC0–24)/MIC90 ratios for selected pathogens suggests that azithromycin concentrations measured in the present study are subinhibitory at all time points in both soft tissues and at the large majority of observed time points in plasma. Hence, it might be speculated that azithromycin's clinical efficacy relies not only on elevated intracellular concentrations but possibly also on its known pleotropic effects, including immunomodulation and influence on bacterial virulence factors. However, prolonged subinhibitory azithromycin concentrations at the target site, as observed in the present study, might favor the emergence of bacterial resistance and should therefore be considered with concern. In conclusion, this study has added important information to the pharmacokinetic profile of the widely used antibiotic drug azithromycin and evidentiates the need for further research on its potential for induction of bacterial resistance. PMID:23357769

  12. Manipulation of host membranes by the bacterial pathogens Listeria, Francisella, Shigella and Yersinia.

    Science.gov (United States)

    Pizarro-Cerdá, Javier; Charbit, Alain; Enninga, Jost; Lafont, Frank; Cossart, Pascale

    2016-12-01

    Bacterial pathogens display an impressive arsenal of molecular mechanisms that allow survival in diverse host niches. Subversion of plasma membrane and cytoskeletal functions are common themes associated to infection by both extracellular and intracellular pathogens. Moreover, intracellular pathogens modify the structure/stability of their membrane-bound compartments and escape degradation from phagocytic or autophagic pathways. Here, we review the manipulation of host membranes by Listeria monocytogenes, Francisella tularensis, Shigella flexneri and Yersinia spp. These four bacterial model pathogens exemplify generalized strategies as well as specific features observed during bacterial infection processes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Opportunistic Premise Plumbing Pathogens: Increasingly Important Pathogens in Drinking Water

    Directory of Open Access Journals (Sweden)

    Joseph O. Falkinham

    2015-06-01

    Full Text Available Opportunistic premise plumbing pathogens are responsible for a significant number of infections whose origin has been traced to drinking water. These opportunistic pathogens represent an emerging water borne disease problem with a major economic cost of at least $1 billion annually. The common features of this group of waterborne pathogens include: disinfectant-resistance, pipe surface adherence and biofilm formation, growth in amoebae, growth on low organic concentrations, and growth at low oxygen levels. Their emergence is due to the fact that conditions resulting from drinking water treatment select for them. As such, there is a need for novel approaches to reduce exposure to these pathogens. In addition to much-needed research, controls to reduce numbers and human exposure can be instituted independently by utilities and homeowners and hospital- and building-operators.

  14. Opportunistic Premise Plumbing Pathogens: Increasingly Important Pathogens in Drinking Water.

    Science.gov (United States)

    Falkinham, Joseph O; Pruden, Amy; Edwards, Marc

    2015-06-09

    Opportunistic premise plumbing pathogens are responsible for a significant number of infections whose origin has been traced to drinking water. These opportunistic pathogens represent an emerging water borne disease problem with a major economic cost of at least $1 billion annually. The common features of this group of waterborne pathogens include: disinfectant-resistance, pipe surface adherence and biofilm formation, growth in amoebae, growth on low organic concentrations, and growth at low oxygen levels. Their emergence is due to the fact that conditions resulting from drinking water treatment select for them. As such, there is a need for novel approaches to reduce exposure to these pathogens. In addition to much-needed research, controls to reduce numbers and human exposure can be instituted independently by utilities and homeowners and hospital- and building-operators.

  15. Trade-Offs of Escherichia coli Adaptation to an Intracellular Lifestyle in Macrophages.

    Directory of Open Access Journals (Sweden)

    M Azevedo

    Full Text Available The bacterium Escherichia coli exhibits remarkable genomic and phenotypic variation, with some pathogenic strains having evolved to survive and even replicate in the harsh intra-macrophage environment. The rate and effects of mutations that can cause pathoadaptation are key determinants of the pace at which E. coli can colonize such niches and become pathogenic. We used experimental evolution to determine the speed and evolutionary paths undertaken by a commensal strain of E. coli when adapting to intracellular life. We estimated the acquisition of pathoadaptive mutations at a rate of 10-6 per genome per generation, resulting in the fixation of more virulent strains in less than a hundred generations. Whole genome sequencing of independently evolved clones showed that the main targets of intracellular adaptation involved loss of function mutations in genes implicated in the assembly of the lipopolysaccharide core, iron metabolism and di- and tri-peptide transport, namely rfaI, fhuA and tppB, respectively. We found a substantial amount of antagonistic pleiotropy in evolved populations, as well as metabolic trade-offs, commonly found in intracellular bacteria with reduced genome sizes. Overall, the low levels of clonal interference detected indicate that the first steps of the transition of a commensal E. coli into intracellular pathogens are dominated by a few pathoadaptive mutations with very strong effects.

  16. Intracellular Polyamines Enhance Astrocytic Coupling

    Science.gov (United States)

    Benedikt, Jan; Inyushin, Mikhail; Kucheryavykh, Yuriy V.; Rivera, Yomarie; Kucheryavykh, Lilia Y.; Nichols, Colin G.; Eaton, Misty J.; Skatchkov, Serguei N.

    2013-01-01

    Spermine (SPM) and spermidine (SPD), endogenous polyamines (PA) with the ability to modulate various ion channels and receptors in the brain, exert neuroprotective, antidepressant, antioxidant and other effects in vivo such as increasing longevity. These PA are preferably accumulated in astrocytes, and we hypothesized that SPM increases glial intercellular communication by interacting with glial gap junctions. Results obtained in situ, using Lucifer yellow propagation in the astrocytic syncitium of 21–25 day old rat CA1 hippocampal slices, showed reduced coupling when astrocytes were dialyzed with standard intracellular solutions (ICS) without SPM. However, there was a robust increase in the spreading of Lucifer yellow via gap junctions to neighboring astrocytes when the cells were patched with ICS containing 1 mM SPM; a physiological concentration in glia. Lucifer yellow propagation was inhibited by gap junction blockers. Our findings show that the glial syncitium propagates SPM via gap junctions and further suggest a new role of polyamines in the regulation of the astroglial network in both normal and pathological conditions. PMID:23076119

  17. Effect of gamma rays in embryogenic callus of agave tequilana weber var. blue and in vitro selection for resistance to pathogens

    International Nuclear Information System (INIS)

    Rubluo, A.; Brunner, I.; Rodriguez-Garay, B.; Rodriguez-Dominguez, J.M.; Santacruz-Ruvalcava, F.; De la Cruz, E.; Gonzalez, J.

    1999-01-01

    Agave tequilana from which tequila is done, suffers genetic erosion leaving the plant susceptible to pathogens. Recently, a pest invades the tequilana fields. To face this problem we designed experiments to induce mutations for disease resistance. Our aims were: to analyse the in vitro responses of embryogenic callus exposed to gamma rays, to determine the LD50 and challenge surviving embryos to crude bacterial extracts (CBE). Axillary buds were cultured in vitro. Leaves from the former plant lets were used as explants to induce embryogenic callus. Samples of 1g embryogenic callus were irradiated from 5 to 40 Gy of CO 60 gamma rays. Effects were recorded at 15, 30 and 45 days after irradiation. The CBE was obtained from isolated bacteria collected from sick field plants. Embryogenic calli were irradiated at 0, 15, 25 and 40 Gy and challenged with the CBE at 0, 10 and 20 % v/v. Embryo expression (globular, white and green cucumber shaped) were affected as a function of the dose. However at low doses (5-15 Gy) globular forms increased its production. On the contrary , green cucumber shaped embryos were severely affected. However, even at high doses (30, 35 and 40 Gy) healthy green forms were recorded. When the embryogenic callus was challenged to the CBE, high mortality was recorded, although, some green forms survived at high radiation doses and high CBE concentrations. Because of the unicellular origin of the somatic embryos, solid mutants can be expected

  18. Biogenic nanosilver incorporated reverse osmosis membrane for antibacterial and antifungal activities against selected pathogenic strains: an enhanced eco-friendly water disinfection approach.

    Science.gov (United States)

    Manjumeena, R; Duraibabu, D; Sudha, J; Kalaichelvan, P T

    2014-01-01

    Reverse osmosis (RO) membranes have been used extensively in water desalination plants, waste water treatment in industries, agricultural farms and drinking water production applications. The objective of this work is to impart antibacterial and antifungal activities to commercially available RO membrane used in water purification systems by incorporating biogenic silver nanoparticles(AgNPs) synthesized using Rosa indica wichuriana hybrid leaf extract. The morphology and surface topography of uncoated and AgNPs-coated RO membrane were studied using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Elemental composition of the AgNPs-coated RO membrane was analyzed by energy-dispersive X-ray spectroscopy (EDAX). The functional groups were identified by Fourier Transform Infrared spectroscopy (FT-IR). Hydrophilicity of the uncoated and AgNPs-coated RO membrane was analyzed using water contact angle measurements. The thermal properties were studied by thermogravimetric analysis (TGA). The AgNPs incorporated RO membrane exhibited good antibacterial and antifungal activities against pathogenic bacterial strains such as E. coli, S. aureus, M. luteus, K. pneumoniae, and P. aeruginosa and fungal strains such as Candida tropicalis, C. krusei, C. glabrata, and C. albicans.

  19. Growth inhibition of pathogenic bacteria and some yeasts by selected essential oils and survival of L. monocytogenes and C. albicans in apple-carrot juice.

    Science.gov (United States)

    Irkin, Reyhan; Korukluoglu, Mihriban

    2009-04-01

    Food safety is a fundamental concern of both consumers and the food industry. The increasing incidence of foodborne diseases increases the demand of using antimicrobials in foods. Spices and plants are rich in essential oils and show inhibition activity against microorganisms, which are composed of many compounds. In this research, effects of garlic, bay, black pepper, origanum, orange, thyme, tea tree, mint, clove, and cumin essential oils on Listeria monocytogenes AUFE 39237, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Proteus mirabilis AUFE 43566, Bacillus cereus AUFE 81154, Saccharomyces uvarum UUFE 16732, Kloeckera apiculata UUFE 10628, Candida albicans ATCC 10231, Candida oleophila UUPP 94365, and Metschnikowia fructicola UUPP 23067 and effects of thyme oil at a concentration of 0.5% on L. monocytogenes and C. albicans in apple-carrot juice during +4 degrees C storage (first to fifth day) were investigated. Strong antibacterial and antifungal activities of some essential oils were found. Thyme, origanum, clove, and orange essential oils were the most inhibitory against bacteria and yeasts. Cumin, tea tree, and mint oils inhibited the yeasts actively. It is concluded that some essential oils could be used as potential biopreservatives capable of controlling foodborne pathogens and food spoilage yeasts.

  20. Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

    Science.gov (United States)

    Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

    2018-01-15

    Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways. IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4 + T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

  1. Lactase persistence and augmented salivary alpha-amylase gene copy numbers might have been selected by the combined toxic effects of gluten and (food born) pathogens

    NARCIS (Netherlands)

    Pruimboom, Leo; Fox, Tom; Muskiet, Frits A. J.

    Various positively selected adaptations to new nutrients have been identified. Lactase persistence is among the best known, conferring the ability for drinking milk at post weaning age. An augmented number of amylase gene (AMY1) copies, giving rise to higher salivary amylase activity, has been

  2. Differentiated THP-1 Cells Exposed to Pathogenic and Nonpathogenic Borrelia Species Demonstrate Minimal Differences in Production of Four Inflammatory Cytokines.

    Science.gov (United States)

    Stokes, John V; Moraru, Gail M; McIntosh, Chelsea; Kummari, Evangel; Rausch, Keiko; Varela-Stokes, Andrea S

    2016-11-01

    Tick-borne borreliae include Lyme disease and relapsing fever agents, and they are transmitted primarily by ixodid (hard) and argasid (soft) tick vectors, respectively. Tick-host interactions during feeding are complex, with host immune responses influenced by biological differences in tick feeding and individual differences within and between host species. One of the first encounters for spirochetes entering vertebrate host skin is with local antigen-presenting cells, regardless of whether the tick-associated Borrelia sp. is pathogenic. In this study, we performed a basic comparison of cytokine responses in THP-1-derived macrophages after exposure to selected borreliae, including a nonpathogen. By using THP-1 cells, differentiated to macrophages, we eliminated variations in host response and reduced the system to an in vitro model to evaluate the extent to which the Borrelia spp. influence cytokine production. Differentiated THP-1 cells were exposed to four Borrelia spp., Borrelia hermsii (DAH), Borrelia burgdorferi (B31), B. burgdorferi (NC-2), or Borrelia lonestari (LS-1), or lipopolysaccharides (LPS) (activated) or media (no treatment) controls. Intracellular and secreted interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured using flow cytometric and Luminex-based assays, respectively, at 6, 24, and 48 h postexposure time points. Using a general linear model ANOVA for each cytokine, treatment (all Borrelia spp. and LPS compared to no treatment) had a significant effect on secreted TNF-α only. Time point had a significant effect on intracellular IFN-γ, TNF-α and IL-6. However, we did not see significant differences in selected cytokines among Borrelia spp. Thus, in this model, we were unable to distinguish pathogenic from nonpathogenic borreliae using the limited array of selected cytokines. While unique immune profiles may be detectable in an in vitro model and may reveal predictors for pathogenicity in borreliae

  3. Experimental evolution of a plant pathogen into a legume symbiont.

    Directory of Open Access Journals (Sweden)

    Marta Marchetti

    2010-01-01

    Full Text Available Rhizobia are phylogenetically disparate alpha- and beta-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis.

  4. Selection of empirical antibiotics for health care-associated pneumonia via integration of pneumonia severity index and risk factors of drug-resistant pathogens.

    Science.gov (United States)

    Wang, Ping-Huai; Wang, Hao-Chien; Cheng, Shih-Lung; Chang, Hou-Tai; Laio, Chun-Hsing

    2016-05-01

    The pneumonia severity index (PSI) both contains some risk factors of drug-resistant pathogens (DRPs) and represents the severity of health care-associated pneumonia. The aim of this study was to investigate whether the PSI could be used to predict DRPs and whether there were risk factors beyond the PSI. A retrospective observational study enrolled 530 patients with health care-associated pneumonia who were admitted from January 2005 to December 2010 in a tertiary care hospital. A total of 206 patients (38.9%) had DRPs, of which the most common was Pseudomonas aeruginosa (24.3%). The incidence of DRPs increased with increasing PSI classes (6.7%, 25.5%, 36.9%, and 44.6% in PSI II, III, IV, and V, respectively). An analysis of the risk factors for DRPs by PSI classes revealed that wound care was associated with methicillin-resistant Staphylococcus aureus (MRSA) infection in PSI V (p = 0.045). Nasogastric tube feeding (odds ratio, 3.88; 95% confidence interval, 1.75-8.60; p = 0.006), and bronchiectasis (odds ratio, 3.12; 95% confidence interval, 0.66-14.69; p = 0.007) were risk factors for DRPs in PSI III and IV. The area under the receiver operating characteristic curve progressed from 0.578 to 0.651 while integrating these risk factors with PSI classes. The findings suggested that PSI plus risk factors predicted the risk of DRPs. PSI II had a low risk of DRPs and could be treated as community-acquired pneumonia. Antibiotics of PSI III and IV with risk factors could be targeted DRPs. PSI V with wound care had a higher risk of MRSA, and empirical anti-MRSA antibiotics could be added. Copyright © 2015. Published by Elsevier B.V.

  5. An intracellular anion channel critical for pigmentation

    Science.gov (United States)

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-01-01

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation. DOI: http://dx.doi.org/10.7554/eLife.04543.001 PMID:25513726

  6. Intracellular calcium homeostasis and signaling.

    Science.gov (United States)

    Brini, Marisa; Calì, Tito; Ottolini, Denis; Carafoli, Ernesto

    2013-01-01

    Ca(2+) is a universal carrier of biological information: it controls cell life from its origin at fertilization to its end in the process of programmed cell death. Ca(2+) is a conventional diffusible second messenger released inside cells by the interaction of first messengers with plasma membrane receptors. However, it can also penetrate directly into cells to deliver information without the intermediation of first or second messengers. Even more distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma membrane receptor to set in motion intracellular signaling pathways that involve Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes an agent that mediates cell distress, or even (toxic) cell death, if its concentration and movements inside cells are not carefully tuned. Ca(2+) is controlled by reversible complexation to specific proteins, which could be pure Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its signal to pass it on to targets. The most important actors in the buffering of cell Ca(2+) are proteins that transport it across the plasma membrane and the membrane of the organelles: some have high Ca(2+) affinity and low transport capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+) uptake system of mitochondria). Between the initial event of fertilization, and the terminal event of programmed cell death, the Ca(2+) signal regulates the most important activities of the cell, from the expression of genes, to heart and muscle contraction and other motility processes, to diverse metabolic pathways involved in the generation of cell fuels.

  7. Ecological niche of plant pathogens

    Directory of Open Access Journals (Sweden)

    Ecaterina Fodor

    2011-02-01

    Full Text Available Disease ecology is a new approach to the understanding of the spread and dynamics of pathogens in natural and man-made environments. Defining and describing the ecological niche of the pathogens is one of the major tasks for ecological theory, as well as for practitioners preoccupied with the control and forecasting of established and emerging diseases. Niche theory has been periodically revised, not including in an explicit way the pathogens. However, many progresses have been achieved in niche modeling of disease spread, but few attempts were made to construct a theoretical frame for the ecological niche of pathogens. The paper is a review of the knowledge accumulated during last decades in the niche theory of pathogens and proposes an ecological approach in research. It quest for new control methods in what concerns forest plant pathogens, with a special emphasis on fungi like organisms of the genus Phytophthora. Species of Phytophthora are the most successful plant pathogens of the moment, affecting forest and agricultural systems worldwide, many of them being invasive alien organisms in many ecosystems. The hyperspace of their ecological niche is defined by hosts, environment and human interference, as main axes. To select most important variables within the hyperspace, is important for the understanding of the complex role of pathogens in the ecosystems as well as for control programs. Biotic relationships within ecosystem of host-pathogen couple are depicted by ecological network and specific metrics attached to this. The star shaped network is characterized by few high degree nodes, by short path lengths and relatively low connectivity, premises for a rapid disturbance spread.

  8. Ecological niche of plant pathogens

    Directory of Open Access Journals (Sweden)

    Ecaterina Fodor

    2011-06-01

    Full Text Available Disease ecology is a new approach to the understanding of the spread and dynamics of pathogens in natural and man-made environments. Defining and describing the ecological niche of the pathogens is one of the major tasks for ecological theory, as well as for practitioners preoccupied with the control and forecasting of established and emerging diseases. Niche theory has been periodically revised, not including in an explicit way the pathogens. However, many progresses have been achieved in niche modeling of disease spread, but few attempts were made to construct a theoretical frame for the ecological niche of pathogens. The paper is a review of the knowledge accumulated during last decades in the niche theory of pathogens and proposes an ecological approach in research. It quest for new control methods in what concerns forest plant pathogens, with a special emphasis on fungi like organisms of the genus Phytophthora. Species of Phytophthora are the most successful plant pathogens of the moment, affecting forest and agricultural systems worldwide, many of them being invasive alien organisms in many ecosystems. The hyperspace of their ecological niche is defined by hosts, environment and human interference, as main axes. To select most important variables within the hyperspace, is important the understanding of the complex role of pathogens in the ecosystems as well as for control programs. Biotic relationships within ecosystem of host-pathogen couple are depicted by ecological network and specific metrics attached to this. The star shaped network is characterized by few high degree nodes, by short path lengths and relatively low connectivity, premises for a rapid disturbance spread. 

  9. Brucella abortus nicotinamidase (PncA) contributes to its intracellular replication and infectivity in mice.

    Science.gov (United States)

    Kim, Suk; Kurokawa, Daisuke; Watanabe, Kenta; Makino, Sou-Ichi; Shirahata, Toshikazu; Watarai, Masahisa

    2004-05-15

    Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. Nicotinamidase/pyrazinamidase mutant (pncA mutant) of Brucella abortus failed to replicate in HeLa cells, and showed a lower rate of intracellular replication than that of wild-type strain in macrophages. Addition of nicotinic acid, but not nicotinamide, into medium supported intracellular replication of pncA mutant in HeLa cells and macrophages. The pncA mutant was not co-localizing with either late endosomes or lysosomes. The B. abortus virB4 mutant was completely cleared from the spleens of mice after 4 weeks, while the pncA mutant showed a 1.5-log reduction of the number of bacteria isolated from spleens after 10 weeks. Although pncA mutant showed reduced virulence in mice and defective intracellular replication, its ability to confer protection against the virulent B. abortus strain 544 was fully retained. These results suggest that PncA does not contribute to intracellular trafficking of B. abortus, but contributes to utilization of nutrients required for intracellular growth. Our results indicate that detailed characterizations of the pncA mutant may help the improvement of currently available live vaccines. Copyright 2004 Federation of European Microbiological Societies

  10. Isolation of pathogen-containing vacuoles.

    Science.gov (United States)

    Shevchuk, Olga; Steinert, Michael

    2013-01-01

    Dictyostelium discoideum cells are "professional phagocytes," as they ingest a large variety of bacteria, yeast, and inert particles. Several bacterial pathogens are able to survive intracellularly within specialized vacuoles of D. discoideum by interfering with host signaling pathways. To better understand the molecular mechanisms underlying these evolutionary conserved processes we have established a method for the isolation of pathogen-containing vacuoles (PCVs). The isolation protocol describes the infection of D. discoideum cells with the intracellular pathogen Legionella pneumophila, loading of the lysosomal compartment with colloidal iron, mechanical lysis of host cells, iodophenylnitrophenyltetrazolium (INT) heavy labeling of mitochondria, removal of nucleic acid by Benzonase treatment, separation of nuclei by low-speed centrifugation, and the magnetic removal of lysosomes. The subcellular fractionation in a discontinuous sucrose density OptiPrep gradient allows the separation of mitochondria and to prepare PCVs with high purity. The proteins isolated from PCVs have been successfully subjected to mass spectrometry and allowed to analyze pathogen-directed maturation processes of vacuoles. The method can also be applied for subsequent protein modification analyses and lipidome comparisons.

  11. Emerging Pathogens Initiative (EPI)

    Data.gov (United States)

    Department of Veterans Affairs — The Emerging Pathogens Initiative (EPI) database contains emerging pathogens information from the local Veterans Affairs Medical Centers (VAMCs). The EPI software...

  12. Novel amidines and analogues as promising agents against intracellular parasites: a systematic review

    Science.gov (United States)

    Soeiro, M. N. C.; Werbovetz, K.; Boykin, D.W.; Wilson, W. D.; Wang, M. Z.; Hemphill, A.

    2013-01-01

    SUMMARY Parasitic protozoa comprise diverse aetiological agents responsible for important diseases in humans and animals including sleeping sickness, Chagas disease, leishmaniasis, malaria, toxoplasmosis and others. They are major causes of mortality and morbidity in tropical and subtropical countries, and are also responsible for important economic losses. However, up to now, for most of these parasitic diseases, effective vaccines are lacking and the approved chemotherapeutic compounds present high toxicity, increasing resistance, limited efficacy and require long periods of treatment. Many of these parasitic illnesses predominantly affect low-income populations of developing countries for which new pharmaceutical alternatives are urgently needed. Thus, very low research funding is available. Amidine-containing compounds such as pentamidine are DNA minor groove binders with a broad spectrum of activities against human and veterinary pathogens. Due to their promising microbicidal activity but their rather poor bioavailability and high toxicity, many analogues and derivatives, including pro-drugs, have been synthesized and screened in vitro and in vivo in order to improve their selectivity and pharmacological properties. This review summarizes the knowledge on amidines and analogues with respect to their synthesis, pharmacological profile, mechanistic and biological effects upon a range of intracellular protozoan parasites. The bulk of these data may contribute to the future design and structure optimization of new aromatic dicationic compounds as novel antiparasitic drug candidates. PMID:23561006

  13. Immune Evasion Strategies of Pathogens in Macrophages: the Potential for Limiting Pathogen Transmission.

    Science.gov (United States)

    Ren, Yuwei; Khan, Faheem Ahmed; Pandupuspitasari, Nuruliarizki Shinta; Zhang, Shujun

    2017-01-01

    Preventing pathogen transmission to a new host is of major interest to the immunologist and could benefit from a detailed investigation of pathogen immune evasion strategies. The first line of defense against pathogen invasion is provided by macrophages. When they sense pathogens, macrophages initiate signals to inflammatory and pro-inflammatory cytokines through pattern recognition receptors (PRRs) subsequently mediating phagocytosis and inflammation. The macrophage immune machinery classically includes two subsets: the activated M1 and the activated M2 that respond accordingly in diverse immune challenges. The lipid and glycogen metabolic pathways work together with the lysosome to help the mature phagosome to degrade and eliminate intracellular pathogens in macrophages. The viral evasion strategies are even more complex due to the interplay between autophagy and apoptosis. However, pathogens evolve several strategies to camouflage themselves against immune responses in order to ensure their survival, replication and transmission. These strategies include the muting of PRRs initiated inflammatory responses, attenuation of M1 and/or induction of M2 macrophages, suppression of autophago-lysosomal formation, interference with lipid and glycogen metabolism, and viral mediation of autophagy and apoptosis cross-talk to enhance viral replication. This review focuses on pathogen immune evasion methods and on the strategies used by the host against camouflaged pathogens.

  14. Independent origins of vectored plant pathogenic bacteria from arthropod-associated Arsenophonus endosymbionts.

    Science.gov (United States)

    Bressan, Alberto; Terlizzi, Federica; Credi, Rino

    2012-04-01

    The genus Arsenophonus (Gammaproteobacteria) is comprised of intracellular symbiotic bacteria that are widespread across the arthropods. These bacteria can significantly influence the ecology and life history of their hosts. For instance, Arsenophonus nasoniae causes an excess of females in the progeny of parasitoid wasps by selectively killing the male embryos. Other Arsenophonus bacteria have been suspected to protect insect hosts from parasitoid wasps or to expand the host plant range of phytophagous sap-sucking insects. In addition, a few reports have also documented some Arsenophonus bacteria as plant pathogens. The adaptation to a plant pathogenic lifestyle seems to be promoted by the infection of sap-sucking insects in the family Cixiidae, which then transmit these bacteria to plants during the feeding process. In this study, we define the specific localization of an Arsenophonus bacterium pathogenic to sugar beet and strawberry plants within the plant hosts and the insect vector, Pentastiridius leporinus (Hemiptera: Cixiidae), using fluorescence in situ hybridization assays. Phylogenetic analysis on 16S rRNA and nucleotide coding sequences, using both maximum likelihood and Bayesian criteria, revealed that this bacterium is not a sister taxon to "Candidatus Phlomobacter fragariae," a previously characterized Arsenophonus bacterium pathogenic to strawberry plants in France and Japan. Ancestral state reconstruction analysis indicated that the adaptation to a plant pathogenic lifestyle likely evolved from an arthropod-associated lifestyle and showed that within the genus Arsenophonus, the plant pathogenic lifestyle arose independently at least twice. We also propose a novel Candidatus status, "Candidatus Arsenophonus phytopathogenicus" novel species, for the bacterium associated with sugar beet and strawberry diseases and transmitted by the planthopper P. leporinus.

  15. Comparative evaluation of the efficacy of a herbal mouthwash and chlorhexidine mouthwash on select periodontal pathogens: An in vitro and ex vivo study

    Directory of Open Access Journals (Sweden)

    Multazim Muradkhan Pathan

    2017-01-01

    Full Text Available Background: Several herbal mouthwash and herbal extracts have been tested in vitro and in vivo in search of a suitable adjunct to mechanical therapy for long-term use. In this study, we aimed to look at the antimicrobial effect of the herbal mouthwash and chlorhexidine (CHX mouthwash on select organisms in in vitro test and an ex vivo model. Materials and Methods: The antimicrobial effects were determined against standard strains of bacteria that are involved in different stages of periodontal diseases. The in vitro tests included determination of minimum inhibitory concentration (MIC using broth dilution and agar diffusion. In the ex vivo part of the study supragingival dental plaque were obtained from 20 periodontally healthy adult volunteers. Descriptive analysis was done for the entire quantitative and qualitative variable recorded. Results: The MIC by broth dilution method found no statistically significant difference between the mouthwashes. The agar dilution method showed CHX was more effective as compared to the herbal mouthwash against standard strains of Streptococcus mutans, Streptococcus sanguinis, and Aggregatibacter actinomycetemcomitans. However, no difference was observed between the mouthwashes for Porphyromonas, Pseudomonas aeruginosa, and Fusobacterium nucleatum. The ex vivo results conclude that none of the selected mouthwashes were statistically significantly different from each other. Conclusion: In the present study, CHX showed higher levels of antimicrobial action than the herbal mouthwash against bacterial species. The results reinforce the earlier findings that the in vitro testing is sensitive to methods and due diligence is needed when extrapolating the data for further use. However, long-term use and in vivo effectiveness against the periopathogens need to be tested in well-planned clinical trials.

  16. Host-microbe interactions: innate pattern recognition of fungal pathogens.

    NARCIS (Netherlands)

    Veerdonk, F.L. van de; Kullberg, B.J.; Meer, J.W.M. van der; Gow, N.A.; Netea, M.G.

    2008-01-01

    The recognition of fungi is mediated by germline pattern recognition receptors (PRRs) such as Toll-like receptors and lectin receptors that interact with conserved structures of the microorganisms, the pathogen-associated molecular patterns (PAMPs). Subsequently, PRRs activate intracellular signals

  17. Investigating Internalization and Intracellular Trafficking of GPCRs

    DEFF Research Database (Denmark)

    Foster, Simon R; Bräuner-Osborne, Hans

    2017-01-01

    for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal...... independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies...... that have been used to investigate internalization and intracellular signaling of GPCRs, with a particular focus on emerging real-time techniques. These recent developments have improved our understanding of the complexities of GPCR internalization and intracellular signaling and suggest that the broader...

  18. Nanoparticles for intracellular-targeted drug delivery

    International Nuclear Information System (INIS)

    Paulo, Cristiana S O; Pires das Neves, Ricardo; Ferreira, Lino S

    2011-01-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  19. Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery.

    Science.gov (United States)

    Labouta, Hagar Ibrahim; Menina, Sara; Kochut, Annika; Gordon, Sarah; Geyer, Rebecca; Dersch, Petra; Lehr, Claus-Michael

    2015-12-28

    Intracellular bacteria invade mammalian cells to establish an infectious niche. The current work models adhesion and subsequent internalization strategy of pathogenic bacteria into mammalian cells to design a bacteriomimetic bioinvasive delivery system. We report on the surface functionalization of liposomes with a C-terminal fragment of invasin (InvA497), an invasion factor in the outer membrane of Yersinia pseudotuberculosis. InvA497-functionalized liposomes adhere to mammalian epithelial HEp-2 cell line at different infection stages with a significantly higher efficiency than liposomes functionalized with bovine serum albumin. Covalent attachment of InvA497 results in higher cellular adhesion than liposomes with physically adsorbed InvA497 with non-specific surface protein alignment. Uptake studies in HEp-2 cells indicate active internalization of InvA497-functionalized liposomes via β1-integrin receptor-mediated uptake mechanism mimicking the natural invasion strategy of Y. pseudotuberculosis. Uptake studies in Caco-2 cells at different polarization states demonstrate specific targeting of the InvA497-functionalized liposomes to less polarized cells reflecting the status of inflamed cells. Moreover, when loaded with the anti-infective agent gentamicin and applied to HEp-2 cells infected with Y. pseudotuberculosis, InvA497-functionalized liposomes are able to significantly reduce the infection load relative to non-functionalized drug-loaded liposomes. This indicates a promising application of such a bacteriomimetic system for drug delivery to intracellular compartments. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Cyclic AMP Pathway Activation and Extracellular Zinc Induce Rapid Intracellular Zinc Mobilization in Candida albicans

    Science.gov (United States)

    Kjellerup, Lasse; Winther, Anne-Marie L.; Wilson, Duncan; Fuglsang, Anja T.

    2018-01-01

    Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this “zinc flux” could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen. PMID:29619016

  1. Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model.

    Science.gov (United States)

    Sánchez, M C; Marín, M J; Figuero, E; Llama-Palacios, A; León, R; Blanc, V; Herrera, D; Sanz, M

    2014-02-01

    Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 μm). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10 . P. gingivalis showed a vitality reduction in the biofilm of 3 log10 , while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction. These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. New intracellular activities of matrix metalloproteinases shine in the moonlight.

    Science.gov (United States)

    Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

    2017-11-01

    Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. T3SS effector VopL inhibits the host ROS response, promoting the intracellular survival of Vibrio parahaemolyticus.

    Science.gov (United States)

    de Souza Santos, Marcela; Salomon, Dor; Orth, Kim

    2017-06-01

    The production of antimicrobial reactive oxygen species by the nicotinamide dinucleotide phosphate (NADPH) oxidase complex is an important mechanism for control of invading pathogens. Herein, we show that the gastrointestinal pathogen Vibrio parahaemolyticus counteracts reactive oxygen species (ROS) production using the Type III Secretion System 2 (T3SS2) effector VopL. In the absence of VopL, intracellular V. parahaemolyticus undergoes ROS-dependent filamentation, with concurrent limited growth. During infection, VopL assembles actin into non-functional filaments resulting in a dysfunctional actin cytoskeleton that can no longer mediate the assembly of the NADPH oxidase at the cell membrane, thereby limiting ROS production. This is the first example of how a T3SS2 effector contributes to the intracellular survival of V. parahaemolyticus, supporting the establishment of a protective intracellular replicative niche.

  4. T3SS effector VopL inhibits the host ROS response, promoting the intracellular survival of Vibrio parahaemolyticus.

    Directory of Open Access Journals (Sweden)

    Marcela de Souza Santos

    2017-06-01

    Full Text Available The production of antimicrobial reactive oxygen species by the nicotinamide dinucleotide phosphate (NADPH oxidase complex is an important mechanism for control of invading pathogens. Herein, we show that the gastrointestinal pathogen Vibrio parahaemolyticus counteracts reactive oxygen species (ROS production using the Type III Secretion System 2 (T3SS2 effector VopL. In the absence of VopL, intracellular V. parahaemolyticus undergoes ROS-dependent filamentation, with concurrent limited growth. During infection, VopL assembles actin into non-functional filaments resulting in a dysfunctional actin cytoskeleton that can no longer mediate the assembly of the NADPH oxidase at the cell membrane, thereby limiting ROS production. This is the first example of how a T3SS2 effector contributes to the intracellular survival of V. parahaemolyticus, supporting the establishment of a protective intracellular replicative niche.

  5. High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

    Directory of Open Access Journals (Sweden)

    Gilbert Greub

    Full Text Available BACKGROUND: With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. METHODS/PRINCIPAL FINDINGS: We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. CONCLUSIONS/SIGNIFICANCE: This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

  6. Chlamydia trachomatis co-opts GBF1 and CERT to acquire host sphingomyelin for distinct roles during intracellular development.

    Directory of Open Access Journals (Sweden)

    Cherilyn A Elwell

    2011-09-01

    Full Text Available The strain designated Chlamydia trachomatis serovar that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis. [corrected]. The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM, a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM, for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic

  7. Viral evasion of intracellular DNA and RNA sensing

    Science.gov (United States)

    Chan, Ying Kai; Gack, Michaela U.

    2016-01-01

    The co-evolution of viruses with their hosts has led to the emergence of viral pathogens that are adept at evading or actively suppressing host immunity. Pattern recognition receptors (PRRs) are key components of antiviral immunity that detect conserved molecular features of viral pathogens and initiate signalling that results in the expression of antiviral genes. In this Review, we discuss the strategies that viruses use to escape immune surveillance by key intracellular sensors of viral RNA or DNA, with a focus on RIG-I-like receptors (RLRs), cyclic GMP–AMP synthase (cGAS) and interferon-γ (IFNγ)-inducible protein 16 (IFI16). Such viral strategies include the sequestration or modification of viral nucleic acids, interference with specific post-translational modifications of PRRs or their adaptor proteins, the degradation or cleavage of PRRs or their adaptors, and the sequestration or relocalization of PRRs. An understanding of viral immune-evasion mechanisms at the molecular level may guide the development of vaccines and antivirals. PMID:27174148

  8. Viral evasion of intracellular DNA and RNA sensing.

    Science.gov (United States)

    Chan, Ying Kai; Gack, Michaela U

    2016-06-01

    The co-evolution of viruses with their hosts has led to the emergence of viral pathogens that are adept at evading or actively suppressing host immunity. Pattern recognition receptors (PRRs) are key components of antiviral immunity that detect conserved molecular features of viral pathogens and initiate signalling that results in the expression of antiviral genes. In this Review, we discuss the strategies that viruses use to escape immune surveillance by key intracellular sensors of viral RNA or DNA, with a focus on RIG-I-like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) and interferon-γ (IFNγ)-inducible protein 16 (IFI16). Such viral strategies include the sequestration or modification of viral nucleic acids, interference with specific post-translational modifications of PRRs or their adaptor proteins, the degradation or cleavage of PRRs or their adaptors, and the sequestration or relocalization of PRRs. An understanding of viral immune-evasion mechanisms at the molecular level may guide the development of vaccines and antivirals.

  9. Intracellular transport: from physics to ... biology.

    Science.gov (United States)

    Roux, Aurélien; Cuvelier, Damien; Bassereau, Patricia; Goud, Bruno

    2008-03-01

    Considerable effort over the past three decades has allowed the identification of the protein families that control the cellular machinery responsible for intracellular transport within eukaryotic cells. These proteins are estimated to represent about 10-20% of the human "proteome." The complexity of intracellular transport makes useful the development of model membranes. We describe here experimental systems based on lipid giant unilamellar vesicles (GUVs), which are attached to kinesin molecules. These systems give rise to thin membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. This type of assay, which mimics key events occurring during intracellular transport, allows physicists and biologists to understand how the unique mechanical properties of lipid membranes could be involved in the budding process, the sorting of cargo proteins and lipids, and the separation of the buds from a donor membrane.

  10. Micro- and nanotechnologies for intracellular delivery.

    Science.gov (United States)

    Yan, Li; Zhang, Jinfeng; Lee, Chun-Sing; Chen, Xianfeng

    2014-11-01

    The majority of drugs and biomolecules need to be delivered into cells to be effective. However, the cell membranes, a biological barrier, strictly resist drugs or biomolecules entering cells, resulting in significantly reduced intracellular delivery efficiency. To overcome this barrier, a variety of intracellular delivery approaches including chemical and physical ways have been developed in recent years. In this review, the focus is on summarizing the nanomaterial routes involved in making use of a collection of receptors for the targeted delivery of drugs and biomolecules and the physical ways of applying micro- and nanotechnologies for high-throughput intracellular delivery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Fluorescent nanothermometers for intracellular thermal sensing.

    Science.gov (United States)

    Jaque, Daniel; Rosal, Blanca Del; Rodríguez, Emma Martín; Maestro, Laura Martínez; Haro-González, Patricia; Solé, José García

    2014-05-01

    The importance of high-resolution intracellular thermal sensing and imaging in the field of modern biomedicine has boosted the development of novel nanosized fluorescent systems (fluorescent nanothermometers) as the next generation of probes for intracellular thermal sensing and imaging. This thermal mapping requires fluorescent nanothermometers with good biocompatibility and high thermal sensitivity in order to obtain submicrometric and subdegree spatial and thermal resolutions, respectively. This review describes the different nanosized systems used up to now for intracellular thermal sensing and imaging. We also include the later advances in molecular systems based on fluorescent proteins for thermal mapping. A critical overview of the state of the art and the future perspective is also included.

  12. Activation of NADPH oxidase is essential, but not sufficient, in controlling intracellular multiplication of Burkholderia pseudomallei in primary human monocytes.

    Science.gov (United States)

    Wikraiphat, Chanthiwa; Pudla, Matsayapan; Baral, Pankaj; Kitthawee, Sangvorn; Utaisincharoen, Pongsak

    2014-06-01

    Burkholderia pseudomallei is a Gram-negative intracellular bacterium and the causative agent of melioidosis. Innate immune mechanisms against this pathogen, which might contribute to outcomes of melioidosis, are little known. We demonstrated here that B. pseudomallei could activate NADPH oxidase in primary human monocytes as judged by production of reactive oxygen species (ROS) and p40(phox) phosphorylation after infection. However, as similar to other intracellular bacteria, this bacterium was able to resist and multiply inside monocytes despite being able to activate NADPH oxidase. In the presence of NADPH oxidase inhibitor, diphenyleneiodonium or apocynin, intracellular multiplication of B. pseudomallei was significantly increased, suggesting that NADPH oxidase-mediated ROS production is essential in suppressing intracellular multiplication of B. pseudomallei. Additionally, interferon-γ (IFN-γ)-mediated intracellular killing of B. pseudomallei requires NADPH oxidase activity, even though ROS level was not detected at higher levels in IFN-γ-treated infected monocytes. Altogether, these results imply that the activation of NADPH plays an essential role in suppressing intracellular multiplication of B. pseudomallei in human monocytes, although this enzyme is not sufficient to stop intracellular multiplication. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  13. Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria.

    Science.gov (United States)

    Ashida, Hiroshi; Sasakawa, Chihiro

    2015-01-01

    Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections.

  14. The Eurasian otter (Lutra lutra as a potential host for rickettsial pathogens in southern Italy.

    Directory of Open Access Journals (Sweden)

    Mario Santoro

    Full Text Available Canine monocytic ehrlichiosis and rickettsiosis are zoonotic tick-borne diseases of canids caused by the intracellular obligate bacteria Ehrlichia canis and Rickettsia species respectively. In this study, we investigated using standard and real-time PCR and sequencing, the occurrence and molecular characterization of E. canis and Rickettsia species in the Eurasian otter (Lutra lutra from the southern Italian population. Samples were screened by using molecular assays also for Neospora caninum, Toxoplasma gondii, Clamydophyla spp., Coxiella burnetii, Leishmania spp., Cryptosporidium spp., and Giardia spp. detection, and helminths were studied by traditional methods. Out of six carcasses tested, three were positive for E. canis and co-infection with Rickettsia sp. occurred in one of those. Sequences of the 16S rRNA E. canis gene were identical to each other but differed from most of those previously found in red foxes (Vulpes vulpes and wolves (Canis lupus from southern Italy. Helminths included just cystacanths of Sphaerirostris spp. from the intestine of two Eurasian otters and the nematode Angiostrongylus vasorum from the lungs of a single Eurasian otter. None of the samples was positive for the other investigated selected pathogens. This study is the first report on the evidence of infection by rickettsial pathogens in the Eurasian otter. The present result prompts some inquiries into the pathogenic role of those bacteria for the isolated sub-populations of the endangered Eurasian otter in southern Italy.

  15. Shigella IpaH family effectors as a versatile model for studying pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Hiroshi eAshida

    2016-01-01

    Full Text Available Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis. Via the type III secretion system (T3SS, Shigella deliver a subset of virulence proteins (effectors that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC. Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections.

  16. The making of a new pathogen

    DEFF Research Database (Denmark)

    Stukenbrock, Eva; Bataillon, Thomas; Dutheil, Julien

    2011-01-01

    The fungus Mycosphaerella graminicola emerged as a new pathogen of cultivated wheat during its domestication ~11,000 yr ago. We assembled 12 high-quality full genome sequences to investigate the genetic footprints of selection in this wheat pathogen and closely related sister species that infect...... that gene-rich regions or regions with low recombination experience stronger effects of natural selection on neutral diversity. Emergence of a new agricultural host selected a highly specialized and fast-evolving pathogen with unique evolutionary patterns compared with its wild relatives. The strong impact....... Recent divergence between pathogen sister species is attested by the high degree of incomplete lineage sorting (ILS) in their genomes. We exploit ILS to generate a genetic map of the species without any crossing data, document recent times of species divergence relative to genome divergence, and show...

  17. Role of intracellular infections in premature childbirth.

    Science.gov (United States)

    Zurabishvili, S; Mamamtavrishvili, I; Apridonidze, K; Shanidze, L

    2005-09-01

    Vaginal Smear taken by sterile Folkman spoon from 15 women with premature birth was studied. The study was performed by the direct immune fluorescence method with the luminescence microscope. We aimed to study the effect of intracellular infections: ureaplasma urealitikum, mycoplasma hominis, Chlamydia trachomatis, herpes simplex virus of I and II type and cytomegalovirus. Intracellular infections were detected in at about 82% of cases, which included mono infections with cytomegalovirus and in 9 cases in the form of bi-component associations. The obtained results may be interesting from the etiologic point of view of premature births in Georgian population.

  18. Reactive oxygen species drive evolution of pro-biofilm variants in pathogens by modulating cyclic-di-GMP levels.

    Science.gov (United States)

    Chua, Song Lin; Ding, Yichen; Liu, Yang; Cai, Zhao; Zhou, Jianuan; Swarup, Sanjay; Drautz-Moses, Daniela I; Schuster, Stephan Christoph; Kjelleberg, Staffan; Givskov, Michael; Yang, Liang

    2016-11-01

    The host immune system offers a hostile environment with antimicrobials and reactive oxygen species (ROS) that are detrimental to bacterial pathogens, forcing them to adapt and evolve for survival. However, the contribution of oxidative stress to pathogen evolution remains elusive. Using an experimental evolution strategy, we show that exposure of the opportunistic pathogen Pseudomonas aeruginosa to sub-lethal hydrogen peroxide (H 2 O 2 ) levels over 120 generations led to the emergence of pro-biofilm rough small colony variants (RSCVs), which could be abrogated by l-glutathione antioxidants. Comparative genomic analysis of the RSCVs revealed that mutations in the wspF gene, which encodes for a repressor of WspR diguanylate cyclase (DGC), were responsible for increased intracellular cyclic-di-GMP content and production of Psl exopolysaccharide. Psl provides the first line of defence against ROS and macrophages, ensuring the survival fitness of RSCVs over wild-type P. aeruginosa Our study demonstrated that ROS is an essential driving force for the selection of pro-biofilm forming pathogenic variants. Understanding the fundamental mechanism of these genotypic and phenotypic adaptations will improve treatment strategies for combating chronic infections. © 2016 The Authors.

  19. Contrasting evolutionary histories of MHC class I and class II loci in grouse—Effects of selection and gene conversion

    Science.gov (United States)

    Minias, Piotr; Bateson, Zachary W.; Whittingham, Linda A.; Johnson, Jeff A.; Oyler-McCance, Sara J.; Dunn, Peter O.

    2016-01-01

    Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and II MHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens.

  20. Intracellular disposition of chitosan nanoparticles in macrophages: intracellular uptake, exocytosis, and intercellular transport

    Directory of Open Access Journals (Sweden)

    Jiang LQ

    2017-08-01

    Full Text Available Li Qun Jiang,1 Ting Yu Wang,1 Thomas J Webster,2 Hua-Jian Duan,1 Jing Ying Qiu,1 Zi Ming Zhao,1 Xiao Xing Yin,1,* Chun Li Zheng3,* 1Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, People’s Republic of China; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3School of Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Biodegradable nanomaterials have been widely used in numerous medical fields. To further improve such efforts, this study focused on the intracellular disposition of chitosan nanoparticles (CsNPs in macrophages, a primary cell of the mononuclear phagocyte system (MPS. Such interactions with the MPS determine the nanoparticle retention time in the body and consequently play a significant role in their own clinical safety. In this study, various dye-labeled CsNPs (about 250 nm were prepared, and a murine macrophage cell line (RAW 264.7 was selected as a model macrophage. The results showed two mechanisms of macrophage incorporation of CsNPs, ie, a clathrin-mediated endocytosis pathway (the primary and phagocytosis. Following internalization, the particles partly dissociated in the cells, indicating cellular digestion of the nanoparticles. It was proved that, after intracellular uptake, a large proportion of CsNPs were exocytosed within 24 h; this excretion induced a decrease in fluorescence intensity in cells by 69%, with the remaining particles possessing difficulty being cleared. Exocytosis could be inhibited by both wortmannin and vacuolin-1, indicating that CsNP uptake was mediated by lysosomal and multivesicular body pathways, and after exocytosis, the reuptake of CsNPs by neighboring cells was verified by further experiments. This study, thus, elucidated the fate of CsNPs in macrophages as well as identified cellular disposition

  1. Hepatitis C virus intracellular host interactions

    NARCIS (Netherlands)

    Liefhebber, Johanna Maaike Pieternella

    2010-01-01

    Hepatitis C virus (HCV) infects about 170 million people worldwide causing a major healthcare problem. The virus lifecycle is greatly dependent on the host-cell for effective replication. In this thesis, the intracellular interactions of the non-structural HCV proteins with the host-cell were

  2. Enhanced production of intracellular dextran dextrinase from ...

    African Journals Online (AJOL)

    Enhanced production of intracellular dextran dextrinase from Gluconobacter oxydans using statistical experimental methods. ... the Plackett-Burman screening. A four-factor five-level central composite design (CCD) was chosen to explain the combined effects of the four medium constituents. The optimum medium consisted ...

  3. Biological synthesis and characterization of intracellular gold ...

    Indian Academy of Sciences (India)

    ... nontoxic, safe, biocompatible and environmentally acceptable. In the present study, Aspergillus fumigatus was used for the intracellular synthesis of gold nanoparticles. Stable nanoparticles were produced when an aqueous solution of chloroauric acid (HAuCl4) was reduced by A. fumigatus biomass as the reducing agent ...

  4. Efficient intracellular delivery of native proteins

    NARCIS (Netherlands)

    D'Astolfo, Diego S; Pagliero, Romina J; Pras, Anita; Karthaus, Wouter R; Clevers, Hans; Prasad, Vikram; Lebbink, Robert Jan; Rehmann, Holger; Geijsen, Niels

    2015-01-01

    Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced

  5. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... [Ganguli P, Chowdhury S, Bhowmick R and Sarkar RR 2015 Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: A ... cells and tissues by studying different signalling pathways, such as Hedgehog ...... Murray JD 2003 On the mechanochemical theory of biological.

  6. Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

    Science.gov (United States)

    Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun

    2018-03-20

    Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane

  7. Public health implications of Acanthamoeba and multiple potential opportunistic pathogens in roof-harvested rainwater tanks.

    Science.gov (United States)

    Hamilton, K A; Ahmed, W; Palmer, A; Sidhu, J P S; Hodgers, L; Toze, S; Haas, C N

    2016-10-01

    A study of six potential opportunistic pathogens (Acanthamoeba spp., Legionella spp., Legionella longbeachae, Pseudomonas aeruginosa, Mycobacterium avium and Mycobacterium intracellulare) and an accidental human pathogen (Legionella pneumophila) in 134 roof-harvested rainwater (RHRW) tank samples was conducted using quantitative PCR (qPCR). All five opportunistic pathogens and accidental pathogen L. pneumophila were detected in rainwater tanks except Legionella longbeachae. Concentrations ranged up to 3.1×10(6) gene copies per L rainwater for Legionella spp., 9.6×10(5) gene copies per L for P. aeruginosa, 6.8×10(5) gene copies per L for M. intracellulare, 6.6×10(5) gene copies per L for Acanthamoeba spp., 1.1×10(5) gene copies per L for M. avium, and 9.8×10(3) gene copies per L for L. pneumophila. Among the organisms tested, Legionella spp. (99% tanks) were the most prevalent followed by M. intracellulare (78%). A survey of tank-owners provided data on rainwater end-uses. Fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp. were enumerated using culture-based methods, and assessed for correlations with opportunistic pathogens and L. pneumophila tested in this study. Opportunistic pathogens did not correlate well with FIB except E. coli vs. Legionella spp. (tau=0.151, P=0.009) and E. coli vs. M. intracellulare (tau=0.14, P=0.015). However, M. avium weakly correlated with both L. pneumophila (Kendall's tau=0.017, P=0.006) and M. intracellulare (tau=0.088, P=0.027), and Legionella spp. also weakly correlated with M. intracellulare (tau=0.128, P=0.028). The presence of these potential opportunistic pathogens in tank water may present health risks from both the potable and non-potable uses documented from the current survey data. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  8. Burkholderia pseudomallei modulates host iron homeostasis to facilitate iron availability and intracellular survival.

    Directory of Open Access Journals (Sweden)

    Imke H E Schmidt

    2018-01-01

    Full Text Available The control over iron homeostasis is critical in host-pathogen-interaction. Iron plays not only multiple roles for bacterial growth and pathogenicity, but also for modulation of innate immune responses. Hepcidin is a key regulator of host iron metabolism triggering degradation of the iron exporter ferroportin. Although iron overload in humans is known to increase susceptibility to Burkholderia pseudomallei, it is unclear how the pathogen competes with the host for the metal during infection. This study aimed to investigate whether B. pseudomallei, the causative agent of melioidosis, modulates iron balance and how regulation of host cell iron content affects intracellular bacterial proliferation.Upon infection of primary macrophages with B. pseudomallei, expression of ferroportin was downregulated resulting in higher iron availability within macrophages. Exogenous modification of iron export function by hepcidin or iron supplementation by ferric ammonium citrate led to increased intracellular iron pool stimulating B. pseudomallei growth, whereas the iron chelator deferoxamine reduced bacterial survival. Iron-loaded macrophages exhibited a lower expression of NADPH oxidase, iNOS, lipocalin 2, cytokines and activation of caspase-1. Infection of mice with the pathogen caused a diminished hepatic ferroportin expression, higher iron retention in the liver and lower iron levels in the serum (hypoferremia. In vivo administration of ferric ammonium citrate tended to promote the bacterial growth and inflammatory response, whereas limitation of iron availability significantly ameliorated bacterial clearance, attenuated serum cytokine levels and improved survival of infected mice.Our data indicate that modulation of the cellular iron balance is likely to be a strategy of B. pseudomallei to improve iron acquisition and to restrict antibacterial immune effector mechanisms and thereby to promote its intracellular growth. Moreover, we provide evidence that

  9. Impact of non-Legionella bacteria on the uptake and intracellular replication of Legionella pneumophila in Acanthamoeba castellanii and Naegleria lovaniensis.

    Science.gov (United States)

    Declerck, P; Behets, J; Delaedt, Y; Margineanu, A; Lammertyn, E; Ollevier, F

    2005-11-01

    In aquatic environments, Legionella pneumophila survives, in association with other bacteria, within biofilms by multiplying in free-living amoebae. The precise mechanisms underlying several aspects of the uptake and intracellular replication of L. pneumophila in amoebae, especially in the presence of other bacteria, remain unknown. In the present study, we examined the competitive effect of selected non-Legionella bacteria (Escherichia coli, Aeromonas hydrophila, Flavobacterium breve, and Pseudomonas aeruginosa) on the uptake of L. pneumophila serogroup 1 by the amoebae Acanthamoeba castellanii and Naegleria lovaniensis. We also investigated their possible influence on the intracellular replication of L. pneumophila in both amoeba species. Our results showed that the non-Legionella bacteria did not compete with L. pneumophila for uptake, suggesting that the amoeba hosts took in L. pneumophila through a specific and presumably highly efficient uptake mechanism. Living and heat-inactivated P. aeruginosa best supported the replication of L. pneumophila in N. lovaniensis and A. castellanii, respectively, whereas for both amoeba species, E. coli yielded the lowest number of replicated L. pneumophila. Furthermore, microscopic examination showed that 100% of the A. castellanii and only 2% of the N. lovaniensis population were infected with L. pneumophila at the end of the experiment. This study clearly shows the influence of some non-Legionella bacteria on the intracellular replication of L. pneumophila in A. castellanii and N. lovaniensis. It also demonstrates the different abilities of the two tested amoeba species to serve as a proper host for the replication and distribution of the human pathogen in man-made aquatic environments such as cooling towers, shower heads, and air conditioning systems with potential serious consequences for human health.

  10. COXIELLA BURNETII PATHOGENICITY MOLECULAR BASIS

    Directory of Open Access Journals (Sweden)

    Yu. A. Panferova

    2016-01-01

    Full Text Available Coxiella burnetii is an obligate intracellular gram-negative bacterial pathogen, an ethiological agent of Q-fever, a zoonotic disease, elapsing as an acute (mostly atypical pneumonia or a chronic (mostly endocarditis form. The host range is represented by wide range of mammal, avian and arthropod species, but the main source of human infection are farm animals. The main route of infection is aerosolic. In case of contact with organism pathogen binds with phagocytal monocytic-macrophagal cell line. C. burnetii promotes maturation of specific phagolysosome-like compartment in host cell, called coxiella-containing vacuole, within this vacuole pathogen becames metabolically activated and actively replicates. Coxiella persists as metabolically inactive spore-like form in environment. Internalisation of C. burnetii occurs using actin-mediated phagocytosis and zipper mechanism. After internalization of bacteria maturation of phagolysosome-like compartment and large coxiella-containing vacuole formation occure, and vacuole can occupy nearly the whole cytoplasm of the host cell. Survivance of infected cells is important for chronic infection with C. burnetii. C. burnetii elongate the viability of host cell by two ways: it actively inhibits apoptotic signal cascades and induce pro-survival factors. Exceptthat C. burnetii involves autophagic pathway during coxiella-containing vacuole formation, and induction of autophagy promotes pathogen replication. During infection C. burnetii translocates effector substrates from bacterial cytosole to euca ryotic host cell cytosole using type IV secretion system, where effectors modulate host cell proteins. Overall approximately 130 secreted effectors of type IV transport system, but function of most of them remains unknown to date. Specific sec reted proteins for variety of strains and isolates were identified, confirmed that certain pathotypes of C. burnetii can exist. Identification and

  11. Immunogenetic variation and differential pathogen exposure in free-ranging cheetahs across Namibian farmlands.

    Directory of Open Access Journals (Sweden)

    Aines Castro-Prieto

    Full Text Available Genes under selection provide ecologically important information useful for conservation issues. Major histocompatibility complex (MHC class I and II genes are essential for the immune defence against pathogens from intracellular (e.g. viruses and extracellular (e.g. helminths origins, respectively. Serosurvey studies in Namibian cheetahs (Acinonyx juabuts revealed higher exposure to viral pathogens in individuals from north-central than east-central regions. Here we examined whether the observed differences in exposure to viruses influence the patterns of genetic variation and differentiation at MHC loci in 88 free-ranging Namibian cheetahs.Genetic variation at MHC I and II loci was assessed through single-stranded conformation polymorphism (SSCP analysis and sequencing. While the overall allelic diversity did not differ, we observed a high genetic differentiation at MHC class I loci between cheetahs from north-central and east-central Namibia. No such differentiation in MHC class II and neutral markers were found.Our results suggest that MHC class I variation mirrors the variation in selection pressure imposed by viruses in free-ranging cheetahs across Namibian farmland. This is of high significance for future management and conservation programs of this species.

  12. A rhodanine agent active against non-replicating intracellular Mycobacterium avium subspecies paratuberculosis

    Directory of Open Access Journals (Sweden)

    Bull Tim J

    2009-12-01

    Full Text Available Abstract Background Antibiotic therapy targeting chronic mycobacterial disease is often ineffective due to problems with the emergence of drug resistance and non-replicating persistent intracellular antibiotic resistant phenotypes. Strategies which include agents able to enhance host cell killing mechanisms could represent an alternative to conventional methods with the potential for host clearance if active against dormant phenotypes. Investigations of agents with potential activity against non-replicating mycobacteria however are restricted due to a need for assays that can assess bacterial viability without having to culture. Results This study describes the development and use of a pre16S ribosomal gene RNA/DNA ratio viability assay which is independent of the need for culture, supported by a novel thin layer accelerated mycobacterial colony forming method for determining viability and culturability of MAP in intracellular environments. We describe the use of these tools to demonstrate intracellular killing activity of a novel rhodanine agent (D157070 against the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (MAP and show that the culturability of MAP decreases relative to its viability on intracellular entry suggesting the induction of a non-culturable phenotype. We further demonstrate that D157070, although having no direct activity against the culturability of extracellular MAP, can bind to cultured MAP cells and has significant influence on the MAP transcriptome, particularly with respect of δL associated genes. D157070 is shown to be taken up by bovine and human cells and able to enhance host cell killing, as measured by significant decreases in both culturability and viability of intracellular MAP. Conclusions This work suggests that pre16srRNA gene ratios represent a viable method for studying MAP viability. In addition, the rhodanine agent D157070 tested is non-toxic and enhances cell killing activity

  13. Lymphocyte apoptosis as an immune subversion strategy of microbial pathogens.

    Science.gov (United States)

    Carrero, Javier A; Unanue, Emil R

    2006-11-01

    Apoptosis is a component of cellular death in several immunological reactions. Lymphocyte apoptosis is a feature of negative selection of thymic lymphocytes. Target cells die by apoptosis during their interaction with cytotoxic T cells. Antigens derived from apoptotic cells can be cross-presented by antigen presenting cells (APCs). In these examples, apoptotic death is a beneficial feature for the individual. The apoptosis of cells also occurs during infection with a variety of microorganisms, but this process can be detrimental to the handling of the infection by the host. Here, we aim to highlight some of the recent advances in understanding why apoptosis can be a detrimental event during infection. We will focus on recent research with the intracellular bacterial pathogen Listeria monocytogenes, which demonstrates how apoptosis is induced, some of the host pathways that are exploited and the immunological consequences of cell death. We propose that L. monocytogenes causes lymphocyte death by enhancing the cell-death programs of the host. The presence of apoptotic lymphocytes downregulates early innate immunity, creating a permissive environment for bacterial growth.

  14. Evaluation of mericon E. coli O157 Screen Plus and mericon E. coli STEC O-Type Pathogen Detection Assays in Select Foods: Collaborative Study, First Action 2017.05.

    Science.gov (United States)

    Bird, Patrick; Benzinger, M Joseph; Bastin, Benjamin; Crowley, Erin; Agin, James; Goins, David; Armstrong, Marcia

    2017-10-18

    QIAGEN mericon Escherichia coli O157 Screen Plus and mericon E. coli Shiga toxin-producing E. coli (STEC) O-Type Pathogen Detection Assays use Real-Time PCR technology for the rapid, accurate detection of E. coli O157 and the “big six” (O26, O45, O103, O111, O121, O145) (non-O157 STEC) in select food types. Using a paired study design, the assays were compared with the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook Chapter 5.09 reference method for the detection of E. coli O157:H7 in raw ground beef. Both mericon assays were evaluated using the manual and an automated DNA extraction method. Thirteen technicians from five laboratories located within the continental United States participated in the collaborative study. Three levels of contamination were evaluated. Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low-inoculum level test portions produced a difference between laboratories POD (dLPOD) value with a 95% confidence interval of 0.00 (–0.12, 0.12) for the mericon E. coli O157 Screen Plus with manual and automated extraction and mericon E. coli STEC O-Type with manual extraction and –0.01 (–0.13, 0.10) for the mericon E. coli STEC O-Type with automated extraction. The dLPOD results indicate equivalence between the candidate methods and the reference method.

  15. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Science.gov (United States)

    Wang, Jinli; Yang, Kun; Zhou, Lin; Minhaowu; Wu, Yongjian; Zhu, Min; Lai, Xiaomin; Chen, Tao; Feng, Lianqiang; Li, Meiyu; Huang, Chunyu; Zhong, Qiu; Huang, Xi

    2013-01-01

    Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  16. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Directory of Open Access Journals (Sweden)

    Jinli Wang

    Full Text Available Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7 reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb, a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  17. Nanomechanical mechanism for lipid bilayer damage induced by carbon nanotubes confined in intracellular vesicles.

    Science.gov (United States)

    Zhu, Wenpeng; von dem Bussche, Annette; Yi, Xin; Qiu, Yang; Wang, Zhongying; Weston, Paula; Hurt, Robert H; Kane, Agnes B; Gao, Huajian

    2016-11-01

    Understanding the behavior of low-dimensional nanomaterials confined in intracellular vesicles has been limited by the resolution of bioimaging techniques and the complex nature of the problem. Recent studies report that long, stiff carbon nanotubes are more cytotoxic than flexible varieties, but the mechanistic link between stiffness and cytotoxicity is not understood. Here we combine analytical modeling, molecular dynamics simulations, and in vitro intracellular imaging methods to reveal 1D carbon nanotube behavior within intracellular vesicles. We show that stiff nanotubes beyond a critical length are compressed by lysosomal membranes causing persistent tip contact with the inner membrane leaflet, leading to lipid extraction, lysosomal permeabilization, release of cathepsin B (a lysosomal protease) into the cytoplasm, and cell death. The precise material parameters needed to activate this unique mechanical pathway of nanomaterials interaction with intracellular vesicles were identified through coupled modeling, simulation, and experimental studies on carbon nanomaterials with wide variation in size, shape, and stiffness, leading to a generalized classification diagram for 1D nanocarbons that distinguishes pathogenic from biocompatible varieties based on a nanomechanical buckling criterion. For a wide variety of other 1D material classes (metal, oxide, polymer), this generalized classification diagram shows a critical threshold in length/width space that represents a transition from biologically soft to stiff, and thus identifies the important subset of all 1D materials with the potential to induce lysosomal permeability by the nanomechanical mechanism under investigation.

  18. A new type of intracellular retention signal identified in a pestivirus structural glycoprotein.

    Science.gov (United States)

    Burrack, Sandra; Aberle, Daniel; Bürck, Jochen; Ulrich, Anne S; Meyers, Gregor

    2012-08-01

    Sorting of membrane proteins into intracellular organelles is crucial for cell function. Viruses exploit intracellular transport and retention systems to concentrate envelope proteins at the site of virus budding. In pestiviruses, a group of important pathogens of pigs and ruminants closely related to human hepatitis C virus, the E(rns) protein translated from the viral RNA is secreted from the infected cells and found in the serum of infected animals. Secretion of the protein is regarded as crucial for its function as a viral virulence factor associated with its RNase activity. However, ∼95% of the E(rns) molecules are retained within the infected cell. Fusion of different E(rns) fragments to the C terminus of CD72 allowed identification of a retention signal within the C-terminal 65 aa of the viral protein. This C-terminal sequence represents its membrane anchor and folds into an amphipathic helix binding in-plane to the membrane surface. Residues L183, I190, and L208 are important for intracellular location of E(rns). Presentation of the retention signal on the cytoplasmic instead of the luminal face of the ER membrane in CD8α fusion proteins still led to retention. Thus, E(rns) contains in its C-terminal amphipathic helix an intracellular retention signal that is active on both faces of the membrane.

  19. Influence of intracellular Na + , K + and Cl - on the salt tolerance in ...

    African Journals Online (AJOL)

    In the process of selection for salt tolerance, it is important to understand the physiological basis of ion management executed by the cells through the exclusion, accumulation or maintenance of ratios of specific ions. Intracellular accumulation of Na+, K+ and Cl- ions in the cells in vitro was studied as a factor in salt ...

  20. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-01-01

    The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or γ-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (author)

  1. Intracellular mechanisms of solar water disinfection

    Science.gov (United States)

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  2. Reduction of intracellular glutathione content and radiosensitivity

    International Nuclear Information System (INIS)

    Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

    1986-05-01

    The intracellular glutathione (GSH) content in HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulfoximine (BSO) or diethyl maleate (DEM). Clonogenicity, single strand DNA breaks (ssb) and double strand DNA breaks (dsb) were used as criteria for radiation induced damage after X- or γ irradiation. In survival experiments DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the OER was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (Auth.)

  3. Intracellular Protein Delivery for Treating Breast Cancer

    Science.gov (United States)

    2014-08-01

    Intracellular delivery of such proteins, including human tumor suppressors (such as p53) (Brown et al., 2009) and exogenous tumor-killing proteins...vivo systems. Nature materials 11, 1038-1043. Chorny, M., Hood, E., Levy, R.J., and Muzykantov, V.R. (2010). Endothelial delivery of antioxidant ...for the ntracellular delivery of such proteins, including human umor suppressors [7] and exogenous tumor-killing proteins 8—10]), is attractive as a

  4. The listeriosis triangle: Pathogen, host and the environment

    Directory of Open Access Journals (Sweden)

    Abram Maja

    2012-01-01

    Full Text Available Listeria monocytogenes is a foodborne pathogen well known for its adaptability to diverse environment and host niches and its high fatality rate among infected immunocompromised populations. Infection in the immunocompetent host occurs but risk factors for the disease primarily points to abnormalities in cell-mediated and innate immunity as major predispositions to listeriosis. After ingestion of contaminated food, this pathogen is able to cross the intestinal, blood-brain and placental barrier and leads to gastroenteritis, meningitis and maternofetal infections which may result in abortion and spontaneous stillbirth. Despite the extensive use of this bacterium in the study of cell-mediated immunity and intracellular growth, our understanding of the host, pathogen and environmental factors that impact the pathogenesis of listeriosis is still incomplete. This review will summarize current knowledge, including our own efforts, about pathogen, host and environmental factors that influence, and contribute to the pathogenesis of Listeria monocytogenes infection.

  5. The listeriosis triangle: Pathogen, host and the environment

    Directory of Open Access Journals (Sweden)

    Abram Maja

    2012-03-01

    Full Text Available Listeria monocytogenes is a foodborne pathogen well known for its adaptability to diverse environment and host niches and its high fatality rate among infected immunocompromised populations. Infection in the immunocompetent host occurs but risk factors for the disease primarily points to abnormalities in cell-mediated and innate immunity as major predispositions to listeriosis. After ingestion of contaminated food, this pathogen is able to cross the intestinal, blood-brain and placental barrier and leads to gastroenteritis, meningitis and maternofetal infections which may result in abortion and spontaneous stillbirth. Despite the extensive use of this bacterium in the study of cell-mediated immunity and intracellular growth, our understanding of the host, pathogen and environmental factors that impact the pathogenesis of listeriosis is still incomplete. This review will summarize current knowledge, including our own efforts, about pathogen, host and environmental factors that influence, and contribute to the pathogenesis of Listeria monocytogenes infection.

  6. Accelerated microevolution in an outer membrane protein (OMP of the intracellular bacteria Wolbachia

    Directory of Open Access Journals (Sweden)

    Russell Jacob A

    2010-02-01

    Full Text Available Abstract Background Outer membrane proteins (OMPs of Gram-negative bacteria are key players in the biology of bacterial-host interactions. However, while considerable attention has been given to OMPs of vertebrate pathogens, relatively little is known about the role of these proteins in bacteria that primarily infect invertebrates. One such OMP is found in the intracellular bacteria Wolbachia, which are widespread symbionts of arthropods and filarial nematodes. Recent experimental studies have shown that the Wolbachia surface protein (WSP can trigger host immune responses and control cell death programming in humans, suggesting a key role of WSP for establishment and persistence of the symbiosis in arthropods. Results Here we performed an analysis of 515 unique alleles found in 831 Wolbachia isolates, to investigate WSP structure, microevolution and population genetics. WSP shows an eight-strand transmembrane β-barrel structure with four extracellular loops containing hypervariable regions (HVRs. A clustering approach based upon patterns of HVR haplotype diversity was used to group similar WSP sequences and to estimate the relative contribution of mutation and recombination during early stages of protein divergence. Results indicate that although point mutations generate most of the new protein haplotypes, recombination is a predominant force triggering diversity since the very first steps of protein evolution, causing at least 50% of the total amino acid variation observed in recently diverged proteins. Analysis of synonymous variants indicates that individual WSP protein types are subject to a very rapid turnover and that HVRs can accommodate a virtually unlimited repertoire of peptides. Overall distribution of WSP across hosts supports a non-random association of WSP with the host genus, although extensive horizontal transfer has occurred also in recent times. Conclusions In OMPs of vertebrate pathogens, large recombination impact, positive

  7. Fluorescent nanoparticles for intracellular sensing: A review

    International Nuclear Information System (INIS)

    Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.

    2012-01-01

    Highlights: ► Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. ► Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. ► Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  8. Fluorescent nanoparticles for intracellular sensing: A review

    Energy Technology Data Exchange (ETDEWEB)

    Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  9. A bacteriophage endolysin that eliminates intracellular streptococci.

    Science.gov (United States)

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-03-15

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB-PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities.

  10. Free-Living Amoebae as Hosts for and Vectors of Intracellular Microorganisms with Public Health Significance

    Science.gov (United States)

    Balczun, Carsten; Scheid, Patrick L.

    2017-01-01

    Free-living amoebae (FLA) are parasites within both humans and animals causing a wide range of symptoms and act as hosts of, and vehicles for phylogenetically diverse microorganisms, called endocytobionts. The interaction of the FLA with sympatric microorganisms leads to an exceptional diversity within FLA. Some of these bacteria, viruses, and even eukaryotes, can live and replicate intracellularly within the FLA. This relationship provides protection to the microorganisms from external interventions and a dispersal mechanism across various habitats. Among those intracellularly-replicating or -residing organisms there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals and are therefore of interest to Public Health Authorities. Mimiviruses, Pandoraviruses, and Pithoviruses are examples for interesting viral endocytobionts within FLA. Future research is expected to reveal further endocytobionts within free-living amoebae and other protozoa through co-cultivation studies, genomic, transcriptomic, and proteomic analyses. PMID:28368313

  11. Intracellular and in vivo oxygen sensing using phosphorescent iridium(III) complexes.

    Science.gov (United States)

    Tobita, Seiji; Yoshihara, Toshitada

    2016-08-01

    Molecular oxygen plays an indispensable role as a terminal electron acceptor in the electron transport chain in mitochondria. Acute or chronic oxygen deprivation (hypoxia) in organisms results in various diseases, and the elucidation of the pathogenic mechanism of hypoxia-related diseases and various cellular responses to hypoxia is an urgent issue. Optical oxygen imaging methods using phosphorescent probes have opened up techniques for noninvasive imaging of the intracellular and tissue oxygen status, and oxygen-sensitive probes play a key role in the development of this approach. We expect that phosphorescent Ir(III) complexes can serve as new oxygen-sensing probes for intracellular and intravascular oxygen imaging in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Measuring immune selection

    OpenAIRE

    Conway, DJ; Polley, SD

    2002-01-01

    Immune responses that kill pathogens or reduce their reproductive rate are generally important in protecting hosts from infection and disease. Pathogens that escape the full impact of such responses will survive, and any heritable genetic basis of this evasion will be selected. Due to the memory component of vertebrate immune responses, pathogens with rare alleles of a target antigen can have an advantage over those with common alleles, leading to the maintenance of a polymorphism. At the gen...

  13. Fluorescent real-time quantitative measurements of intracellular peroxynitrite generation and inhibition.

    Science.gov (United States)

    Luo, Zhen; Zhao, Qin; Liu, Jixiang; Liao, Jinfang; Peng, Ruogu; Xi, Yunting; Diwu, Zhenjun

    2017-03-01

    Peroxynitrite (ONOO - ), a strong oxidant species, is produced by the reaction of nitric oxide (NO) and superoxide (O 2 .- ) radicals. It plays an important role as a biological regulator in numbers of physiological and pathological processes. In this study, we developed fluorescence-based real-time quantitative measurements to detect intracellular ONOO - . The probe DAX-J2 PON Green showed high selectivity toward ONOO - over other competing species, and has been successfully applied in microplate reader and flow cytometer to quantitatively measure endogenous ONOO - production. Moreover, the results demonstrated the inhibitory effects of curcumin on intracellular ONOO - generation. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Use of magnetic nanobeads to study intracellular antigen processing

    Energy Technology Data Exchange (ETDEWEB)

    Perrin-Cocon, Laure A.; Chesne, Serge; Pignot-Paintrand, Isabelle; Marche, Patrice N.; Villiers, Christian L. E-mail: christian.villiers@cea.fr

    2001-07-01

    Magnetic nanobeads were covalently linked to antigens and used as a tool to simultaneously follow their intracellular transport into the cells and specifically purify the intracellular compartments implicated in antigen processing. The protein content of these vesicles was analysed by 2D-electrophoresis. Furthermore, nanobeads allowed intracellular localisation of the antigen in electron and fluorescence microscopy.

  15. Use of magnetic nanobeads to study intracellular antigen processing

    International Nuclear Information System (INIS)

    Perrin-Cocon, Laure A.; Chesne, Serge; Pignot-Paintrand, Isabelle; Marche, Patrice N.; Villiers, Christian L.

    2001-01-01

    Magnetic nanobeads were covalently linked to antigens and used as a tool to simultaneously follow their intracellular transport into the cells and specifically purify the intracellular compartments implicated in antigen processing. The protein content of these vesicles was analysed by 2D-electrophoresis. Furthermore, nanobeads allowed intracellular localisation of the antigen in electron and fluorescence microscopy

  16. Food-borne pathogens

    International Nuclear Information System (INIS)

    Niemand, J.G.

    1985-01-01

    The Salmonella scare reinforced the importance of never taking chances when it comes to controlling pathogens. The issue has been resolved by radurisation. The article deals with the various pathogens that can effect food and argues the case for radurisation in dealing with them. It also looks at some of the other food products that can be treated using this process

  17. Plant pathogen resistance

    Science.gov (United States)

    Greenberg, Jean T; Jung, Ho Won; Tschaplinski, Timothy

    2012-11-27

    Azelaic acid or its derivatives or analogs induce a robust and a speedier defense response against pathogens in plants. Azelaic acid treatment alone does not induce many of the known defense-related genes but activates a plant's defense signaling upon pathogen exposure.

  18. Potatoes, pathogens and pests

    NARCIS (Netherlands)

    Lazebnik, Jenny

    2017-01-01

    Currently, fungicides are necessary to protect potato crops against late blight, Phytophthora infestans, one of the world’s most damaging crop pathogens. The introgression of plant resistance genes from wild potato species targeted specifically to the late blight pathogen into

  19. Hypothesis: apoptosis caused by cytotoxins represents a defensive response that evolved to combat intracellular pathogens.

    Science.gov (United States)

    Vaux, D L; Häcker, G

    1995-11-01

    1. Over 100 different agents have been shown, under certain circumstances, to cause apoptosis, a form of cell death with characteristic morphology. In most cases, the mechanism of cell death is likely to be the same, as expression of the cell death inhibitory gene bcl-2 can frequently prevent apoptotic changes and/or delay cell death. 2. These observations raise the question of how and why cells detect these agents and why they respond by implementing the suicide mechanism that bcl-2 can control. Our hypothesis is that apoptosis is used as an anti-viral strategy, and that cells interpret any metabolic disturbance as evidence of infection by a virus and thereby kill themselves in response to these toxins before they are killed by the action of the toxin itself. 3. Experiments on the effect of sodium azide upon growth factor-dependent cells support this idea. Bcl-2 can delay cell death caused by azide, and inhibit apoptotic changes seen by electron microscopy, but cannot prevent the eventual death of the cells. 4. These ideas suggest that drugs designed to regulate cell death may be useful for the treatment of ischaemic or neoplastic diseases. For example, human cells may activate a suicide pathway in response to sub-lethal amounts of anoxia following a stroke or heart attack and so blocking apoptosis may be a useful therapy to limit tissue damage. On the other hand, increasing the propensity of cells to activate their physiological cell death mechanisms may enhance the effectiveness of toxins designed to kill tumour cells.

  20. Arylthiazole antibiotics targeting intracellular methicillin-resistant Staphylococcus aureus (MRSA) that interfere with bacterial cell wall synthesis.

    Science.gov (United States)

    Eid, Islam; Elsebaei, Mohamed M; Mohammad, Haroon; Hagras, Mohamed; Peters, Christine E; Hegazy, Youssef A; Cooper, Bruce; Pogliano, Joe; Pogliano, Kit; Abulkhair, Hamada S; Seleem, Mohamed N; Mayhoub, Abdelrahman S

    2017-10-20

    The promising antibacterial potency of arylthiazole antibiotics is offset by their limited activity against intracellular bacteria (namely methicillin-resistant Staphylococcus aureus (MRSA)), similar to many clinically-approved antibiotics. The failure to target these hidden pathogens is due to the compounds' lack of proper characteristics to accumulate intracellularly. Fine tuning of the size and polar-surface-area of the linking heteroaromatic ring provided a new series of 5-thiazolylarylthiazoles with balanced properties that allow them to sufficiently cross and accumulate inside macrophages infected with MRSA. The most promising compound 4i exhibited rapid bactericidal activity, good metabolic stability and produced over 80% reduction of intracellular MRSA in infected macrophages. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. The intracellular cholesterol landscape: dynamic integrator of the immune response

    Science.gov (United States)

    Fessler, Michael B.

    2016-01-01

    Cholesterol has typically been considered an exogenous, disease-related factor in immunity; however, recent literature suggests that a paradigm shift is in order. Sterols are now recognized to ligate several immune receptors. Altered flux through the mevalonic acid synthesis pathway also appears to be a required event in the antiviral interferon response of macrophages and in the activation, proliferation, and differentiation of T cells. In this review, evidence is discussed that suggests an intrinsic, ‘professional’ role for sterols and oxysterols in macrophage and T cell immunity. Host defense may have been the original selection pressure behind the development of mechanisms for intracellular cholesterol homeostasis. Functional coupling between sterol metabolism and immunity has fundamental implications for health and disease. PMID:27692616

  2. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    Science.gov (United States)

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-05-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (A549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment.

  3. Processes for managing pathogens.

    Science.gov (United States)

    Godfree, Alan; Farrell, Joseph

    2005-01-01

    Wastewater contains human, animal, and plant pathogens capable of causing viral, bacterial, or parasitic infections. There are several routes whereby sewage pathogens may affect human health, including direct contact, contamination of food crops, zoonoses, and vectors. The range and numbers of pathogens in municipal wastewater vary with the level of endemic disease in the community, discharges from commercial activities, and seasonal factors. Regulations to control pathogen risk in the United States and Europe arising from land application of biosolids are based on the concept of multiple barriers to the prevention of transmission. The barriers are (i) treatment to reduce pathogen content and vector attraction, (ii) restrictions on crops grown on land to which biosolids have been applied, and (iii) minimum intervals following application and grazing or harvesting. Wastewater treatment reduces number of pathogens in the wastewater by concentrating them with the solids in the sludge. Although some treatment processes are designed specifically to inactivate pathogens, many are not, and the actual mechanisms of microbial inactivation are not fully understood for all processes. Vector attraction is reduced by stabilization (reduction of readily biodegradable material) and/or incorporation immediately following application. Concerns about health risks have renewed interest in the effects of treatment (on pathogens) and advanced treatment methods, and work performed in the United States suggests that Class A pathogen reduction can be achieved less expensively than previously thought. Effective pathogen risk management requires control to the complete chain of sludge treatment, biosolids handling and application, and post-application activities. This may be achieved by adherence to quality management systems based on hazard analysis critical control point (HACCP) principles.

  4. Comparative genomics of transport proteins in probiotic and pathogenic Escherichia coli and Salmonella enterica strains.

    Science.gov (United States)

    Do, Jimmy; Zafar, Hassan; Saier, Milton H

    2017-06-01

    Escherichia coli is a genetically diverse species that can be pathogenic, probiotic, commensal, or a harmless laboratory strain. Pathogenic strains of E. coli cause urinary tract infections, diarrhea, hemorrhagic colitis, and pyelonephritis, while the two known probiotic E. coli strains combat inflammatory bowel disease and play a role in immunomodulation. Salmonella enterica, a close relative of E. coli, includes two important pathogenic serovars, Typhi and Typhimurium, causing typhoid fever and enterocolitis in humans, respectively, with the latter strain also causing a lethal typhoid fever-like disease in mice. In this study, we identify the transport systems and their substrates within seven E. coli strains: two probiotic strains, two extracellular pathogens, two intracellular pathogens, and K-12, as well as the two intracellular pathogenic S. enterica strains noted above. Transport systems characteristic of each probiotic or pathogenic species were thus identified, and the tabulated results obtained with all of these strains were compared. We found that the probiotic and pathogenic strains generally contain more iron-siderophore and sugar transporters than E. coli K-12. Pathogens have increased numbers of pore-forming toxins, protein secretion systems, decarboxylation-driven Na + exporters, electron flow-driven monovalent cation exporters, and putative transporters of unknown function compared to the probiotic strains. Both pathogens and probiotic strains encode metabolite transporters that reflect their intracellular versus extracellular environments. The results indicate that the probiotic strains live extracellularly. It seems that relatively few virulence factors can convert a beneficial or commensal microorganism into a pathogen. Taken together, the results reveal the distinguishing features of these strains and provide a starting point for future engineering of beneficial enteric bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity.

    Science.gov (United States)

    Chiaraviglio, Lucius; Kang, Yoon-Suk; Kirby, James E

    2016-11-16

    Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1 st generation assay) or fluorescent protein reporters (2 nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these

  6. Pathogen pollution and the emergence of a deadly amphibian pathogen.

    Science.gov (United States)

    McKenzie, Valerie J; Peterson, Anna C

    2012-11-01

    ). In a new study published in this issue of Molecular Ecology, Schloegel et al. (2012) identify an additional unique Bd lineage that is endemic to the Atlantic Brazilian rainforests (Bd-Brazil) and provide striking evidence that the Bd-Brazil lineage has sexually recombined with the BdGPL lineage in an area where the two lineages likely came into contact as a result of classic anthropogenically mediated 'pathogen pollution'(see below). Fungal pathogens, including Bd, have the propensity to form recombinant lineages when allopatric populations that have not yet formed genetic reproductive barriers are provided with opportunities to intermingle, and virulent strains may be selected for because they tend to be highly transmissible (Fisher et al. 2012). As Schloegel et al. (2012) point out, the demonstrated ability for Bd to undergo meiosis may also mean that it has the capacity to form a resistant spore stage (as yet undiscovered), based on extrapolation from other sexually reproducing chytrids that all have spore stages. © 2012 Blackwell Publishing Ltd.

  7. Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.

    Directory of Open Access Journals (Sweden)

    Helene Tournu

    Full Text Available The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs. Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.

  8. Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

    Directory of Open Access Journals (Sweden)

    Martin Meyer

    2016-08-01

    Full Text Available We here compared pathogenic (p and non-pathogenic (np isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12 derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12 derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

  9. Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

    Science.gov (United States)

    Meyer, Martin; Fehling, Helena; Matthiesen, Jenny; Lorenzen, Stephan; Schuldt, Kathrin; Bernin, Hannah; Zaruba, Mareen; Lender, Corinna; Ernst, Thomas; Ittrich, Harald; Roeder, Thomas; Tannich, Egbert; Lotter, Hannelore; Bruchhaus, Iris

    2016-08-01

    We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12) derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

  10. Host gene response to endosymbiont and pathogen in the cereal weevil Sitophilus oryzae

    Directory of Open Access Journals (Sweden)

    Vigneron Aurélien

    2012-01-01

    Full Text Available Abstract Background Insects thriving on nutritionally poor habitats have integrated mutualistic intracellular symbiotic bacteria (endosymbionts in a bacteria-bearing tissue (the bacteriome that isolates the endosymbionts and protects them against a host systemic immune response. Whilst the metabolic and physiological features of long-term insect associations have been investigated in detail over the past decades, cellular and immune regulations that determine the host response to endosymbionts and pathogens have attracted interest more recently. Results To investigate bacteriome cellular specificities and weevil immune responses to bacteria, we have constructed and sequenced 7 cDNA libraries from Sitophilus oryzae whole larvae and bacteriomes. Bioinformatic analysis of 26,886 ESTs led to the generation of 8,941 weevil unigenes. Based on in silico analysis and on the examination of genes involved in the cellular pathways of potential interest to intracellular symbiosis (i.e. cell growth and apoptosis, autophagy, immunity, we have selected and analyzed 29 genes using qRT-PCR, taking into consideration bacteriome specificity and symbiosis impact on the host response to pathogens. We show that the bacteriome tissue accumulates transcripts from genes involved in cellular development and survival, such as the apoptotic inhibitors iap2 and iap3, and endosomal fusion and trafficking, such as Rab7, Hrs, and SNARE. As regards our investigation into immunity, we first strengthen the bacteriome immunomodulation previously reported in S. zeamais. We show that the sarcotoxin, the c-type lysozyme, and the wpgrp2 genes are downregulated in the S. oryzae bacteriome, when compared to aposymbiotic insects and insects challenged with E. coli. Secondly, transcript level comparison between symbiotic and aposymbiotic larvae provides evidence that the immune systemic response to pathogens is decreased in symbiotic insects, as shown by the relatively high expression of

  11. Episodic positive selection in the evolution of avian toll-like receptor innate immunity genes.

    Science.gov (United States)

    Grueber, Catherine E; Wallis, Graham P; Jamieson, Ian G

    2014-01-01

    Toll-like receptors (TLRs) are a family of conserved pattern-recognition molecules responsible for initiating innate and acquired immune responses. Because they play a key role in host defence, these genes have received increasing interest in the evolutionary and population genetics literature, as their variation represents a potential target of adaptive evolution. However, the role of pathogen-mediated selection (i.e. episodic positive selection) in the evolution of these genes remains poorly known and has not been examined outside of mammals. A recent increase in the number of bird species for which TLR sequences are available has enabled us to examine the selective processes that have influenced evolution of the 10 known avian TLR genes. Specifically, we tested for episodic positive selection to identify codons that experience purifying selection for the majority of their evolution, interspersed with bursts of positive selection that may occur only in restricted lineages. We included up to 23 species per gene (mean = 16.0) and observed that, although purifying selection was evident, an average of 4.5% of codons experienced episodic positive selection across all loci. For four genes in which sequence coverage traversed both the extracellular leucine-rich repeat region (LRR) and transmembrane/intracellular domains of the proteins, increased positive selection was observed at the extracellular domain, consistent with theoretical predictions. Our results provide evidence that episodic positive selection has played an important role in the evolution of most avian TLRs, consistent with the role of these loci in pathogen recognition and a mechanism of host-pathogen coevolution.

  12. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics

    Science.gov (United States)

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-01-01

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design. PMID:26488648

  13. Intracellular bacteria: the origin of dinoflagellate toxicity.

    Science.gov (United States)

    Silva, E S

    1990-01-01

    Dinoflagellate blooms of the same species have been registered either as toxic or nontoxic and, in the latter case, toxicity may be of different types. A hypothesis has been formulated according to which the bacteria having in some way taken part in the toxin formation are either inside the dinoflagellate cell or in the nutritive liquid. The presence of intracellular bacteria in those microorganisms has been studied mainly in material from cultures, a few from the sea, and several strains were isolated from different species. Experiments with crossed inoculations have shown that the bacterial strain from Gonyaulax tamarensis caused the cells of some other species to become toxic. From nontoxic clonal cultures of Prorocentrum balticum, Glenodinium foliaceum, and Gyrodinium instriatum, after inoculation of that bacterial strain, cultures were obtained whose cell extracts showed the same kind of toxicity as G. tamarensis. No toxic action could be found in the extracts of the bacterial cells form the assayed strains. The interference of intracellular bacteria in the metabolism of dinoflagellates must be the main cause of their toxicity.

  14. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    Science.gov (United States)

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. Intracellular accumulation of norfloxacin in Mycobacterium smegmatis.

    Science.gov (United States)

    Corti, S; Chevalier, J; Cremieux, A

    1995-01-01

    To evaluate the intracellular accumulation of norfloxacin in mycobacteria, two methods were used with Mycobacterium smegmatis. A radiometric method (K. V. Cundy, C. E. Fasching, K. E. Willard, and L. R. Peterson, J. Antimicrob. Chemother. 28:491-497, 1991) was used without great modification, but the fluorometric method (P. G. S. Mortimer and L. J. V. Piddock, J. Antimicrob. Chemother. 28:639-653, 1991) was changed considerably. Indeed, adsorption of the quinolone to the bacterial surface was characterized by measuring the level of accumulation of 0 degree C. Taking into account the adsorption, the pH of the washing buffer was increased from 7.0 to 9.0 to improve the desorption of norfloxacin from the cell surface. Both the fluorometric method, with the technical improvement, and the radiometric method could be used to estimate the intracellular accumulation of norfloxacin, which resulted from the difference between the whole uptake measured at 37 degrees C and the adsorption measured at 0 degrees C. A total of 35 ng of norfloxacin per mg of cells (dry weight) penetrated into the M. smegmatis cell, and the steady state was achieved in 5 min. Use of inhibitors of the proton motive force revealed that transport of norfloxacin was energy independent. Thus, the same mechanisms of quinolone accumulation that occur in eubacteria seem to occur in mycobacteria, at least in M. smegmatis. PMID:8585727

  16. Fluorescent nanoparticles for intracellular sensing: a review.

    Science.gov (United States)

    Ruedas-Rama, Maria J; Walters, Jamie D; Orte, Angel; Hall, Elizabeth A H

    2012-11-02

    Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. [Intracellular signaling mechanisms in thyroid cancer].

    Science.gov (United States)

    Mondragón-Terán, Paul; López-Hernández, Luz Berenice; Gutiérrez-Salinas, José; Suárez-Cuenca, Juan Antonio; Luna-Ceballos, Rosa Isela; Erazo Valle-Solís, Aura

    2016-01-01

    Thyroid cancer is the most common malignancy of the endocrine system, the papillary variant accounts for 80-90% of all diagnosed cases. In the development of papillary thyroid cancer, BRAF and RAS genes are mainly affected, resulting in a modification of the system of intracellular signaling proteins known as «protein kinase mitogen-activated» (MAPK) which consist of «modules» of internal signaling proteins (Receptor/Ras/Raf/MEK/ERK) from the cell membrane to the nucleus. In thyroid cancer, these signanling proteins regulate diverse cellular processes such as differentiation, growth, development and apoptosis. MAPK play an important role in the pathogenesis of thyroid cancer as they are used as molecular biomarkers for diagnostic, prognostic and as possible therapeutic molecular targets. Mutations in BRAF gene have been correlated with poor response to treatment with traditional chemotherapy and as an indicator of poor prognosis. To review the molecular mechanisms involved in intracellular signaling of BRAF and RAS genes in thyroid cancer. Molecular therapy research is in progress for this type of cancer as new molecules have been developed in order to inhibit any of the components of the signaling pathway (RET/PTC)/Ras/Raf/MEK/ERK; with special emphasis on the (RET/PTC)/Ras/Raf section, which is a major effector of ERK pathway. Copyright © 2016 Academia Mexicana de Cirugía A.C. Publicado por Masson Doyma México S.A. All rights reserved.

  18. Stochastic models of intracellular calcium signals

    Energy Technology Data Exchange (ETDEWEB)

    Rüdiger, Sten, E-mail: sten.ruediger@physik.hu-berlin.de

    2014-01-10

    Cellular signaling operates in a noisy environment shaped by low molecular concentrations and cellular heterogeneity. For calcium release through intracellular channels–one of the most important cellular signaling mechanisms–feedback by liberated calcium endows fluctuations with critical functions in signal generation and formation. In this review it is first described, under which general conditions the environment makes stochasticity relevant, and which conditions allow approximating or deterministic equations. This analysis provides a framework, in which one can deduce an efficient hybrid description combining stochastic and deterministic evolution laws. Within the hybrid approach, Markov chains model gating of channels, while the concentrations of calcium and calcium binding molecules (buffers) are described by reaction–diffusion equations. The article further focuses on the spatial representation of subcellular calcium domains related to intracellular calcium channels. It presents analysis for single channels and clusters of channels and reviews the effects of buffers on the calcium release. For clustered channels, we discuss the application and validity of coarse-graining as well as approaches based on continuous gating variables (Fokker–Planck and chemical Langevin equations). Comparison with recent experiments substantiates the stochastic and spatial approach, identifies minimal requirements for a realistic modeling, and facilitates an understanding of collective channel behavior. At the end of the review, implications of stochastic and local modeling for the generation and properties of cell-wide release and the integration of calcium dynamics into cellular signaling models are discussed.

  19. Strain-specific impact of the high-pathogenicity island on virulence in extra-intestinal pathogenic Escherichia coli.

    Science.gov (United States)

    Smati, Mounira; Magistro, Giuseppe; Adiba, Sandrine; Wieser, Andreas; Picard, Bertrand; Schubert, Sören; Denamur, Erick

    2017-01-01

    In order to clarify the role of the high-pathogenicity island (HPI) in the experimental virulence of Escherichia coli, we constructed different deletion mutants of the entire HPI and of three individual genes (irp2, fyuA and ybtA), encoding for three main functions within the HPI. Those mutants were constructed for three phylogroup B2 strains (536-STc127, CFT073-STc73, and NU14-STc95), representative of the main B2 subgroups causing extra-intestinal infections. Transcriptional profiles obtained for the selected HPI genes irp2, fyuA and ybtA revealed similar patterns for all strains, both under selective iron-deplete conditions and in intracellular bacterial communities in vitro, with a high expression of irp2. Deletion of irp2 and ybtA abrogated yersiniabactin production, whereas the fyuA knockout was only slightly impaired for siderophore synthesis. The experimental virulence of the strains was then tested in amoeba Dictyostelium discoideum and mouse septicaemia models. No effect of any HPI mutant was observed for the two more virulent strains 536 and CFT073. In contrast, the virulence of the less virulent NU14 strain was dramatically diminished by the complete deletion of the HPI and irp2 gene whereas a lesser reduction in virulence was observed for the fyuA and ybtA deletion mutants. The two experimental virulence models gave similar results. It appears that the role of the HPI in experimental virulence is depending on the genetic background of the strains despite similar inter-strain transcriptional patterns of HPI genes, as well as of the functional class of the studied gene. Altogether, these data indicate that the intrinsic extra-intestinal virulence in the E. coli species is multigenic, with epistatic interactions between the genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

    Directory of Open Access Journals (Sweden)

    Norkin Leonard C

    2004-07-01

    Full Text Available Abstract Background Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function. Methods We utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions. Results We show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp, C. caviae (GPIC, and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi. Conclusion Caveolin-2 associates with the chlamydial

  1. Shigella IpaH family effectors as a versatile model for studying pathogenic bacteria

    OpenAIRE

    Hiroshi eAshida; Chihiro eSasakawa; Chihiro eSasakawa; Chihiro eSasakawa

    2016-01-01

    Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens...

  2. Microbial pathogens in source and treated waters from drinking water treatment plants in the United States and implications for human health

    Science.gov (United States)

    King, Dawn N.; Donohue, Maura J.; Vesper, Stephen J.; Villegas, Eric N.; Ware, Michael W.; Vogel, Megan E.; Furlong, Edward; Kolpin, Dana W.; Glassmeyer, Susan T.; Pfaller, Stacy

    2016-01-01

    An occurrence survey was conducted on selected pathogens in source and treated drinking water collected from 25 drinking water treatment plants (DWTPs) in the United States. Water samples were analyzed for the protozoa Giardia and Cryptosporidium (EPA Method 1623); the fungi Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus (quantitative PCR [qPCR]); and the bacteria Legionella pneumophila (qPCR), Mycobacterium avium, M. avium subspecies paratuberculosis, and Mycobacterium intracellulare (qPCR and culture). Cryptosporidium and Giardia were detected in 25% and in 46% of the source water samples, respectively (treated waters were not tested). Aspergillus fumigatus was the most commonly detected fungus in source waters (48%) but none of the three fungi were detected in treated water. Legionella pneumophila was detected in 25% of the source water samples but in only 4% of treated water samples. M. avium and M. intracellulare were both detected in 25% of source water, while all three mycobacteria were detected in 36% of treated water samples. Five species of mycobacteria, Mycobacterium mucogenicum, Mycobacterium phocaicum, Mycobacterium triplex, Mycobacterium fortuitum, and Mycobacterium lentiflavum were cultured from treated water samples. Although these DWTPs represent a fraction of those in the U.S., the results suggest that many of these pathogens are widespread in source waters but that treatment is generally effective in reducing them to below detection limits. The one exception is the mycobacteria, which were commonly detected in treated water, even when not detected in source waters.

  3. Microbial pathogens in source and treated waters from drinking water treatment plants in the United States and implications for human health.

    Science.gov (United States)

    King, Dawn N; Donohue, Maura J; Vesper, Stephen J; Villegas, Eric N; Ware, Michael W; Vogel, Megan E; Furlong, Edward F; Kolpin, Dana W; Glassmeyer, Susan T; Pfaller, Stacy

    2016-08-15

    An occurrence survey was conducted on selected pathogens in source and treated drinking water collected from 25 drinking water treatment plants (DWTPs) in the United States. Water samples were analyzed for the protozoa Giardia and Cryptosporidium (EPA Method 1623); the fungi Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus (quantitative PCR [qPCR]); and the bacteria Legionella pneumophila (qPCR), Mycobacterium avium, M. avium subspecies paratuberculosis, and Mycobacterium intracellulare (qPCR and culture). Cryptosporidium and Giardia were detected in 25% and in 46% of the source water samples, respectively (treated waters were not tested). Aspergillus fumigatus was the most commonly detected fungus in source waters (48%) but none of the three fungi were detected in treated water. Legionella pneumophila was detected in 25% of the source water samples but in only 4% of treated water samples. M. avium and M. intracellulare were both detected in 25% of source water, while all three mycobacteria were detected in 36% of treated water samples. Five species of mycobacteria, Mycobacterium mucogenicum, Mycobacterium phocaicum, Mycobacterium triplex, Mycobacterium fortuitum, and Mycobacterium lentiflavum were cultured from treated water samples. Although these DWTPs represent a fraction of those in the U.S., the results suggest that many of these pathogens are widespread in source waters but that treatment is generally effective in reducing them to below detection limits. The one exception is the mycobacteria, which were commonly detected in treated water, even when not detected in source waters. Published by Elsevier B.V.

  4. Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

    DEFF Research Database (Denmark)

    Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...

  5. Social barriers to pathogen transmission in wild animal populations

    Energy Technology Data Exchange (ETDEWEB)

    Loehle, C.

    1995-03-01

    Diseases and pathogens are receiving increasing recognition as sources of mortality in animal populations. Immune system strength is clearly important in fending off pathogen attack. Physical barriers to pathogen entry are also important. Various individual behaviors are efficacious in reducing contact with diseases and pests. This paper focuses on a fourth mode of defense: social barriers to transmission. Various social behaviors have pathogen transmission consequences. Selective pressures on these social behaviors may therefore exist. Effects on pathogen transmission of mating strategies, social avoidance, group size, group isolation, and other behaviors are explored. It is concluded that many of these behaviors may have been affected by selection pressures to reduce transmission of pathogens. 84 refs., 1 tab.

  6. Indicators for waterborne pathogens

    National Research Council Canada - National Science Library

    Committee on Indicators for Waterborne Pathogens; Board on Life Sciences; Water Science and Technology Board; Division on Earth and Life Studies; National Research Council; National Academy of Sciences

    2004-01-01

    ... not practical or feasible to monitor for the complete spectrum of microorganisms that may occur in water, and many known pathogens are difficult to detect directly and reliably in water samples.Â...

  7. Evolution of microbial pathogens

    National Research Council Canada - National Science Library

    DiRita, Victor J; Seifert, H. Steven

    2006-01-01

    ... A. Hogan vvi ■ CONTENTS 8. Evolution of Pathogens in Soil Rachel Muir and Man-Wah Tan / 131 9. Experimental Models of Symbiotic Host-Microbial Relationships: Understanding the Underpinnings of ...

  8. Host–Pathogen Interactions

    NARCIS (Netherlands)

    Smits, M.A.; Schokker, D.J.

    2011-01-01

    The outcome of an infection is determined by numerous interactions between hosts and pathogens occurring at many different biological levels, ranging from molecule to population. To develop new control strategies for infectious diseases in livestock species, appropriate methodologies are needed

  9. Sub-10-nm intracellular bioelectronic probes from nanowire-nanotube heterostructures.

    Science.gov (United States)

    Fu, Tian-Ming; Duan, Xiaojie; Jiang, Zhe; Dai, Xiaochuan; Xie, Ping; Cheng, Zengguang; Lieber, Charles M

    2014-01-28

    The miniaturization of bioelectronic intracellular probes with a wide dynamic frequency range can open up opportunities to study biological structures inaccessible by existing methods in a minimally invasive manner. Here, we report the design, fabrication, and demonstration of intracellular bioelectronic devices with probe sizes less than 10 nm. The devices are based on a nanowire-nanotube heterostructure in which a nanowire field-effect transistor detector is synthetically integrated with a nanotube cellular probe. Sub-10-nm nanotube probes were realized by a two-step selective etching approach that reduces the diameter of the nanotube free-end while maintaining a larger diameter at the nanowire detector necessary for mechanical strength and electrical sensitivity. Quasi-static water-gate measurements demonstrated selective device response to solution inside the nanotube, and pulsed measurements together with numerical simulations confirmed the capability to record fast electrophysiological signals. Systematic studies of the probe bandwidth in different ionic concentration solutions revealed the underlying mechanism governing the time response. In addition, the bandwidth effect of phospholipid coatings, which are important for intracellular recording, was investigated and modeled. The robustness of these sub-10-nm bioelectronics probes for intracellular interrogation was verified by optical imaging and recording the transmembrane resting potential of HL-1 cells. These ultrasmall bioelectronic probes enable direct detection of cellular electrical activity with highest spatial resolution achieved to date, and with further integration into larger chip arrays could provide a unique platform for ultra-high-resolution mapping of activity in neural networks and other systems.

  10. Plastic Transcriptomes Stabilize Immunity to Pathogen Diversity

    DEFF Research Database (Denmark)

    Zhang, Wei; Corwin, Jason A; Copeland, Daniel

    2017-01-01

    To respond to pathogen attack, selection and associated evolution has led to the creation of plant immune system that are a highly effective and inducible defense system. Central to this system are the plant defense hormones jasmonic acid (JA) and salicylic acid (SA) and crosstalk between the two...

  11. Staphylococcus lugdunensis, a serious pathogen in periprosthetic joint infections: comparison to Staphylococcus aureus and Staphylococcus epidermidis

    Directory of Open Access Journals (Sweden)

    J. Lourtet-Hascoët

    2016-10-01

    Conclusion: S. lugdunensis is an emerging pathogen with a pathogenicity quite similar to that of S. aureus. This coagulase-negative Staphylococcus must be identified precisely in PJI, in order to select the appropriate surgical treatment and antibiotics .

  12. Liposome-based Formulation for Intracellular Delivery of Functional Proteins

    Directory of Open Access Journals (Sweden)

    Benoît Chatin

    2015-01-01

    Full Text Available The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal enzyme and the anti-cytokeratin8 (K8 antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium-tren-cholesterol (BGTC (bis (guanidinium-tren-cholesterol combined to the colipid dioleoyl phosphatidylethanolamine (DOPE (dioleoyl phosphatidylethanolamine was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

  13. Intracellular pH in sperm physiology.

    Science.gov (United States)

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Drosophila VAMP7 regulates Wingless intracellular trafficking.

    Science.gov (United States)

    Gao, Han; He, Fang; Lin, Xinhua; Wu, Yihui

    2017-01-01

    Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. Here, we showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, we found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. We hypothesize that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling.

  15. Intracellular Signalling by C-Peptide

    Directory of Open Access Journals (Sweden)

    Claire E. Hills

    2008-01-01

    Full Text Available C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.

  16. Intracellular Na⁺ and cardiac metabolism.

    Science.gov (United States)

    Bay, Johannes; Kohlhaas, Michael; Maack, Christoph

    2013-08-01

    In heart failure, alterations of excitation-contraction underlie contractile dysfunction. One important defect is an elevation of the intracellular Na(+) concentration in cardiac myocytes ([Na(+)]i), which has an important impact on cytosolic and mitochondrial Ca(2+) homeostasis. While elevated [Na(+)]i is thought to compensate for decreased Ca(2+) load of the sarcoplasmic reticulum (SR), it yet negatively affects energy supply-and-demand matching and can even induce mitochondrial oxidative stress. Here, we review the mechanisms underlying these pathophysiological changes. The chain of events may constitute a vicious cycle of ion dysregulation, oxidative stress and energetic deficit, resembling characteristic cellular deficits that are considered key hallmarks of the failing heart. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes". Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Fastidious intracellular bacteria as causal agents of community-acquired pneumonia.

    Science.gov (United States)

    Lamoth, Frédéric; Greub, Gilbert

    2010-07-01

    Intracellular bacteria are common causes of community-acquired pneumonia that grow poorly or not at all on standard culture media and do not respond to beta-lactam antibiotic therapy. Apart from well-established agents of pneumonia such as Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia psittaci and Coxiella burnetii, some new emerging pathogens have recently been recognized, mainly Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-related bacteria. Most of them are causes of benign and self-limited infections. However, they may cause severe pneumonia in some cases (i.e., Legionnaires' disease) and they may cause outbreaks representing a public health problem deserving prompt recognition and appropriate therapy. Although extrapulmonary manifestations are often present, no clinical features allow them to be distinguished from classical bacterial agents of pneumonia such as Streptococcus pneumoniae. Thus, specific molecular diagnostic tools are very helpful for early recognition of the offending bacteria, whereas serology often only allows retrospective or late diagnosis. Macrolides remain the best empirical treatment of intracellular respiratory pathogens, although some observational studies suggest that quinolones may be superior for the treatment of legionellosis.

  18. Disturbance in forest ecosystems caused by pathogens and insects

    Science.gov (United States)

    Philip M. Wargo; Philip M. Wargo

    1995-01-01

    Pathogens and insects are major driving forces of processes in forested ecosystems. Disturbances caused by them are as intimately involved in ecosystem dynamics as the more sudden and obvious abiotic disturbances, for example, those caused by wind or fire. However, because pathogens and insects are selective and may affect only one or several related species of...

  19. The burden, antibiogram and pathogenicity of bacteria found in ...

    African Journals Online (AJOL)

    Municipal wastes may harbour microbial pathogens but the quantum and diseasecausing capacities of such organisms are rarely investigated. This study sought to establish the burden, antibiogram and pathogenicity of bacteria in 4 selected waste dumpsites and those found on hands and clothings of the respective waste ...

  20. Autophagy response: manipulating the mTOR-controlled machinery by amino acids and pathogens.

    Science.gov (United States)

    Fader, Claudio Marcelo; Aguilera, Milton Osmar; Colombo, María Isabel

    2015-10-01

    Macroautophagy is a self-degradative process that normally maintains cellular homeostasis via a lysosomal pathway. It is induced by different stress signals, including nutrients and growth factors' restriction as well as pathogen invasions. These stimuli are modulated by the serine/threonine protein kinase mammalian target of rapamycin (mTOR) which control not only autophagy but also protein translation and gene expression. This review focuses on the important role of mTOR as a master regulator of cell growth and the autophagy pathway. Here, we have discussed the role of intracellular amino acid availability and intracellular pH in the redistribution of autophagic structures, which may contribute to mammalian target of rapamycin complex 1 (mTORC1) activity regulation. We have also discussed that mTORC1 complex and components of the autophagy machinery are localized at the lysosomal surface, representing a fascinating mechanism to control the metabolism, cellular clearance and also to restrain invading intracellular pathogens.

  1. Identification of host-dependent survival factors for intracellular Mycobacterium tuberculosis through an siRNA screen.

    Directory of Open Access Journals (Sweden)

    Shilpi Jayaswal

    2010-04-01

    Full Text Available The stable infection of host macrophages by Mycobacterium tuberculosis (Mtb involves, and depends on, the attenuation of the diverse microbicidal responses mounted by the host cell. This is primarily achieved through targeted perturbations of the host cellular signaling machinery. Therefore, in view of the dependency of the pathogen on host molecules for its intracellular survival, we wanted to test whether targeting such factors could provide an alternate route for the therapeutic management of tuberculosis. To first identify components of the host signaling machinery that regulate intracellular survival of Mtb, we performed an siRNA screen against all known kinases and phosphatases in murine macrophages infected with the virulent strain, H37Rv. Several validated targets could be identified by this method where silencing led either to a significant decrease, or enhancement in the intracellular mycobacterial load. To further resolve the functional relevance of these targets, we also screened against these identified targets in cells infected with different strains of multiple drug-resistant mycobacteria which differed in terms of their intracellular growth properties. The results obtained subsequently allowed us to filter the core set of host regulatory molecules that functioned independently of the phenotypic variations exhibited by the pathogen. Then, using a combination of both in vitro and in vivo experimentation, we could demonstrate that at least some of these host factors provide attractive targets for anti-TB drug development. These results provide a "proof-of-concept" demonstration that targeting host factors subverted by intracellular Mtb provides an attractive and feasible strategy for the development of anti-tuberculosis drugs. Importantly, our findings also emphasize the advantage of such an approach by establishing its equal applicability to infections with Mtb strains exhibiting a range of phenotypic diversifications, including

  2. Candida albicans pathogenicity mechanisms.

    Science.gov (United States)

    Mayer, François L; Wilson, Duncan; Hube, Bernhard

    2013-02-15

    The polymorphic fungus Candida albicans is a member of the normal human microbiome. In most individuals, C. albicans resides as a lifelong, harmless commensal. Under certain circumstances, however, C. albicans can cause infections that range from superficial infections of the skin to life-threatening systemic infections. Several factors and activities have been identified which contribute to the pathogenic potential of this fungus. Among them are molecules which mediate adhesion to and invasion into host cells, the secretion of hydrolases, the yeast-to-hypha transition, contact sensing and thigmotropism, biofilm formation, phenotypic switching and a range of fitness attributes. Our understanding of when and how these mechanisms and factors contribute to infection has significantly increased during the last years. In addition, novel virulence mechanisms have recently been discovered. In this review we present an update on our current understanding of the pathogenicity mechanisms of this important human pathogen.

  3. Candida albicans pathogenicity mechanisms

    Science.gov (United States)

    Mayer, François L.; Wilson, Duncan; Hube, Bernhard

    2013-01-01

    The polymorphic fungus Candida albicans is a member of the normal human microbiome. In most individuals, C. albicans resides as a lifelong, harmless commensal. Under certain circumstances, however, C. albicans can cause infections that range from superficial infections of the skin to life-threatening systemic infections. Several factors and activities have been identified which contribute to the pathogenic potential of this fungus. Among them are molecules which mediate adhesion to and invasion into host cells, the secretion of hydrolases, the yeast-to-hypha transition, contact sensing and thigmotropism, biofilm formation, phenotypic switching and a range of fitness attributes. Our understanding of when and how these mechanisms and factors contribute to infection has significantly increased during the last years. In addition, novel virulence mechanisms have recently been discovered. In this review we present an update on our current understanding of the pathogenicity mechanisms of this important human pathogen. PMID:23302789

  4. Models of Caenorhabditis elegans infection by bacterial and fungal pathogens.

    Science.gov (United States)

    Powell, Jennifer R; Ausubel, Frederick M

    2008-01-01

    The nematode Caenorhabditis elegans is a simple model host for studying the relationship between the animal innate immune system and a variety of bacterial and fungal pathogens. Extensive genetic and molecular tools are available in C. elegans, facilitating an in-depth analysis of host defense factors and pathogen virulence factors. Many of these factors are conserved in insects and mammals, indicating the relevance of the nematode model to the vertebrate innate immune response. Here, we describe pathogen assays for a selection of the most commonly studied bacterial and fungal pathogens using the C. elegans model system.

  5. Safety and Waste Management for SAM Pathogen Methods

    Science.gov (United States)

    The General Safety and Waste Management page offers section-specific safety and waste management details for the pathogens included in EPA's Selected Analytical Methods for Environmental Remediation and Recovery (SAM).

  6. Development of a multi-pathogen enrichment broth for simultaneous growth of five common foodborne pathogens.

    Science.gov (United States)

    Chen, Juan; Tang, Junni; Bhunia, Arun K; Tang, Cheng; Wang, Changting; Shi, Hui

    2015-01-01

    The objective of the present study was to formulate a multi-pathogen enrichment broth which could support the simultaneous growth of five common foodborne pathogens (Salmonella enterica, Staphylococcus aureus, Shigella flexneri, Listeria monocytogenes and Escherichia coli O157:H7). The formulated broth SSSLE was composed of potassium tellurite, bile salt, lithium chloride, and sodium chloride as growth-inhibitors; glucose, esculin, mannitol and sodium pyruvate as growth-promoters. Compared with the respective specific selective enrichment broths, the individual growth pattern of each target pathogen in SSSLE was equal, or even better, except in the case of S. flexneri. In mixed-culture experiments, the gram-negative bacteria showed higher growth capabilities than the gram-positive bacteria after 8-h enrichment; however, the cell numbers after 24-h enrichment indicated that SSSLE could support the concurrent growth of five target pathogens irrespective of whether pathogens were inoculated initially at equal or unequal levels. For natural food samples under the high background flora, the final cell numbers enriched in SSSLE for five targets were enough to be detected by multiplex PCR. In conclusion, SSSLE was capable of supporting the growth of five target pathogens concurrently. The new broth formulated in this study has the potential of saving time, efforts and costs in multi-pathogen enrichment procedures.

  7. Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

    2017-08-24

    Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

  8. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    International Nuclear Information System (INIS)

    Majumdar, S.; Basu, S.K.

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections

  9. New insights into valve-related intramural and intracellular bacterial diversity in infective endocarditis.

    Science.gov (United States)

    Oberbach, Andreas; Schlichting, Nadine; Feder, Stefan; Lehmann, Stefanie; Kullnick, Yvonne; Buschmann, Tilo; Blumert, Conny; Horn, Friedemann; Neuhaus, Jochen; Neujahr, Ralph; Bagaev, Erik; Hagl, Christian; Pichlmaier, Maximilian; Rodloff, Arne Christian; Gräber, Sandra; Kirsch, Katharina; Sandri, Marcus; Kumbhari, Vivek; Behzadi, Armirhossein; Behzadi, Amirali; Correia, Joao Carlos; Mohr, Friedrich Wilhelm; Friedrich, Maik

    2017-01-01

    In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE. Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM). Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified. The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may

  10. New insights into valve-related intramural and intracellular bacterial diversity in infective endocarditis.

    Directory of Open Access Journals (Sweden)

    Andreas Oberbach

    Full Text Available In infective endocarditis (IE, a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE.Using next-generation sequencing (NGS of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM.Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified.The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic

  11. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    International Nuclear Information System (INIS)

    Levy, J.R.; Olefsky, J.M.

    1988-01-01

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 0 C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 0 C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation

  12. Identification of Diverse Mycoviruses through Metatranscriptomics Characterization of the Viromes of Five Major Fungal Plant Pathogens

    Science.gov (United States)

    Nelson, Berlin D.; Ajayi-Oyetunde, Olutoyosi; Bradley, Carl A.; Hughes, Teresa J.; Hartman, Glen L.; Eastburn, Darin M.

    2016-01-01

    ABSTRACT Mycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant-pathogenic fungi, Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembled de novo, and contigs with predicted amino acid sequence similarities to viruses in the nonredundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages: Barnaviridae, Benyviridae, Chrysoviridae, Endornaviridae, Fusariviridae, Hypoviridae, Mononegavirales, Narnaviridae, Ophioviridae, Ourmiavirus, Partitiviridae, Tombusviridae, Totiviridae, Tymoviridae, and Virgaviridae. More than half of the viral sequences were predicted to be members of the Mitovirus genus in the family Narnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae, Ophioviridae, and Virgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts. IMPORTANCE Plant-pathogenic fungi reduce crop yields, which affects food security worldwide. Plant host resistance is considered a sustainable disease management option but may often be incomplete or lacking for some crops to certain fungal pathogens

  13. Intracellular Shuttle: The Lactate Aerobic Metabolism

    Directory of Open Access Journals (Sweden)

    Rogério Santos de Oliveira Cruz

    2012-01-01

    Full Text Available Lactate is a highly dynamic metabolite that can be used as a fuel by several cells of the human body, particularly during physical exercise. Traditionally, it has been believed that the first step of lactate oxidation occurs in cytosol; however, this idea was recently challenged. A new hypothesis has been presented based on the fact that lactate-to-pyruvate conversion cannot occur in cytosol, because the LDH enzyme characteristics and cytosolic environment do not allow the reaction in this way. Instead, the Intracellular Lactate Shuttle hypothesis states that lactate first enters in mitochondria and only then is metabolized. In several tissues of the human body this idea is well accepted but is quite resistant in skeletal muscle. In this paper, we will present not only the studies which are protagonists in this discussion, but the potential mechanism by which this oxidation occurs and also a link between lactate and mitochondrial proliferation. This new perspective brings some implications and comes to change our understanding of the interaction between the energy systems, because the product of one serves as a substrate for the other.

  14. Intracellular sphingosine releases calcium from lysosomes

    Science.gov (United States)

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

  15. Intracellular recording from a spider vibration receptor.

    Science.gov (United States)

    Gingl, Ewald; Burger, Anna-M; Barth, Friedrich G

    2006-05-01

    The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ's cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.

  16. LIPID SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION

    Science.gov (United States)

    Stein, Olga; Stein, Yechezkiel

    1967-01-01

    In the mammary glands of lactating albino mice injected intravenously with 9, 10-oleic acid-3H or 9, 10-palmitic acid-3H, it has been shown that the labeled fatty acids are incorporated into mammary gland glycerides. The labeled lipid in the mammary gland 1 min after injection was in esterified form (> 95%), and the radioautographic reaction was seen over the rough endoplasmic reticulum and over lipid droplets, both intracellular and intraluminal. At 10–60 min after injection, the silver grains were concentrated predominantly over lipid droplets. There was no concentration of radioactivity over the granules in the Golgi apparatus, at any time interval studied. These findings were interpreted to indicate that after esterification of the fatty acid into glycerides in the rough endoplasmic reticulum an in situ aggregation of lipid occurs, with acquisition of droplet form. The release of the lipid into the lumen proceeds directly and not through the Golgi apparatus, in contradistinction to the mode of secretion of casein in the mammary gland or of lipoprotein in the liver. The presence of strands of endoplasmic reticulum attached to intraluminal lipid droplets provides a structural counterpart to the milk microsomes described in ruminant milk. PMID:6033535

  17. On the Computing Potential of Intracellular Vesicles.

    Science.gov (United States)

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing.

  18. Human pathogen avoidance adaptations

    NARCIS (Netherlands)

    Tybur, J.M.; Lieberman, D.

    2016-01-01

    Over the past few decades, researchers have become increasingly interested in the adaptations guiding the avoidance of disease-causing organisms. Here we discuss the latest developments in this area, including a recently developed information-processing model of the adaptations underlying pathogen

  19. Pathogenicity and virulence

    Science.gov (United States)

    Many pathogenic microorganisms are host-specific in that they parasitize only one or a few animal species. For example, the cause of equine strangles, Streptococcus equi subspecies equi, is essentially limited to infection of horses. Others—certain Salmonella serotypes, for example—have a broad host...

  20. Modeling HIV-1 intracellular replication: two simulation approaches

    NARCIS (Netherlands)

    Zarrabi, N.; Mancini, E.; Tay, J.; Shahand, S.; Sloot, P.M.A.

    2010-01-01

    Many mathematical and computational models have been developed to investigate the complexity of HIV dynamics, immune response and drug therapy. However, there are not many models which consider the dynamics of virus intracellular replication at a single level. We propose a model of HIV intracellular

  1. Pico gauges for minimally invasive intracellular hydrostatic pressure measurements

    DEFF Research Database (Denmark)

    Knoblauch, Jan; Mullendore, Daniel L.; Jensen, Kaare Hartvig

    2014-01-01

    Intracellular pressure has a multitude of functions in cells surrounded by a cell wall or similar matrix in all kingdoms of life. The functions include cell growth, nastic movements, and penetration of tissue by parasites. The precise measurement of intracellular pressure in the majority of cells...

  2. Intracellular angiotensin II inhibits heterologous receptor stimulated Ca2+ entry

    NARCIS (Netherlands)

    Filipeanu, CM; Brailoiu, E; Henning, RH; Deelman, LE; de Zeeuw, D; Nelemans, SA

    2001-01-01

    Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngII(i)) is unclear. Besides direct effects of AngII(i) on cellular

  3. Development of bacterial cell-based system for intracellular ...

    African Journals Online (AJOL)

    Development of bacterial cell-based system for intracellular antioxidant activity screening assay using green fluorescence protein (GFP) reporter. ... Both strains demonstrated that quercetin and α- tocopherol exhibited the most potent and significant antioxidant activity with more than 60% reduction of intracellular superoxide ...

  4. Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols.

    Science.gov (United States)

    Pinu, Farhana R; Villas-Boas, Silas G; Aggio, Raphael

    2017-10-23

    Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

  5. Genome expression analysis of nonproliferating intracellular Salmonella enterica serovar Typhimurium unravels an acid pH-dependent PhoP-PhoQ response essential for dormancy.

    Science.gov (United States)

    Núñez-Hernández, Cristina; Tierrez, Alberto; Ortega, Alvaro D; Pucciarelli, M Graciela; Godoy, Marta; Eisman, Blanca; Casadesús, Josep; García-del Portillo, Francisco

    2013-01-01

    Genome-wide expression analyses have provided clues on how Salmonella proliferates inside cultured macrophages and epithelial cells. However, in vivo studies show that Salmonella does not replicate massively within host cells, leaving the underlying mechanisms of such growth control largely undefined. In vitro infection models based on fibroblasts or dendritic cells reveal limited proliferation of the pathogen, but it is presently unknown whether these phenomena reflect events occurring in vivo. Fibroblasts are distinctive, since they represent a nonphagocytic cell type in which S. enterica serovar Typhimurium actively attenuates intracellular growth. Here, we show in the mouse model that S. Typhimurium restrains intracellular growth within nonphagocytic cells positioned in the intestinal lamina propria. This response requires a functional PhoP-PhoQ system and is reproduced in primary fibroblasts isolated from the mouse intestine. The fibroblast infection model was exploited to generate transcriptome data, which revealed that ∼2% (98 genes) of the S. Typhimurium genome is differentially expressed in nongrowing intracellular bacteria. Changes include metabolic reprogramming to microaerophilic conditions, induction of virulence plasmid genes, upregulation of the pathogenicity islands SPI-1 and SPI-2, and shutdown of flagella production and chemotaxis. Comparison of relative protein levels of several PhoP-PhoQ-regulated functions (PagN, PagP, and VirK) in nongrowing intracellular bacteria and extracellular bacteria exposed to diverse PhoP-PhoQ-inducing signals denoted a regulation responding to acidic pH. These data demonstrate that S. Typhimurium restrains intracellular growth in vivo and support a model in which dormant intracellular bacteria could sense vacuolar acidification to stimulate the PhoP-PhoQ system for preventing intracellular overgrowth.

  6. Genome Expression Analysis of Nonproliferating Intracellular Salmonella enterica Serovar Typhimurium Unravels an Acid pH-Dependent PhoP-PhoQ Response Essential for Dormancy

    Science.gov (United States)

    Núñez-Hernández, Cristina; Tierrez, Alberto; Ortega, Álvaro D.; Pucciarelli, M. Graciela; Godoy, Marta; Eisman, Blanca; Casadesús, Josep

    2013-01-01

    Genome-wide expression analyses have provided clues on how Salmonella proliferates inside cultured macrophages and epithelial cells. However, in vivo studies show that Salmonella does not replicate massively within host cells, leaving the underlying mechanisms of such growth control largely undefined. In vitro infection models based on fibroblasts or dendritic cells reveal limited proliferation of the pathogen, but it is presently unknown whether these phenomena reflect events occurring in vivo. Fibroblasts are distinctive, since they represent a nonphagocytic cell type in which S. enterica serovar Typhimurium actively attenuates intracellular growth. Here, we show in the mouse model that S. Typhimurium restrains intracellular growth within nonphagocytic cells positioned in the intestinal lamina propria. This response requires a functional PhoP-PhoQ system and is reproduced in primary fibroblasts isolated from the mouse intestine. The fibroblast infection model was exploited to generate transcriptome data, which revealed that ∼2% (98 genes) of the S. Typhimurium genome is differentially expressed in nongrowing intracellular bacteria. Changes include metabolic reprogramming to microaerophilic conditions, induction of virulence plasmid genes, upregulation of the pathogenicity islands SPI-1 and SPI-2, and shutdown of flagella production and chemotaxis. Comparison of relative protein levels of several PhoP-PhoQ-regulated functions (PagN, PagP, and VirK) in nongrowing intracellular bacteria and extracellular bacteria exposed to diverse PhoP-PhoQ-inducing signals denoted a regulation responding to acidic pH. These data demonstrate that S. Typhimurium restrains intracellular growth in vivo and support a model in which dormant intracellular bacteria could sense vacuolar acidification to stimulate the PhoP-PhoQ system for preventing intracellular overgrowth. PMID:23090959

  7. Structure and Biological Activity of Pathogen-like Synthetic Nanomedicines

    Science.gov (United States)

    Lőrincz, Orsolya; Tőke, Enikő R.; Somogyi, Eszter; Horkay, Ferenc; Chandran, Preethi; Douglas, Jack F.; Szebeni, János; Lisziewicz, Julianna

    2011-01-01

    Here we characterize the structure, stability and intracellular mode-of-action of DermaVir nanomedicine that is under clinical development for the treatment of HIV/AIDS. This nanomedicine is comprised of pathogen-like pDNA/PEIm nanoparticles (NPs) having the structure and function resembling spherical viruses that naturally evolved to deliver nucleic acids to the cells. Atomic force microscopy demonstrated spherical 100–200nm NPs with a smooth polymer surface protecting the pDNA in the core. Optical-absorption determined both the NP structural stability and biological activity relevant to their ability to escape from the endosome and release the pDNA at the nucleus. Salt, pH and temperature influence the nanomedicine shelf-life and intracellular stability. This approach facilitates the development of diverse polyplex nanomedicines where the delivered pDNA-expressed antigens induce immune responses to kill infected cells. PMID:21839051

  8. Autophagy in plant pathogenic fungi.

    Science.gov (United States)

    Liu, Xiao-Hong; Xu, Fei; Snyder, John Hugh; Shi, Huan-Bin; Lu, Jian-Ping; Lin, Fu-Cheng

    2016-09-01

    Autophagy is a conserved cellular process that degrades cytoplasmic constituents in vacuoles. Plant pathogenic fungi develop special infection structures and/or secrete a range of enzymes to invade their plant hosts. It has been demonstrated that monitoring autophagy processes can be extremely useful in visualizing the sequence of events leading to pathogenicity of plant pathogenic fungi. In this review, we introduce the molecular mechanisms involved in autophagy. In addition, we explore the relationship between autophagy and pathogenicity in plant pathogenic fungi. Finally, we discuss the various experimental strategies available for use in the study of autophagy in plant pathogenic fungi. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. AMP-activated Protein Kinase As a Target For Pathogens: Friends Or Foes?

    Science.gov (United States)

    Moreira, Diana; Silvestre, Ricardo; Cordeiro-da-Silva, Anabela; Estaquier, Jérôme; Foretz, Marc; Viollet, Benoit

    2016-01-01

    Intracellular pathogens are known to manipulate host cell regulatory pathways to establish an optimal environment for their growth and survival. Pathogens employ active mechanisms to hijack host cell metabolism and acquire existing nutrient and energy store. The role of the cellular energy sensor AMP-activated protein kinase (AMPK) in the regulation of cellular energy homeostasis is well documented. Here, we highlight recent advances showing the importance of AMPK signaling in pathogen-host interactions. Pathogens interact with AMPK by a variety of mechanisms aimed at reprogramming host cell metabolism to their own benefit. Stimulation of AMPK activity provides an efficient process to rapidly adapt pathogen metabolism to the major nutritional changes often encountered during the different phases of infection. However, inhibition of AMPK is also used by pathogens to manipulate innate host response, indicating that AMPK appears relevant to restriction of pathogen infection. We also document the effects of pharmacological AMPK modulators on pathogen proliferation and survival. This review illustrates intricate pathogen-AMPK interactions that may be exploited to the development of novel anti-pathogen therapies.

  10. Filamentous pathogen effector functions: of pathogens, hosts and microbiomes

    NARCIS (Netherlands)

    Rövenich, H.; Boshoven, J.C.; Thomma, B.

    2014-01-01

    Microorganisms play essential roles in almost every environment on earth. For instance, microbes decompose organic material, or establish symbiotic relationships that range from pathogenic to mutualistic. Symbiotic relationships have been particularly well studied for microbial plant pathogens and

  11. Intracellular transport of pancreatic zymogens during caerulein supramaximal stimulation

    International Nuclear Information System (INIS)

    Saito, I.; Hashmimoto, S.; Saluja, A.; Steer, M.L.; Meldolesi, J.

    1987-01-01

    Rats infused with a dose of the secretagogue caerulein that is in excess of that which stimulates a maximal rate of pancreatic digestive enzyme secretion develop acute edematous pancreatitis. The authors have previously noted that infusion of this dose of caerulein induces the appearance of large heterogeneous vacuoles in acinar cell, blockage of exocytosis, and intracellular accumulation of digestive zymogens. The current studies were performed to further elucidate these phenomena at the electron microscopic level of resolution and employed the techniques of pulse labeling, radioautography, and immunolocalization. Rats were infused with caerulein for 1 h, given a pulse of [ 3 H]phenylalanine, and killed at selected times during the subsequent 5- to 180-min postpulse period during which caerulein infusion was continued. Transport from the endoplasmic reticulum to the Golgi cisternae was not altered by supramaximal stimulation, but transport through post-Golgi elements was altered. In particular, the maturation of condensing vacuoles into zymogen granules was found to be impaired. Thus these studies indicate that the large heterogeneous vacuoles that appear during supramaximal secretagogue stimulation and that contain admixed digestive zymogens and lysosomal hydrolases arise by at least two mechanisms, impaired condensing vacuole maturation and crinophagy

  12. Comparison of the intracellular trafficking itinerary of ctla-4 orthologues.

    Directory of Open Access Journals (Sweden)

    Satdip Kaur

    Full Text Available CTLA-4 is an essential inhibitor of T cell immune responses. At steady state, most CTLA-4 resides in intracellular compartments due to constitutive internalisation mediated via a tyrosine based endocytic motif (YVKM within the cytoplasmic domain. This domain is highly conserved in mammals suggesting strong selective pressure. In contrast, the C-terminal domain varies considerably in non-mammals such as fish, xenopus and birds. We compared the ability of the C-terminus of these species to direct the trafficking of CTLA-4 with human CTLA-4. Using a chimeric approach, endocytosis was found to be conserved between human, xenopus and chicken CTLA-4 but was reduced substantially in trout CTLA-4, which lacks the conserved YXXM motif. Nevertheless, we identified an alternative YXXF motif in trout CTLA-4 that permitted limited endocytosis. Post-internalisation, CTLA-4 was either recycled or targeted for degradation. Human and chicken CTLA-4, which contain a YVKM motif, showed efficient recycling compared to xenopus CTLA-4 which contains a less efficient YEKM motif. Specific mutation of this motif in human CTLA-4 reduced