de Barsy, Marie; Greub, Gilbert
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.
Full Text Available Abstract Background Molecular oxygen (O2 is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues. Results We developed a genetically encoded Förster resonance energy transfer (FRET-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen consists of the yellow fluorescent protein (YFP that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP. Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation. Conclusions FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (pathophysiological processes in living cells.
Weiss, Günter; Schaible, Ulrich E
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
Weiss, Günter; Schaible, Ulrich E
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
Silva, E S
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.
Wingreen, Ned S; Huang, Kerwyn Casey
With the realization that bacteria achieve exquisite levels of spatiotemporal organization has come the challenge of discovering the underlying mechanisms. In this review, we describe three classes of such mechanisms, each of which has physical origins: the use of landmarks, the creation of higher-order structures that enable geometric sensing, and the emergence of length scales from systems of chemical reactions coupled to diffusion. We then examine the diversity of geometric cues that exist even in cells with relatively simple geometries, and end by discussing both new technologies that could drive further discovery and the implications of our current knowledge for the behavior, fitness, and evolution of bacteria. The organizational strategies described here are employed in a wide variety of systems and in species across all kingdoms of life; in many ways they provide a general blueprint for organizing the building blocks of life.
Endres Robert G
Full Text Available Abstract Advances in computational biology allow systematic investigations to ascertain whether internal chemical gradients can be maintained in bacteria – an open question at the resolution limit of fluorescence microscopy. While it was previously believed that the small bacterial cell size and fast diffusion in the cytoplasm effectively remove any such gradient, a new computational study published in BMC Biophysics supports the emerging view that gradients can exist. The study arose from the recent observation that phosphorylated CtrA forms a gradient prior to cell division in Caulobacter crescentus, a bacterium known for its complicated cell cycle. Tropini et al. (2012 postulate that such gradients can provide an internal chemical compass, directing protein localization, cell division and cell development. More specifically, they describe biochemical and physical constraints on the formation of such gradients and explore a number of existing bacterial cell morphologies. These chemical gradients may limit in vitro analyses, and may ensure timing control and robustness to fluctuations during critical stages in cell development.
Mu, Haibo; Tang, Jiangjiang; Liu, Qianjin; Sun, Chunli; Wang, Tingting; Duan, Jinyou
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.
Mu, Haibo; Niu, Hong; Wang, Dongdong; Sun, Feifei; Sun, Yuelin; Duan, Jinyou
Most chronic infections are difficult to eradicate because bacteria capable of surviving in host-infected cells may be protected from the killing actions of antibiotics, leading to therapy failures and disease relapses. Here we demonstrated that covalent-coupling chitosan to streptomycin significantly improved intracellular bactericidal capacity towards multiple organisms within phagocytic or nonphagocytic cells. Structure-activity relationship investigations indicated that antibiotic contents, molecular size and positive charges of the conjugate were the key to retain this intracellular bactericidal activity. Mechanistic insight demonstrated the conjugate was capable to target and eliminate endocytic or endosomal escaped bacteria through facilitating the direct contact between the antibiotic and intracellular organism. In vivo acute infection models indicated that compared to equal dose of the antibiotic, chitosan-streptomycin (C-S) conjugate and especially the human serum album binding chitosan-streptomycin conjugate (HCS) complex formed by human serum album and C-S conjugate greatly decreased the bacteria burden in the spleen and liver in both wild type and immuno-suppressive mice. Furthermore, the HCS complex remarkably reduced mortality of infected TLR2 deficient mice, mimicking immune-compromised persons who were more susceptible to bacterial infections. These findings might open up a new avenue to combat intracellular bacterial infection by aminoglycosides antibiotics at a lower effective dose. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: email@example.com.
Mu, Haibo; Tang, Jiangjiang; Liu, Qianjin; Sun, Chunli; Wang, Tingting; Duan, Jinyou
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...
de Souza Santos, Marcela; Orth, Kim
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
Sui, Bao Quan; Dutta, Pinaki R.; Nataro, James P.
The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a serine protease autotransporter that acts as an enterotoxin and cytotoxin. When applied to epithelial cells in culture, purified toxin induces cell elongation and rounding, followed by exfoliation of cells from the substratum. These effects are accompanied by loss of actin stress fibers and electrophysiologic changes. Although it has been hypothesized that Pet has an intracellular site of action, evidence for this is...
Full Text Available Proteins secreted by the type V secretion system (T5SS, known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of 'Candidatus Liberibacter asiaticus' (Las, and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB, a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.
O'Neill, E A; Kiely, G M; Bender, R A
Strains of Caulobacter crescentus, Pseudomonas putida, Acinetobacter calcoaceticus, Rhizobium meliloti, and Rhodopseudomonas sphaeroides carrying the kanamycin resistance-encoding transposon Tn5 were 15 to 500 times more resistant to streptomycin than transposon-free strains. The streptomycin resistance determinant, which is separable from the kanamycin resistance determinant of Tn5, was not expressed in Escherichia coli or Klebsiella aerogenes.
Hanski, Leena; Genina, Natalja; Uvell, Hanna
Given the established role of Chlamydia spp. as causative agents of both acute and chronic diseases, search for new antimicrobial agents against these intracellular bacteria is required to promote human health. Isoflavones are naturally occurring phytoestrogens, antioxidants and efflux pump...
Kim, Soo-Jin; Lee, Chang-Muk; Han, Bo-Ram; Kim, Min-Young; Yeo, Yun-Soo; Yoon, Sang-Hong; Koo, Bon-Sung; Jun, Hong-Ki
To detect cellulases encoded by uncultured microorganisms, we constructed metagenomic libraries from Korean soil DNAs. Screenings of the libraries revealed a clone pCM2 that uses carboxymethyl cellulose (CMC) as a sole carbon source. Further analysis of the insert showed two consecutive ORFs (celM2 and xynM2) encoding proteins of 226 and 662 amino acids, respectively. A multiple sequence analysis with the deduced amino acid sequences of celM2 showed 36% sequence identity with cellulase from the Synechococcus sp., while xynM2 had 59% identity to endo-1,4-beta-xylanase A from Cellulomonas pachnodae. The highest enzymatic CMC hydrolysis was observable at pH 4.0 and 45 degrees C with recombinant CelM2 protein. Although the enzyme CelM2 additionally hydrolyzed avicel and xylan, no substrate hydrolysis was observed on oligosaccharides such as cellobiose, pNP-beta-cellobioside, pNP-beta-glucoside, and pNP-beta-xyloside. These results showed that CelM2 is a novel endo-type cellulase.
Full Text Available Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.
Lamoth, Frédéric; Greub, Gilbert
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.
Käser, T; Pasternak, J A; Hamonic, G; Rieder, M; Lai, K; Delgado-Ortega, M; Gerdts, V; Meurens, F
Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.
Huang, Beijing K; Stein, Kassi T; Sikes, Hadley D
Reactive oxygen species (ROS) such as H 2 O 2 play paradoxical roles in mammalian physiology. It is hypothesized that low, baseline levels of H 2 O 2 are necessary for growth and differentiation, while increased intracellular H 2 O 2 concentrations are associated with pathological phenotypes and genetic instability, eventually reaching a toxic threshold that causes cell death. However, the quantities of intracellular H 2 O 2 that lead to these different responses remain an unanswered question in the field. To address this question, we used genetically encoded constructs that both generate and quantify H 2 O 2 in a dose-response study of H 2 O 2 -mediated toxicity. We found that, rather than a simple concentration-response relationship, a combination of intracellular concentration and the cumulative metric of H 2 O 2 concentration multiplied by time (i.e., the area under the curve) determined the occurrence and level of cell death. Establishing the quantitative relationship between H 2 O 2 and cell toxicity promotes a deeper understanding of the intracellular effects of H 2 O 2 specifically as an individual reactive oxygen species, and it contributes to an understanding of its role in various redox-related diseases.
Stöckli, Martina; Lin, Chia-Wei; Sieber, Ramon; Plaza, David F; Ohm, Robin A; Künzler, Markus
Biofilm formation on fungal hyphae and production of antifungal molecules are strategies of bacteria in their competition with fungi for nutrients. Since these strategies are often coordinated and under control of quorum sensing by the bacteria, interference with this bacterial communication system can be used as a counter-strategy by the fungi in this competition. Hydrolysis of N-acyl-homoserine lactones (HSL), a quorum sensing molecule used by Gram-negative bacteria, by fungal cultures has been demonstrated. However, the enzymes that are responsible for this activity, have not been identified. In this study, we identified and characterized two paralogous HSL hydrolyzing enzymes from the coprophilous fungus Coprinopsis cinerea. The C. cinerea HSL lactonases belong to the metallo-β-lactamase family and show sequence homology to and a similar biochemical activity as the well characterized lactonase AiiA from Bacillus thuringiensis. We show that the fungal lactonases, similar to the bacterial enzymes, are kept intracellularly and act as a sink for the bacterial quorum sensing signals both in C. cinerea and in Saccharomyces cerevisiae expressing C. cinerea lactonases, due to the ability of these signal molecules to diffuse over the fungal cell wall and plasma membrane. The two isogenes coding for the C. cinerea HSL lactonases are arranged in the genome as a tandem repeat and expressed preferentially in vegetative mycelium. The occurrence of orthologous genes in genomes of other basidiomycetes appears to correlate with a saprotrophic lifestyle. Copyright © 2016 Elsevier Inc. All rights reserved.
Hansen, L J; Chalker, D L; Orlinsky, K J; Sandmeyer, S B
Ty3 is a Saccharomyces cerevisiae retrotransposon that integrates near the transcription initiation sites of polymerase III-transcribed genes. It is distinct from the copialike Ty1 and Ty2 retrotransposons of S. cerevisiae in both the sequences of encoded proteins and gene order. It is a member of the gypsylike family of retrotransposons which resemble animal retroviruses. This study was undertaken to investigate the nucleocapsid particle of a transpositionally active gypsylike retrotransposo...
Full Text Available Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is mainly transmitted to humans through an aerosol route. A spore-like form allows C. burnetii to resist different environmental conditions. Because of this, analysis of the survival strategies used by this bacterium to adapt to new environmental conditions is critical for our understanding of C. burnetii pathogenicity. Here, we report the early transcriptional response of C. burnetii under temperature stresses. Our data show that C. burnetii exhibited minor changes in gene regulation under short exposure to heat or cold shock. While small differences were observed, C. burnetii seemed to respond similarly to cold and heat shock. The expression profiles obtained using microarrays produced in-house were confirmed by quantitative RT-PCR. Under temperature stresses, 190 genes were differentially expressed in at least one condition, with a fold change of up to 4. Globally, the differentially expressed genes in C. burnetii were associated with bacterial division, (pppGpp synthesis, wall and membrane biogenesis and, especially, lipopolysaccharide and peptidoglycan synthesis. These findings could be associated with growth arrest and witnessed transformation of the bacteria to a spore-like form. Unexpectedly, clusters of neighboring genes were differentially expressed. These clusters do not belong to operons or genetic networks; they have no evident associated functions and are not under the control of the same promoters. We also found undescribed but comparable clusters of regulation in previously reported transcriptomic analyses of intracellular bacteria, including Rickettsia sp. and Listeria monocytogenes. The transcriptomic patterns of C. burnetii observed under temperature stresses permits the recognition of unpredicted clusters of regulation for which the trigger mechanism remains unidentified but which may be the result of a new mechanism of epigenetic regulation.
Hansen, L J; Chalker, D L; Orlinsky, K J; Sandmeyer, S B
Ty3 is a Saccharomyces cerevisiae retrotransposon that integrates near the transcription initiation sites of polymerase III-transcribed genes. It is distinct from the copialike Ty1 and Ty2 retrotransposons of S. cerevisiae in both the sequences of encoded proteins and gene order. It is a member of the gypsylike family of retrotransposons which resemble animal retroviruses. This study was undertaken to investigate the nucleocapsid particle of a transpositionally active gypsylike retrotransposon. Characterization of extracts from cells in which Ty3 expression was induced showed the presence of Ty3 nucleoprotein complexes, or viruslike particles, that migrated on linear sucrose gradients with a size of 156S. These particles are composed of Ty3 RNA, full-length, linear DNA, and proteins. In this study, antibodies raised against peptides predicted from the Ty3 sequence were used to identify Ty3-encoded proteins. These include the capsid (26 kDa), nucleocapsid (9 kDa), and reverse transcriptase (55 kDa) proteins. Ty3 integrase proteins of 61 and 58 kDa were identified previously (L. J. Hansen and S. B. Sandmeyer, J. Virol. 64:2599-2607, 1990). Reverse transcriptase activity associated with the particles was measured by using exogenous and endogenous primer-templates. Immunofluorescence studies of cells overexpressing Ty3 revealed cytoplasmic clusters of immunoreactive proteins. Transmission electron microscopy showed that Ty3 viruslike particles are about 50 nm in diameter. Thus, despite the unusual position specificity of Ty3 upstream of tRNA-coding regions, aspects of the Ty3 life cycle are fundamentally similar to those of retroviruses.
Jack L Leonard
Full Text Available Members of the Dickkopf (Dkk family of Wnt antagonists interrupt Wnt-induced receptor assembly and participate in axial patterning and cell fate determination. One family member, DKK3, does not block Wnt receptor activation. Loss of Dkk3 expression in cancer is associated with hyperproliferation and dysregulated ß-catenin signaling, and ectopic expression of Dkk3 halts cancer growth. The molecular events mediating the DKK3-dependent arrest of ß-catenin-driven cell proliferation in cancer cells are unknown. Here we report the identification of a new intracellular gene product originating from the Dkk3 locus. This Dkk3b transcript originates from a second transcriptional start site located in intron 2 of the Dkk3 gene. It is essential for early mouse development and is a newly recognized regulator of ß-catenin signaling and cell proliferation. Dkk3b interrupts nuclear translocation ß-catenin by capturing cytoplasmic, unphosphorylated ß-catenin in an extra-nuclear complex with ß-TrCP. These data reveal a new regulator of one of the most studied signal transduction pathways in metazoans and provides a novel, completely untapped therapeutic target for silencing the aberrant ß-catenin signaling that drives hyperproliferation in many cancers.
Russell Jacob A
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
Full Text Available Mutations in the immunoglobulin superfamily, member 1 gene (IGSF1/Igsf1 cause an X-linked form of central hypothyroidism. The canonical form of IGSF1 is a transmembrane glycoprotein with 12 immunoglobulin (Ig loops. The protein is co-translationally cleaved into two sub-domains. The carboxyl-terminal domain (CTD, which contains the last 7 Ig loops, is trafficked to the plasma membrane. Most pathogenic mutations in IGSF1 map to the portion of the gene encoding the CTD. IGSF1/Igsf1 encodes a variety of transcripts. A little studied, but abundant splice variant encodes a truncated form of the protein, predicted to contain the first 2 Ig loops of the full-length IGSF1. The protein (hereafter referred to as IGSF1 isoform 2 or IGSF1-2 is likely retained in most individuals with IGSF1 mutations. Here, we characterized basic biochemical properties of the protein as a foray into understanding its potential function. IGSF1-2, like the IGSF1-CTD, is a glycoprotein. In both mouse and rat, the protein is N-glycosylated at a single asparagine residue in the first Ig loop. Contrary to earlier predictions, neither the murine nor rat IGSF1-2 is secreted from heterologous or homologous cells. In addition, neither protein associates with the plasma membrane. Rather, IGSF1-2 appears to be retained in the endoplasmic reticulum. Whether the protein plays intracellular functions or is trafficked through the secretory pathway under certain physiologic or pathophysiologic conditions has yet to be determined.
Heissenberger, A.; Leppard, G. G.; Herndl, G. J.
The integrity of the intracellular structures and the presence and dimension of the capsular envelope were investigated in marine snow-associated and marine free-living bacteria by transmission electron microscopy and special fixation techniques. Three categories depending on the presence of internal structures were differentiated. In marine snow, 51% of the marine snow-associated bacterial community was considered intact, 26% had a partly degraded internal structure, and 23% were empty with ...
Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc
In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Langerak Ankie A
Full Text Available Abstract Background The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease. Results A collection of 26 strains of C. trachomatis of different serovars and clinical presentation and 18 strains of C. pneumoniae were included in the study. For comparison, sequences of C. abortus, C. psittaci, C. caviae, C. felis, C. pecorum (Chlamydophila, C. muridarum (Chlamydia and of Candidatus protochlamydia and Simkania negevensis were also included. Sequences of fragments (400 – 500 base pairs from seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX, oppA were analysed. Analysis of allelic profiles by eBurst revealed three non-overlapping clonal complexes among the C. trachomatis strains, while the C. pneumoniae strains formed a single group. An UPGMA tree produced from the allelic profiles resulted in three groups of sequence types. The LGV strains grouped in a single cluster, while the urogenital strains were distributed over two separated groups, one consisted solely of strains with frequent occurring serovars (E, D and F. The distribution of the different serovars over the three groups was not consistent, suggesting exchange of serovar encoding ompA sequences. In one instance, exchange of fumC sequences between strains of different groups was observed. Cluster analyses of concatenated sequences of the Chlamydophila and Chlamydia species together with those of Candidatus Protochlamydia amoebophila and Simkania
Pannekoek, Yvonne; Morelli, Giovanna; Kusecek, Barica; Morré, Servaas A; Ossewaarde, Jacobus M; Langerak, Ankie A; van der Ende, Arie
The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST) scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease. A collection of 26 strains of C. trachomatis of different serovars and clinical presentation and 18 strains of C. pneumoniae were included in the study. For comparison, sequences of C. abortus, C. psittaci, C. caviae, C. felis, C. pecorum (Chlamydophila), C. muridarum (Chlamydia) and of Candidatus protochlamydia and Simkania negevensis were also included. Sequences of fragments (400 - 500 base pairs) from seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX, oppA) were analysed. Analysis of allelic profiles by eBurst revealed three non-overlapping clonal complexes among the C. trachomatis strains, while the C. pneumoniae strains formed a single group. An UPGMA tree produced from the allelic profiles resulted in three groups of sequence types. The LGV strains grouped in a single cluster, while the urogenital strains were distributed over two separated groups, one consisted solely of strains with frequent occurring serovars (E, D and F). The distribution of the different serovars over the three groups was not consistent, suggesting exchange of serovar encoding ompA sequences. In one instance, exchange of fumC sequences between strains of different groups was observed. Cluster analyses of concatenated sequences of the Chlamydophila and Chlamydia species together with those of Candidatus Protochlamydia amoebophila and Simkania negevensis resulted in a tree identical to that
Full Text Available Memory CD8(+ T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m. Memory CD8(+ T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8(+ T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8(+ T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8(+ T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8(+ T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8(+ T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8(+ T produce inflammatory cytokines such as IFN-gamma and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8(+ T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8(+ T cells provide a local response by secreting effector molecules around infected cells.
Pannekoek, Yvonne; Morelli, Giovanna; Kusecek, Barica; Morré, Servaas A.; Ossewaarde, Jacobus M.; Langerak, Ankie A.; van der Ende, Arie
BACKGROUND: The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome.
Tung, Quach Ngoc; Linzner, Nico; Loi, Vu Van; Antelmann, Haike
Gram-negative bacteria utilize glutathione (GSH) as their major LMW thiol. However, most Gram-positive bacteria do not encode enzymes for GSH biosynthesis and produce instead alternative LMW thiols, such as bacillithiol (BSH) and mycothiol (MSH). BSH is utilized by Firmicutes and MSH is the major LMW thiol of Actinomycetes. LMW thiols are required to maintain the reduced state of the cytoplasm, but are also involved in virulence mechanisms in human pathogens, such as Staphylococcus aureus, Mycobacterium tuberculosis, Streptococcus pneumoniae, Salmonella enterica subsp. Typhimurium and Listeria monocytogenes. Infection conditions often cause perturbations of the intrabacterial redox balance in pathogens, which is further affected under antibiotics treatments. During the last years, novel glutaredoxin-fused roGFP2 biosensors have been engineered in many eukaryotic organisms, including parasites, yeast, plants and human cells for dynamic live-imaging of the GSH redox potential in different compartments. Likewise bacterial roGFP2-based biosensors are now available to measure the dynamic changes in the GSH, BSH and MSH redox potentials in model and pathogenic Gram-negative and Gram-positive bacteria. In this review, we present an overview of novel functions of the bacterial LMW thiols GSH, MSH and BSH in pathogenic bacteria in virulence regulation. Moreover, recent results about the application of genetically encoded redox biosensors are summarized to study the mechanisms of host-pathogen interactions, persistence and antibiotics resistance. In particularly, we highlight recent biosensor results on the redox changes in the intracellular food-borne pathogen Salmonella Typhimurium as well as in the Gram-positive pathogens S. aureus and M. tuberculosis during infection conditions and under antibiotics treatments. These studies established a link between ROS and antibiotics resistance with the intracellular LMW thiol-redox potential. Future applications should be directed
Galperin Michael Y
Full Text Available Abstract Background Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. Results This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. Conclusion The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the
Galperin, Michael Y
Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. This paper presents results of a comprehensive census of signal transduction proteins--histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases--encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the current leader Wolinella succinogenes
Giuditta Fiorella Schiavano
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.
Esposito-Polesi, Natalia Pimentel; de Abreu-Tarazi, Monita Fiori; de Almeida, Cristina Vieira; Tsai, Siu Mui; de Almeida, Marcílio
Asepsis, defined as the absence of microbial contamination, is one of the most important requirements of plant micropropagation. In long-term micropropagated cultures, there may occasionally occur scattered microorganism growth in the culture medium. These microorganisms are common plant components and are known as latent endophytes. Thus, the aim of this research was to investigate the presence of endophytic bacteria in asymptomatic pineapple and orchid microplants, which were cultivated in three laboratories for 1 year. Isolation and characterization of bacterial isolates, PCR-DGGE from total genomic DNA of microplants and ultrastructural analysis of leaves were performed. In the culture-dependent technique, it was only possible to obtain bacterial isolates from pineapple microplants. In this case, the bacteria genera identified in the isolation technique were Bacillus, Acinetobacter, and Methylobacterium. The scanning electron microscopy and transmission electron microscopy (SEM and TEM) analyses revealed the presence of endophytic bacteria in intracellular spaces in the leaves of pineapple and orchid microplants, independent of the laboratory or cultivation protocol. Our results strongly indicate that there are endophytic bacterial communities inhabiting the microplants before initiation of the in vitro culture and that some of these endophytes persist in their latent form and can also grow in the culture medium even after long-term micropropagation, thus discarding the concept of "truly axenic plants."
Koskimäki, Janne J; Pirttilä, Anna Maria; Ihantola, Emmi-Leena; Halonen, Outi; Frank, A Carolin
Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside
Omar, Atef; Zhang, Qianqian; Zou, Songbao; Gong, Jun
The morphology and infraciliature of a new ciliate, Metopus yantaiensis n. sp., discovered in coastal soil of northern China, were investigated. It is distinguished from its congeners by a combination of the following features: nuclear apparatus situated in the preoral dome; 18-21 somatic ciliary rows, of which three extend onto the preoral dome (dome kineties); three to five distinctly elongated caudal cilia, and 21-29 adoral polykinetids. The 18S rRNA genes of this new species and two congeners, Metopus contortus and Metopus hasei, were sequenced and phylogenetically analyzed. The new species is more closely related to M. hasei and the clevelandellids than to other congeners; both the genus Metopus and the order Metopida are not monophyletic. In addition, the digestion-resistant bacteria in the cytoplasm of M. yantaiensis were identified, using a 16S rRNA gene clone library, sequencing, and fluorescence in situ hybridization. The detected intracellular bacteria are affiliated with Sphingomonadales, Rhizobiales, Rickettsiales (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria), Rhodocyclales (Betaproteobacteria), Clostridiales (Firmicutes), and Flavobacteriales (Bacteroidetes). © 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.
Marion Le Coadic
Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.
Hackmann, Timothy J; Ngugi, David Kamanda; Firkins, Jeffrey L; Tao, Junyi
Bacteria have been thought to follow only a few well-recognized biochemical pathways when fermenting glucose or other hexoses. These pathways have been chiseled in the stone of textbooks for decades, with most sources rendering them as they appear in the classic 1986 text by Gottschalk. Still, it is unclear how broadly these pathways apply, given that they were established and delineated biochemically with only a few model organisms. Here, we show that well-recognized pathways often cannot explain fermentation products formed by bacteria. In the most extensive analysis of its kind, we reconstructed pathways for glucose fermentation from genomes of 48 species and subspecies of bacteria from one environment (the rumen). In total, 44% of these bacteria had atypical pathways, including several that are completely unprecedented for bacteria or any organism. In detail, 8% of bacteria had an atypical pathway for acetate formation; 21% of bacteria had an atypical pathway for propionate or succinate formation; 6% of bacteria had an atypical pathway for butyrate formation and 33% of bacteria had an atypical or incomplete Embden-Meyerhof-Parnas pathway. This study shows that reconstruction of metabolic pathways - a common goal of omics studies - could be incorrect if well-recognized pathways are used for reference. Furthermore, it calls for renewed efforts to delineate fermentation pathways biochemically. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.
Plourde-Owobi, Lucile; Durner, Sophie; Parrou, Jean-Luc; Wieczorke, Roman; Goma, Gerard; François, Jean
The trehalose content in Saccharomyces cerevisiae can be significantly manipulated by including trehalose at an appropriate level in the growth medium. Its uptake is largely dependent on the expression of AGT1, which encodes an α-glucoside transporter. The trehalose found in a tps1 mutant of trehalose synthase may therefore largely reflect its uptake from the enriched medium that was employed.
Full Text Available Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm infection model. Our data show that Hdac6-/- bone marrow-derived dendritic cells (BMDCs have a higher bacterial load than Hdac6+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR. Compared with Hdac6+/+ counterparts, Hdac6-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88, and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection.
The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages.
Auricelio A Macedo
Full Text Available Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi. In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment.
Hackmann, Timothy J.
Bacteria have been thought to follow only a few well-recognized biochemical pathways when fermenting glucose or other hexoses. These pathways have been chiseled in the stone of textbooks for decades, with most sources rendering them as they appear in the classic 1986 text by Gottschalk. Still, it is unclear how broadly these pathways apply, given that they were established and delineated biochemically with only a few model organisms. Here we show that well-recognized pathways often cannot explain fermentation products formed by bacteria. In the most extensive analysis of its kind, we reconstructed pathways for glucose fermentation from genomes of 48 species and subspecies of bacteria from one environment (the rumen). In total, 44% of these bacteria had atypical pathways, including several that are completely unprecedented for bacteria or any organism. In detail, 8% of bacteria had an atypical pathway for acetate formation; 21% for propionate or succinate formation; 6% for butyrate formation; and 33% had an atypical or incomplete Embden-Meyerhof-Parnas pathway. This study shows that reconstruction of metabolic pathways-a common goal of omics studies-could be incorrect if well-recognized pathways are used for reference. Further, it calls for renewed efforts to delineate fermentation pathways biochemically. This article is protected by copyright. All rights reserved.
Declerck, P; Behets, J; Delaedt, Y; Margineanu, A; Lammertyn, E; Ollevier, F
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.
Nishith Kumar Pal
Full Text Available The present study investigates the occurrence of R-plasmid in Salmonella typhi isolates from enteric fever cases in and around Kolkata (1991-2001, India following in vitro conjugation experiments, isolation of plasmid DNAs and agarose gel electrophoretic analysis. The multidrug resistant (MDR S. typhi strains contained a transferable plasmid conferring resistance to ampicillin, chloramphenicol, cotrimoxazole and tetracycline. The plasmid encoding ACCoT-resistance of Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris were conjugative and co-migrated with the plasmid of MDR S. typhi isolates. The antibiotic sensitive S. typhi isolates did not contain any plasmid. Thus the findings of the present study confirmed the instability of the R-plasmid in S. typhi, and that the antibiotic sensitive S. typhi strains acquired the R-plasmid from other enteric bacteria such as E. coli, K. pneumoniae and P.vulgaris to undergo a suitable adaptation for survival in the changing antibiotic environment.
Das, Debaditya; Bishayi, Biswadev
To determine the interrelationship between the hydrogen peroxide (H(2)O(2)) mediated killing and the potential role of bacterial catalase and SOD in the evasion of host defense, we examined three clinical isolates of Staphylococcus aureus and evaluated their intracellular survival mechanism within murine peritoneal macrophages. Fluorescent microscopy and bacterial colony-forming unit (cfu) count revealed that phagocytic capacity of murine peritoneal macrophages was highest after 2h of in vitro infection with S. aureus. To understand whether catalase and SOD contributing in the intracellular survival, were of bacterial origin or not, 3 amino 1,2,4 triazole (ATZ) and Diethyldithiocarbamic acid (DDC) were used to inhibit specifically macrophage derived catalase and SOD respectively. Catalase activity from the whole staphylococcal cell in presence of ATZ suggested that the released catalase were of extracellular origin. Scanning electron microscopy revealed the degraded host cell membrane integrity during prolonged infection. Purified bacterial catalase from the intracellularly survived S. aureus recovered after 5h of infection and its inhibition by ATZ in the zymography strengthened the scope of involvement of these anti-oxidants in the intracellular survival of S. aureus.
Vahling-Armstrong, Cheryl M.; Zhou, Huasong; Benyon, Lesley; Morgan, J. Kent; Duan, Yongping
The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems w...
Mariza M. Coêlho
Full Text Available Endophytic bacteria from three arboreal species native to the Amazon (Carapa guianenses, Ceiba pentandra, and Swietenia macrophylla, were isolated and identified, through partial sequencing of the 16S rRNA encoding gene. From these, 16 isolates were obtained, although, when compared to sequences deposited in GenBank, only seven had produced identifiable fragments. Bacillus, Pantoea and two non-culturable samples were identified. Results obtained through sequence analysis revealed low genetic diversity across the isolates, even when analyzing different species and plant structures. This is the first report concerning the isolation and identification of endophytic bacteria in these plant species.
Wood, R M; Egan, J R; Hall, I M
In a novel approach, the standard birth-death process is extended to incorporate a fundamental mechanism undergone by intracellular bacteria, phagocytosis. The model accounts for stochastic interaction between bacteria and cells of the immune system and heterogeneity in susceptibility to infection of individual hosts within a population. Model output is the dose-response relation and the dose-dependent distribution of time until response, where response is the onset of symptoms. The model is thereafter parametrized with respect to the highly virulent Schu S4 strain of Francisella tularensis, in the first such study to consider a biologically plausible mathematical model for early human infection with this bacterium. Results indicate a median infectious dose of about 23 organisms, which is higher than previously thought, and an average incubation period of between 3 and 7 days depending on dose. The distribution of incubation periods is right-skewed up to about 100 organisms and symmetric for larger doses. Moreover, there are some interesting parallels to the hypotheses of some of the classical dose-response models, such as independent action (single-hit model) and individual effective dose (probit model). The findings of this study support experimental evidence and postulations from other investigations that response is, in fact, influenced by both in-host and between-host variability.
Gong, Jun; Qing, Yao; Guo, Xiaohong; Warren, Alan
An intracellular bacterium was discovered in an isolate of Paramecium bursaria from a freshwater pond in Yantai, China. The bacteria were abundant and exclusively found in the cytoplasm of the host which, along with the green alga Chlorella, formed a three-partner consortium that could survive in pure water for at least one week. Cloning, sequencing and phylogenetic analysis of the bacterial 16S rRNA gene showed that the bacterium belonged to the uncultured candidate division OD1, which usually forms part of the rare biosphere. Transmission electron microscopy and fluorescence in situ hybridization (FISH) with specific probes showed that the bacteria were usually located close to the perialgal membranes of endosymbiotic Chlorella cells, and occasionally irregularly distributed throughout the host cytoplasm. The name "Candidatus Sonnebornia yantaiensis" gen. nov., sp. nov. is proposed for the new bacterium. A strongly supported monophyletic subclade, OD1-p, which included the new species, was recognized and this study highlights that protists can be important hosts for rare bacterial taxa. Copyright © 2013 Elsevier GmbH. All rights reserved.
Full Text Available Abstract Background Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components. Results A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters. Conclusion We propose that the CscA, CscB, CscC and Csc
Pham, Oanh H; O'Donnell, Hope; Al-Shamkhani, Aymen; Kerrinnes, Tobias; Tsolis, Renée M; McSorley, Stephen J
Th1 cells can be activated by TCR-independent stimuli, but the importance of this pathway in vivo and the precise mechanisms involved require further investigation. Here, we used a simple model of non-cognate Th1 cell stimulation in Salmonella-infected mice to examine these issues. CD4 Th1 cell expression of both IL-18R and DR3 was required for optimal IFN-γ induction in response to non-cognate stimulation, while IL-15R expression was dispensable. Interestingly, effector Th1 cells generated by immunization rather than live infection had lower non-cognate activity despite comparable IL-18R and DR3 expression. Mice lacking T cell intrinsic expression of MyD88, an important adapter molecule in non-cognate T cell stimulation, exhibited higher bacterial burdens upon infection with Salmonella, Chlamydia or Brucella, suggesting that non-cognate Th1 stimulation is a critical element of efficient bacterial clearance. Thus, IL-18R and DR3 are critical players in non-cognate stimulation of Th1 cells and this response plays an important role in protection against intracellular bacteria.
Andrade, Angel; Valvano, Miguel A
Burkholderia cenocepacia infects patients with cystic fibrosis. We have previously shown that B. cenocepacia can survive in macrophages within membrane vacuoles [B. cenocepacia-containing vacuoles (BcCVs)] that preclude fusion with the lysosome. The bacterial factors involved in B. cenocepacia intracellular survival are not fully elucidated. We report here that deletion of BCAM0628, encoding a predicted low molecular weight protein tyrosine phosphatase (LMW-PTP) that is restricted to B. cenocepacia strains of the transmissible ET-12 clone, accelerates the maturation of the BcCVs. Compared to the parental strain and deletion mutants in other LMW-PTPs that are widely conserved in Burkholderia species, a greater proportion of BcCVs containing the ΔBCAM0628 mutant were targeted to the lysosome. Accelerated BcCV maturation was not due to reduced intracellular viability since ΔBCAM0628 survived and replicated in macrophages similarly to the parental strain. Therefore, BCAM0628 was referred to as dpm (delayed phagosome maturation). We provide evidence that the Dpm protein is secreted during growth in vitro and upon macrophage infection. Dpm secretion requires an N-terminal signal peptide. Heterologous expression of Dpm in Burkholderia multivorans confers to this bacterium a similar phagosomal maturation delay to that found with B. cenocepacia. We demonstrate that Dpm is an inactive phosphatase, suggesting that its contribution to phagosomal maturation arrest must be unrelated to tyrosine phosphatase activity. © 2014 The Authors.
Singh, M B; Hough, T; Theerakulpisut, P; Avjioglu, A; Davies, S; Smith, P M; Taylor, P; Simpson, R J; Ward, L D; McCluskey, J
We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE...
Singh, M B; Hough, T; Theerakulpisut, P; Avjioglu, A; Davies, S; Smith, P M; Taylor, P; Simpson, R J; Ward, L D; McCluskey, J
We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain. Images PMID:1671715
Halio, S B; Blumentals, I I; Short, S A; Merrill, B M; Kelly, R M
A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95 degrees C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95 degrees C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98 degrees C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5' end of the transcript. There are only 10
Núñez Hernández, Cristina; Alonso, Ana; Pucciarelli, María Graciela; Casadesús Pursals, Josep; García del Portillo, Francisco
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...
Sebastien P Faucher
Full Text Available Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot Type IVB secretion system (TFBSS, which is used to translocate more than 150 effector proteins to host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called SCOTS to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to L-arginine, L-histidine and L-proline as well as many transport systems involved in amino acid and iron uptake. Gene involved in catabolism of glycerol is also induced during intracellular growth and could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.
Bessoles, Stéphanie; Ni, Ming; Garcia-Jimenez, Sara; Sanchez, Françoise; Lafont, Virginie
Human Vγ9Vδ2 T cells play a crucial role in early immune response to intracellular pathogens. Their number is drastically increased in the peripheral blood of patients during the acute phase of brucellosis. In vitro, Vγ9Vδ2 T cells exhibit strong cytolytic activity against Brucella-infected cells and impair intracellular growth of Brucella suis in autologous macrophages. Vγ9Vδ2 T cells use cell contact-dependent mechanisms such as the release of lytic granules and Fas-mediated signals to lyse infected macrophages and decrease the development of intracellular Brucella. Although the involvement of the T-cell receptor (TCR) in the triggering of these responses is known, other surface receptors can modulate Vγ9Vδ2 T-cell response. In this study, we have investigated a potential role of NKG2D and its ligands in the anti-infectious activity of human Vγ9Vδ2 T cells against B. suis. We show that the recruitment of NKG2D by its ligands is sufficient to induce cytokine production and the release of lytic granules through PI3K-dependent pathways, but can also increase the TCR-triggered responses of Vγ9Vδ2 T cells. We also demonstrate that the interaction between NKG2D and its main ligand expressed on Brucella-infected macrophages, UL16-binding protein 1 (ULBP1), is involved in the inhibition of bacterium development. Altogether, these results suggest a direct contribution of NKG2D and its ligands to the anti-infectious activity of Vγ9Vδ2 T cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Hongo, Yuki; Ikuta, Tetsuro; Takaki, Yoshihiro; Shimamura, Shigeru; Shigenobu, Shuji; Maruyama, Tadashi; Yoshida, Takao
Deep-sea vesicomyid clams, including the genus Phreagena (formerly Calyptogena), harbor thioautotrophic bacterial symbionts in the host symbiosome, which consists of cytoplasmic vacuoles in gill epithelial cells called bacteriocytes. The symbiont requires inorganic carbon (Ci), such as CO2, HCO3(-), and CO3(2-), to synthesize organic compounds, which are utilized by the host clam. The dominant Ci in seawater is HCO3(-), which is impermeable to cell membranes. Within the bacteriocyte, cytoplasmic carbonic anhydrase (CA) from the host, which catalyzes the inter-conversion between CO2 and HCO3(-), has been shown to be abundant and is thought to supply intracellular CO2 to symbionts in the symbiosome. However, the mechanism of Ci uptake by the host gill from seawater is poorly understood. To elucidate the influx pathway of Ci into the bacteriocyte, we isolated the genes related to Ci uptake via the pyrosequencing of cDNA from the gill of Phreagena okutanii, and investigated their expression patterns. Using phylogenetic and amino acid sequence analyses, three solute carrier family 4 (SLC4) bicarbonate transporters (slc4co1, slc4co2, and slc4co4) and two membrane-associated CAs (mcaco1 and mcaco2) were identified as candidate genes for Ci uptake. In an in situ hybridization analysis of gill sections, the expression of mcaco1 and mcaco2 was detected in the bacteriocytes and asymbiotic non-ciliated cells, respectively, and the expression of slc4co1 and slc4co2 was detected in the asymbiotic cells, including the intermediate cells of the inner area and the non-ciliated cells of the external area. Although subcellular localizations of the products of these genes have not been fully elucidated, they may play an important role in the uptake of Ci into the bacteriocytes. These findings will improve our understanding of the Ci transport system in the symbiotic relationships of chemosynthetic bivalves. Copyright © 2016 Elsevier B.V. All rights reserved.
Núñez-Hernández, Cristina; Alonso, Ana; Pucciarelli, M Graciela; Casadesús, Josep; García-del Portillo, Francisco
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.
Russell, Jay H.; Keiler, Kenneth C.
Many bacterial proteins are localized to precise intracellular locations, but in most cases the mechanism for encoding localization information is not known. Screening libraries of peptides fused to green fluorescent protein identified sequences that directed the protein to helical structures or to midcell. These peptides indicate that protein localization can be encoded in 20-amino-acid peptides instead of complex protein-protein interactions and raise the possibility that the location of a ...
Færgeman, Nils J.; Black, P N; Zhao, X D
CoA levels were defined following incubation of wild type and mutant cells with limiting concentrations of exogenous oleate. These studies demonstrated oleoyl CoA levels were reduced to less than 10% wild-type levels in faa1 Delta and faa1 Delta faa4 Delta strains. Defects in metabolic utilization...... and intracellular trafficking were also found in the fatty acyl-CoA synthetase-deficient strains. The faa1 Delta faa4 Delta strain had a marked reduction in endogenous acyl-CoA pools, suggesting these enzymes play a role in maintenance of endogenous acyl-CoA pools, metabolism and trafficking. In addition...
Wen, Zhifeng; Yao, Liping; Wan, Ran; Li, Zhi; Liu, Chonghuai; Wang, Xiping
Plant resistance proteins mediate pathogen recognition and activate innate immune responses to restrict pathogen proliferation. One common feature of these proteins is an NB-ARC domain. In this study, we characterized a gene encoding a protein with an NB-ARC domain from wild Chinese grapevine Vitis pseudoreticulata accession "Baihe-35-1," which was identified in a transcriptome analysis of the leaves following inoculation with Erysiphe necator (Schw.), a causal agent of powdery mildew. Transcript levels of this gene, designated VpCN (GenBank accession number KT265084), increased strongly after challenge of grapevine leaves with E. necator. The deduced amino acid sequence was predicted to contain an NB-ARC domain in the C-terminus and an RxCC-like domain similar to CC domain of Rx protein in the N-terminus. Ectopic expression of VpCN in Arabidopsis thaliana resulted in either a wild-type phenotype or a dwarf phenotype. The phenotypically normal transgenic A. thaliana showed enhance resistance to A. thaliana powdery mildew Golovinomyces cichoracearum, as well as to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Moreover, promoter::GUS (β-glucuronidase) analysis revealed that powdery mildew infection induced the promoter activity of VpCN in grapevine leaves. Finally, a promoter deletion analysis showed that TC rich repeat elements likely play an important role in the response to E. necator infection. Taken together, our results suggest that VpCN contribute to powdery mildew disease resistant in grapevine.
Full Text Available Plant resistance proteins mediate pathogen recognition and activate innate immune responses to restrict pathogen proliferation. One common feature of these proteins is an NB-ARC domain. In this study, we characterized a gene encoding a protein with an NB-ARC domain from wild Chinese grapevine Vitis pseudoreticulata accession Baihe-35-1, which was identified in a transcriptome analysis of the leaves following inoculation with Erysiphe necator (Schw., a causal agent of powdery mildew. Transcript levels of this gene, designated VpCN (GenBank accession number KT265084, increased strongly after challenge of grapevine leaves with E. necator. The deduced amino acid sequence was predicted to contain an NB-ARC domain in the C-terminus and an RxCC-like domain similar to CC domain of Rx protein in the N-terminus. Ectopic expression of VpCN in Arabidopsis thaliana resulted in either a wild-type phenotype or a dwarf phenotype. The phenotypically normal transgenic A. thaliana showed enhance resistance to A. thaliana powdery mildew Golovinomyces cichoracearum, as well as to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Moreover, promoter::GUS (β-glucuronidase analysis revealed that powdery mildew infection induced the promoter activity of VpCN in grapevine leaves. Finally, a promoter deletion analysis showed that TC rich repeat elements likely play an important role in the response to E. necator infection. Taken together, our results suggest that VpCN contribute to powdery mildew disease resistant in grapevine.
García-Angulo, Víctor Antonio
Riboflavin derivatives are essential cofactors for a myriad of flavoproteins. In bacteria, flavins importance extends beyond their role as intracellular protein cofactors, as secreted flavins are a key metabolite in a variety of physiological processes. Bacteria obtain riboflavin through the endogenous riboflavin biosynthetic pathway (RBP) or by the use of importer proteins. Bacteria frequently encode multiple paralogs of the RBP enzymes and as for other micronutrient supply pathways, biosynthesis and uptake functions largely coexist. It is proposed that bacteria shut down biosynthesis and would rather uptake riboflavin when the vitamin is environmentally available. Recently, the overlap of riboflavin provisioning elements has gained attention and the functions of duplicated paralogs of RBP enzymes started to be addressed. Results point towards the existence of a modular structure in the bacterial riboflavin supply pathways. Such structure uses subsets of RBP genes to supply riboflavin for specific functions. Given the importance of riboflavin in intra and extracellular bacterial physiology, this complex array of riboflavin provision pathways may have developed to contend with the various riboflavin requirements. In riboflavin-prototrophic bacteria, riboflavin transporters could represent a module for riboflavin provision for particular, yet unidentified processes, rather than substituting for the RBP as usually assumed.
Herrick, J B; Haynes, R; Heringa, S; Brooks, J M; Sobota, L T
Transmissible plasmids captured from stream and soil bacteria conferring resistance to tetracycline in Pseudomonas were evaluated for linked resistance to antibiotics used in the treatment of human infections. Cells released from stream sediments and soils were conjugated with a rifampicin-resistant, plasmid-free Pseudomonas putida recipient and selected on tetracycline and rifampicin. Each transconjugant contained a single 50-80 kb plasmid. Resistance to 11 antibiotics, in addition to tetracycline, was determined for the stream transconjugants using a modification of the Stokes disc diffusion antibiotic susceptibility assay. Nearly half of plasmids conferred resistance to six or more antibiotics. Resistance to streptomycin, gentamicin, and/or ticarcillin was conferred by a majority of the plasmids, and resistance to additional human clinical use antibiotics such as piperacillin/tazobactam, ciprofloxacin and aztreonam was observed. MICs of 16 antibiotics for representative sediment and soil transconjugants revealed large increases, relative to the Ps. putida recipient, for 11 of 16 antibiotics tested, including the expanded spectrum antibiotics cefotaxime and ceftazidime, as well as piperacillin/tazobactam, lomefloxacin and levofloxacin. Resistance to multiple antibiotics-including those typically used in clinical Pseudomonas and enterobacterial infections-can be conferred by transmissible plasmids in streams and soils. Selective pressure exerted by the use of one antibiotic, such as the common agricultural antibiotic tetracycline, may result in the persistence of linked genes conferring resistance to important human clinical antibiotics. This may impact the spread of resistance to human use antibiotics even in the absence of direct selection. © 2014 The Society for Applied Microbiology.
D'Astolfo, Diego S; Pagliero, Romina J; Pras, Anita; Karthaus, Wouter R; Clevers, Hans; Prasad, Vikram; Lebbink, Robert Jan; Rehmann, Holger; Geijsen, Niels
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
Jun 6, 2011 ... Bioplastics are naturally occurring biodegradable polymers made from polyhydroxyalkanoates (PHA) of which poly 3-hydroxy butyric acid ... The plastic polymers accumulate intracellularly as light- refracting amorphous ... study focuses on the isolation and identification of novel species of bacteria capable ...
Hanson, Brett R; Tan, Ming
Bacteria encode heat shock proteins that aid in survival during stressful growth conditions. In addition, the major heat shock proteins of the intracellular bacterium Chlamydia trachomatis have been associated with immune pathology and disease. We developed a ChIP-qPCR method to study the regulation of chlamydial heat shock gene regulation during an intracellular infection. This approach allowed us to show that chlamydial heat shock genes are regulated by the transcription factor HrcA within an infected cell, providing validation for previous in vitro findings. Induction of chlamydial heat shock gene expression by elevated temperature was due to loss of HrcA binding to heat shock promoters, supporting a mechanism of derepression. This heat shock response was rapid, whereas recovery of HrcA binding and return to non-stress transcript levels occurred more slowly. We also found that control of heat shock gene expression was differentially regulated over the course of the intracellular Chlamydia infection. There was evidence of HrcA-mediated regulation of heat shock genes throughout the chlamydial developmental cycle, but the level of repression was lower at early times. This is the first study of Chlamydia-infected cells showing the effect of an environmental signal on transcription factor-DNA binding and target gene expression in the bacterium. © 2015 John Wiley & Sons Ltd.
Christopher C. Thompson
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.
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.
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.
Faherty, Christina S; Faherty, Christina; Harper, Jill M; Shea-Donohue, Terez; Barry, Eileen M; Kaper, James B; Fasano, Alessio; Nataro, James P
Shigella flexneri is a Gram-negative, facultative intracellular pathogen that causes millions of cases of watery or bloody diarrhea annually, resulting in significant global mortality. Watery diarrhea is thought to arise in the jejunum, and subsequent bloody diarrhea occurs as a result of invasion of the colonic epithelium. Previous literature has demonstrated that Shigella encodes enterotoxins, both chromosomally and on the 220 kilobase virulence plasmid. The ShigellaEnterotoxins 1 and 2 (ShET1 and ShET2) have been shown to increase water accumulation in the rabbit ileal loop model. In addition, these toxins increase the short circuit current in rabbit tissue mounted in Ussing chambers, which is a model for the ion exchange that occurs during watery diarrhea. In this study, we sought to validate the use of mouse jejunum in Ussing chamber as an alternative, more versatile model to study bacterial pathogenesis. In the process, we also identified enterotoxins in addition to ShET1 and ShET2 encoded by S. flexneri. Through analysis of proteins secreted from wildtype bacteria and various deletion mutants, we have identified four factors responsible for enterotoxin activity: ShET1 and Pic, which are encoded on the chromosome; ShET2 (encoded by sen or ospD3), which requires the type-III secretion system for secretion; and SepA, an additional factor encoded on the virulence plasmid. The use of mouse jejunum serves as a reliable and reproducible model to identify the enterotoxins elaborated by enteric bacteria. Moreover, the identification of all Shigella proteins responsible for enterotoxin activity is vital to our understanding of Shigella pathogenicity and to our success in developing safe and effective vaccine candidates.
Christina S Faherty
Full Text Available Shigella flexneri is a Gram-negative, facultative intracellular pathogen that causes millions of cases of watery or bloody diarrhea annually, resulting in significant global mortality. Watery diarrhea is thought to arise in the jejunum, and subsequent bloody diarrhea occurs as a result of invasion of the colonic epithelium. Previous literature has demonstrated that Shigella encodes enterotoxins, both chromosomally and on the 220 kilobase virulence plasmid. The ShigellaEnterotoxins 1 and 2 (ShET1 and ShET2 have been shown to increase water accumulation in the rabbit ileal loop model. In addition, these toxins increase the short circuit current in rabbit tissue mounted in Ussing chambers, which is a model for the ion exchange that occurs during watery diarrhea. In this study, we sought to validate the use of mouse jejunum in Ussing chamber as an alternative, more versatile model to study bacterial pathogenesis. In the process, we also identified enterotoxins in addition to ShET1 and ShET2 encoded by S. flexneri. Through analysis of proteins secreted from wildtype bacteria and various deletion mutants, we have identified four factors responsible for enterotoxin activity: ShET1 and Pic, which are encoded on the chromosome; ShET2 (encoded by sen or ospD3, which requires the type-III secretion system for secretion; and SepA, an additional factor encoded on the virulence plasmid. The use of mouse jejunum serves as a reliable and reproducible model to identify the enterotoxins elaborated by enteric bacteria. Moreover, the identification of all Shigella proteins responsible for enterotoxin activity is vital to our understanding of Shigella pathogenicity and to our success in developing safe and effective vaccine candidates.
Selman, Mohammed; Pombert, Jean-François; Solter, Leellen; Farinelli, Laurent; Weiss, Louis M.; Keeling, Patrick; Corradi, Nicolas
Parasites have adapted to their specialised way of life by a number of means, including the acquisition of genes by horizontal gene transfer. These newly acquired genes seem to come from a variety of sources, but seldom from the host, even in the most intimate associations between obligate intracellular parasite and host . Microsporidian intracellular parasites have acquired a handful of genes, mostly from bacteria, that help them take energy from their hosts or protect them from the envir...
Full Text Available It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term “protozoan-priming.” We sought to identify L.p. virulence factors that were required for replication in amoebae in order to highlight the genes necessary for production of the most infectious form of the bacterium. Using a transposon mutagenesis screen, we successfully identified 12 insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icm type IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages, revealing attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 were required for survival and replication in amoebae and macrophage host cells. Both genes are conserved among numerous important human pathogenic bacteria that can persist or replicate in amoebae. Each gene encodes a component of an ATP binding cassette (ABC transport complex of unknown function. We demonstrate the lpg0730 ortholog in Francisella tularensis subsp. novicida to be essential for colonization of both protozoan and mammalian host cells, highlighting conserved survival mechanisms employed by bacteria that utilize protozoa as an environmental reservoir for replication.
Bahnsen, Jesper S; Franzyk, Henrik; Sayers, Edward J
. 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...
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
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.
Barisch, Caroline; Soldati, Thierry
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
Durmaz, Evelyn; Hu, Yan; Aroian, Raffi V; Klaenhammer, Todd R
The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe. Copyright © 2016 Durmaz et al.
Durmaz, Evelyn; Hu, Yan; Aroian, Raffi V.
The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe. PMID:26682852
Swe, Pearl M; Heng, Nicholas C K; Ting, Yi-Tian; Baird, Hayley J; Carne, Alan; Tauch, Andreas; Tagg, John R; Jack, Ralph W
Unlike the colicins, microcins and related peptide antibiotics, little is known about antibiotic proteins (M(r)>10,000) from Gram-positive bacteria, since only few examples have been described to date. In this study we used heterologous expression of recombinant proteins to access the 17 kDa antibiotic protein SA-M57 from Streptococcus pyogenes, along with two proteins of unknown function identified in publicly available databases: EF1097 from Enterococcus faecalis and YpkK from Corynebacterium jeikeium. Here we show that all three are antibiotic proteins with different spectra of antimicrobial activity that kill sensitive bacteria at nanomolar concentrations. In silico structure predictions indicate that although the three proteins share little sequence similarity, they may be composed of conserved secondary structural elements: a relatively unstructured, acidic N-terminal portion and a basic C-terminal portion characterized by two helical elements separated by a loop structure and stabilized by an essential disulphide. Expression of individual segments as well as protein chimaeras revealed that, at least in the case of YpkK, the C-terminal portion is responsible for the killing action of the protein, whereas the role of the N-terminal portion remains unclear. Both scnM57 and ef1097 appear to be widely distributed in Strep. pyogenes and Ent. faecalis (respectively), whereas ypkK is found only rarely amongst clinical isolates of C. jeikeium. Finally, we determined that the proteins kill sensitive bacteria without lysis, a feature that distinguishes them from known murolytic proteins.
Pareja, Maria Eugenia Mansilla; Colombo, Maria I
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.
Horner, T. J.; Lee, R. B.; Henderson, G. M.; Rickaby, R. E.
Recent stable isotope studies into the biological utilization of transition metals (e.g. Cu, Fe, Zn, Cd) suggest several stepwise cellular processes can fractionate isotopes in both culture and nature. However, the determination of fractionation factors is often unsatisfactory, as significant variability can exist - even between different organisms with the same cellular functions. Thus, it has not been possible to adequately understand the source and mechanisms of metal isotopic fractionation. In order to address this problem, we investigated the biological fractionation of Cd isotopes within genetically-modified bacteria (E. coli). There is currently only one known biological use or requirement of Cd, a Cd/Zn carbonic anhydrase (CdCA, from the marine diatom T. weissfloggii), which we introduce into the E. coli genome. We have also developed a cleaning procedure that allows for the treating of bacteria so as to study the isotopic composition of different cellular components. We find that whole cells always exhibit a preference for uptake of the lighter isotopes of Cd. Notably, whole cells appear to have a similar Cd isotopic composition regardless of the expression of CdCA within the E. coli. However, isotopic fractionation can occur within the genetically modified E. coli during Cd use, such that Cd bound in CdCA can display a distinct isotopic composition compared to the cell as a whole. Thus, the externally observed fractionation is independent of the internal uses of Cd, with the largest Cd isotope fractionation occurring during cross-membrane transport. A general implication of these experiments is that trace metal isotopic fractionation most likely reflects metal transport into biological cells (either actively or passively), rather than relating to expression of specific physiological function and genetic expression of different metalloenzymes.
Hincha Dirk K
Full Text Available Abstract Background LEA (late embryogenesis abundant proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown. Results We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE and/or low temperature response (LTRE elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded. Conclusion The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for
Constitutive heterologous overexpression of a TIR-NB-ARC-LRR gene encoding a putative disease resistance protein from wild Chinese Vitis pseudoreticulata in Arabidopsis and tobacco enhances resistance to phytopathogenic fungi and bacteria.
Wen, Zhifeng; Yao, Liping; Singer, Stacy D; Muhammad, Hanif; Li, Zhi; Wang, Xiping
Plants use resistance (R) proteins to detect pathogen effector proteins and activate their innate immune response against the pathogen. The majority of these proteins contain an NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain along with a leucine-rich repeat (LRR), and some also bear a toll interleukin 1 receptor (TIR) domain. In this study, we characterized a gene encoding a TIR-NB-ARC-LRR R protein (VpTNL1) (GenBank accession number KX649890) from wild Chinese grapevine Vitis pseudoreticulata accession "Baihe-35-1", which was identified previously from a transcriptomic analysis of leaves inoculated with powdery mildew (PM; Erysiphe necator (Schw.)). The VpTNL1 transcript was found to be highly induced in V. pseudoreticulata following inoculation with E. necator, as well as treatment with salicylic acid (SA). Sequence analysis demonstrated that the deduced amino acid sequence contained a TIR domain at the N-terminus, along with an NB-ARC and four LRRs domains within the C-terminus. Constitutive expression of VpTNL1 in Arabidopsis thaliana resulted in either a wild-type or dwarf phenotype. Intriguingly, the phenotypically normal transgenic lines displayed enhanced resistance to Arabidopsis PM, Golovinomyces cichoracearum, as well as to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Similarly, constitutive expression of VpTNL1 in Nicotiana tabacum was found to confer enhanced resistance to tobacco PM, Erysiphe cichoacearum DC. Subsequent isolation of the VpTNL1 promoter and deletion analysis indicated that TC-rich repeats and TCA elements likely play an important role in its response to E. necator and SA treatment, respectively. Taken together, these results indicate that VpTNL1 contributes to PM resistance in grapevine and provide an interesting gene target for the future amelioration of grape via breeding and/or biotechnology. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
Full Text Available Huanglongbing (HLB is presently the most devastating citrus disease worldwide. As an intracellular plant pathogen and insect symbiont, the HLB bacterium, 'Candidatus Liberibacter asiaticus' (Las, retains the entire flagellum-encoding gene cluster in its significantly reduced genome. Las encodes a flagellin and hook-associated protein (Fla of 452 amino acids that contains a conserved 22 amino acid domain (flg22 at positions 29 to 50 in the N-terminus. The phenotypic alteration in motility of a Sinorhizobium meliloti mutant lacking the fla genes was partially restored by constitutive expression of Fla(Las. Agrobacterium-mediated transient expression in planta revealed that Fla(Las induced cell death and callose deposition in Nicotiana benthamiana, and that the transcription of BAK1 and SGT1, which are associated with plant innate immunity, was upregulated. Amino acid substitution experiments revealed that residues 38 (serine and 39 (aspartate of Fla(Las were essential for callose induction. The synthetic flg22(Las peptide could not induce plant cell death but retained the ability to induce callose deposition at a concentration of 20 µM or above. This demonstrated that the pathogen-associated molecular pattern (PAMP activity of flg22 in Las was weaker than those in other well-studied plant pathogenic bacteria. These results indicate that Fla(Las acts as a PAMP and may play an important role in triggering host plant resistance to the HLB bacteria.
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.
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
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.
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
Full Text Available Abstract Background Peptidase family A1, to which pepsin belongs, had been assumed to be restricted to eukaryotes. The tertiary structure of pepsin shows two lobes with similar folds and it has been suggested that the gene has arisen from an ancient duplication and fusion event. The only sequence similarity between the lobes is restricted to the motif around the active site aspartate and a hydrophobic-hydrophobic-Gly motif. Together, these contribute to an essential structural feature known as a psi-loop. There is one such psi-loop in each lobe, and so each lobe presents an active Asp. The human immunodeficiency virus peptidase, retropepsin, from peptidase family A2 also has a similar fold but consists of one lobe only and has to dimerize to be active. All known members of family A1 show the bilobed structure, but it is unclear if the ancestor of family A1 was similar to an A2 peptidase, or if the ancestral retropepsin was derived from a half-pepsin gene. The presence of a pepsin homologue in a prokaryote might give insights into the evolution of the pepsin family. Results Homologues of the aspartic peptidase pepsin have been found in the completed genomic sequences from seven species of bacteria. The bacterial homologues, unlike those from eukaryotes, do not possess signal peptides, and would therefore be intracellular acting at neutral pH. The bacterial homologues have Thr218 replaced by Asp, a change which in renin has been shown to confer activity at neutral pH. No pepsin homologues could be detected in any archaean genome. Conclusion The peptidase family A1 is found in some species of bacteria as well as eukaryotes. The bacterial homologues fall into two groups, one from oceanic bacteria and one from plant symbionts. The bacterial homologues are all predicted to be intracellular proteins, unlike the eukaryotic enzymes. The bacterial homologues are bilobed like pepsin, implying that if no horizontal gene transfer has occurred the duplication
Full Text Available Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations > 60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2-3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations.
Kamp, Anja; Stief, Peter; Bristow, Laura A.; Thamdrup, Bo; Glud, Ronnie N.
Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations >60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2–3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations. PMID:27847498
William A. McEwan
Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.
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
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.
Nelson, Jane Bray; Nelson, Jim
Describes the history of Richard Blakemore's discovery of magnetotaxic organisms. Discusses possible reasons why the magnetic response in bacteria developed. Proposes research experiments integrating biology and physics in which students investigate problems using cultures of magnetotaxic organisms. (MDH)
Schulz, HN; Jørgensen, BB
A small number of prokaryotic species have a unique physiology or ecology related to their development of unusually large size. The biomass of bacteria varies over more than 10 orders of magnitude, from the 0.2 mum wide nanobacteria to the largest cells of the colorless sulfur bacteria......, Thiomargarita namibiensis, with a diameter of 750 mum. All bacteria, including those that swim around in the environment, obtain their food molecules by molecular diffusion. Only the fastest and largest swimmers known, Thiovulum majus, are able to significantly increase their food supply by motility...... and by actively creating an advective flow through the entire population. Diffusion limitation generally restricts the maximal size of prokaryotic cells and provides a selective advantage for mum-sized cells at the normally low substrate concentrations in the environment. The largest heterotrophic bacteria...
Schulz, HN; Jørgensen, BB
A small number of prokaryotic species have a unique physiology or ecology related to their development of unusually large size. The biomass of bacteria varies over more than 10 orders of magnitude, from the 0.2 mum wide nanobacteria to the largest cells of the colorless sulfur bacteria...... and by actively creating an advective flow through the entire population. Diffusion limitation generally restricts the maximal size of prokaryotic cells and provides a selective advantage for mum-sized cells at the normally low substrate concentrations in the environment. The largest heterotrophic bacteria......, the 80 x 600 mum large Epulopiscium sp. from the gut of tropical fish, are presumably living in a very nutrient-rich medium. Many large bacteria contain numerous inclusions in the cells that reduce the volume of active cytoplasm. The most striking examples of competitive advantage from large cell size...
Moffat, J F; Tompkins, L S
A model of intracellular growth for Legionella pneumophila in Acanthamoeba castellanii has been developed and provides a quantitative measure of survival and replication after entry. In this model, Acanthamoeba monolayers were incubated with bacteria in tissue culture plates under nutrient-limiting conditions. Gentamicin was used to kill extracellular bacteria following the period of incubation, and the number of intracellular bacteria was determined following lysis of amebae. Intracellular growth of virulent L. pneumophila and other wild-type Legionella species was observed when the assay was performed at 37 degrees C. At room temperature, none of the Legionella strains tested grew intracellularly, while an avirulent L. pneumophila strain was unable to replicate in this assay at either temperature. The effect of nutrient limitation on A. castellanii during the assay prevented multiplication of the amebae and increased the level of infection by Legionella spp. The level of infection of the amebae was directly proportional to the multiplicity of infection with bacteria; at an inoculum of 1.03 x 10(7) bacteria added to wells containing 1.10 x 10(5) amebae (multiplicity of infection of 100), approximately 4.4% of A. castellanii cells became infected. Cytochalasin D reduced the uptake of bacteria by the amebae primarily by causing amebae to lift off the culture dish, reducing the number of target hosts; methylamine also reduced the level of initial infection, yet neither inhibitor was able to prevent intracellular replication of Legionella spp. Consequently, once the bacteria entered the cell, only lowered temperature could restrict replication. This model of intracellular growth provides a one-step growth curve and should be useful to study the molecular basis of the host-parasite interaction. PMID:1729191
Kamp, Anja; Stief, Peter; Bristow, Laura A.
Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species......-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly....... Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59...
sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival. Conclusions Mass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.
Gonzalo-Asensio, Jesús; Ortega, Alvaro D; Rico-Pérez, Gadea; Pucciarelli, M Graciela; García-Del Portillo, Francisco
Bacterial small RNAs (sRNAs) are regulatory molecules playing relevant roles in response to environmental changes, stressful conditions and pathogenesis. The intracellular bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is known to regulate expression of some sRNAs during colonization of fibroblasts. Here, we characterize a previously unknown sRNA encoded in the S. Typhimurium pSLT virulence plasmid that is specifically up-regulated by non-growing dormant bacteria persisting inside fibroblasts. This sRNA was inferred in microarray expression analyses, which unraveled enhanced transcriptional activity in the PSLT047- PSLT046 (mig5) intergenic region. The sRNA transcript was further identified as a 597-nucleotide molecule, which we named IesR-1, for 'Intracellular-expressed-sRNA-1'. IesR-1 expression is low in bacteria growing in axenic cultures across a variety of experimental conditions but displays a marked increase (∼200-300 fold) following bacterial entry into fibroblasts. Remarkably, induction of IesR-1 expression is not prominent in bacteria proliferating within epithelial cells. IesR-1 deletion affects the control of bacterial growth in defined fibroblast cell lines and impairs virulence in a mouse infection model. Expression analyses performed in the PSLT047-iesR-1-PSLT046 (mig5) region support a cis-acting regulatory mechanism of IesR-1 as antisense RNA over the PSLT047 transcript involving interaction at their respective 3' ends and modulation of PSLT047 protein levels. This model is sustained by the scarce production of PSLT047 protein observed in non-growing intracellular bacteria and the high amount of PSLT047 protein produced by bacteria carrying a truncated IesR-1 version with separated 5' and 3' regions. Taken together, these data reveal that S. Typhimurium sRNAs encoded in the pSLT virulence plasmid respond to a state of persistence inside the host cell. As exemplified by IesR-1, some of these sRNAs may contribute to
Full Text Available Bacterial small RNAs (sRNAs are regulatory molecules playing relevant roles in response to environmental changes, stressful conditions and pathogenesis. The intracellular bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium is known to regulate expression of some sRNAs during colonization of fibroblasts. Here, we characterize a previously unknown sRNA encoded in the S. Typhimurium pSLT virulence plasmid that is specifically up-regulated by non-growing dormant bacteria persisting inside fibroblasts. This sRNA was inferred in microarray expression analyses, which unraveled enhanced transcriptional activity in the PSLT047- PSLT046 (mig5 intergenic region. The sRNA transcript was further identified as a 597-nucleotide molecule, which we named IesR-1, for 'Intracellular-expressed-sRNA-1'. IesR-1 expression is low in bacteria growing in axenic cultures across a variety of experimental conditions but displays a marked increase (∼200-300 fold following bacterial entry into fibroblasts. Remarkably, induction of IesR-1 expression is not prominent in bacteria proliferating within epithelial cells. IesR-1 deletion affects the control of bacterial growth in defined fibroblast cell lines and impairs virulence in a mouse infection model. Expression analyses performed in the PSLT047-iesR-1-PSLT046 (mig5 region support a cis-acting regulatory mechanism of IesR-1 as antisense RNA over the PSLT047 transcript involving interaction at their respective 3' ends and modulation of PSLT047 protein levels. This model is sustained by the scarce production of PSLT047 protein observed in non-growing intracellular bacteria and the high amount of PSLT047 protein produced by bacteria carrying a truncated IesR-1 version with separated 5' and 3' regions. Taken together, these data reveal that S. Typhimurium sRNAs encoded in the pSLT virulence plasmid respond to a state of persistence inside the host cell. As exemplified by IesR-1, some of these sRNAs may
Brown, N L; Rouch, D A; Lee, B T
Copper is an essential trace element that is utilized in a number of oxygenases and electron transport proteins, but it is also a highly toxic heavy metal, against which all organisms must protect themselves. Known bacterial determinants of copper resistance are plasmid-encoded. The mechanisms which confer resistance must be integrated with the normal metabolism of copper. Different bacteria have adopted diverse strategies for copper resistance, and this review outlines what is known about bacterial copper resistance mechanisms and their genetic regulation.
Cortes-Bratti, Ximena; Brasseres, Eugenie; Herrera-Rodriquez, Fabiola
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...
Nieth, Anita; Verseux, Cyprien; Barnert, Sabine; Süss, Regine; Römer, Winfried
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.
Marleau, Peter; Brubaker, Erik
This report provides a short overview of the DNN R&D funded project, Time-Encoded Imagers. The project began in FY11 and concluded in FY14. The Project Description below provides the overall motivation and objectives for the project as well as a summary of programmatic direction. It is followed by a short description of each task and the resulting deliverables.
Persis, Claudio De
Monitoring large-scale systems is of fundamental importance in modern infrastructures. Many of these large-scale systems are complex interconnections of sub-components which interact by means of communication channels with limited bandwidth. Therefore the information must be encoded in order to be
Pucciarelli, M Graciela; García-Del Portillo, Francisco
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.
Full Text Available Horizontal gene transfer (HGT plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR, with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10-90% in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (kcat/KM, correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the
Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.
The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.
Johnson, Erin E; Srikanth, Chittur V; Sandgren, Andreas
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...
Bressloff, Paul C.
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.
Thornton Ruth B
Full Text Available Abstract Background Bacteria which are metabolically active yet unable to be cultured and eradicated by antibiotic treatment are present in the middle ear effusion of children with chronic otitis media with effusion (COME and recurrent acute otitis media (rAOM. These observations are suggestive of biofilm presence or intracellular sequestration of bacteria and may play a role in OM pathogenesis. The aim of this project is to provide evidence for the presence of otopathogenic bacteria intracellularly or within biofilm in the middle ear mucosa of children with COME or rAOM. Methods Middle ear mucosal biopsies from 20 children with COME or rAOM were examined for otopathogenic bacteria (either in biofilm or located intracellularly using transmission electron microscopy (TEM or species specific fluorescent in situ hybridisation (FISH and confocal laser scanning microscopy (CLSM. One healthy control biopsy from a child undergoing cochlear implant surgery was also examined. Results No bacteria were observed in the healthy control sample. In 2 of the 3 biopsies imaged using TEM, bacteria were observed in mucus containing vacuoles within epithelial cells. Bacterial species within these could not be identified and biofilm was not observed. Using FISH with CLSM, bacteria were seen in 15 of the 17 otitis media mucosal specimens. In this group, 11 (65% of the 17 middle ear mucosal biopsies showed evidence of bacterial biofilm and 12 demonstrated intracellular bacteria. 52% of biopsies were positive for both biofilm and intracellular bacteria. At least one otopathogen was identified in 13 of the 15 samples where bacteria were present. No differences were observed between biopsies from children with COME and those with rAOM. Conclusion Using FISH and CLSM, bacterial biofilm and intracellular infection with known otopathogens are demonstrated on/in the middle ear mucosa of children with COME and/or rAOM. While their role in disease pathogenesis remains to be
Munkhdalai, Tsendsuren; Yu, Hong
We present a memory augmented neural network for natural language understanding: Neural Semantic Encoders. NSE is equipped with a novel memory update rule and has a variable sized encoding memory that evolves over time and maintains the understanding of input sequences through read , compose and write operations. NSE can also access multiple and shared memories. In this paper, we demonstrated the effectiveness and the flexibility of NSE on five different natural language tasks: natural language inference, question answering, sentence classification, document sentiment analysis and machine translation where NSE achieved state-of-the-art performance when evaluated on publically available benchmarks. For example, our shared-memory model showed an encouraging result on neural machine translation, improving an attention-based baseline by approximately 1.0 BLEU.
Full Text Available Small non-coding RNAs (sRNAs that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA and small interfering RNA (siRNA in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.
Deann T. Snyder
Full Text Available Type I interferons represent a unique and complex group of cytokines, serving many purposes during innate and adaptive immunity. Discovered in the context of viral infections, type I IFNs are now known to have myriad effects in infectious and autoimmune disease settings. Type I IFN signaling during bacterial infections is dependent on many factors including whether the infecting bacterium is intracellular or extracellular, as different signaling pathways are activated. As such, the repercussions of type I IFN induction can positively or negatively impact the disease outcome. This review focuses on type I IFN induction and downstream consequences during infection with the following intracellular bacteria: Chlamydia trachomatis, Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Francisella tularensis, Brucella abortus, Legionella pneumophila, and Coxiella burnetii. Intracellular bacterial infections are unique because the bacteria must avoid, circumvent, and even co-opt microbial “sensing” mechanisms in order to reside and replicate within a host cell. Furthermore, life inside a host cell makes intracellular bacteria more difficult to target with antibiotics. Because type I IFNs are important immune effectors, modulating this pathway may improve disease outcomes. But first, it is critical to understand the context-dependent effects of the type I IFN pathway in intracellular bacterial infections.
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.
Full Text Available Abstract Background Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results Here we show that both the prion domain of Sup35 (Sup35-NM and the Ure2 protein (Ure2p form inclusion bodies (IBs displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.
Studies of phonological disturbances in aphasic speech are reviewed. It is argued that failure to test for error consistency in individual patients makes it generally improper to draw inferences about specific disorders of phonological encoding. A minimalist interpretation of available data on phonological errors is therefore proposed that involves variable loss of information in transmission between processing subsystems. Proposals for systematic loss or corruption of phonological information in lexical representations or in translation subsystems is shown to be inadequately grounded. The review concludes with some simple methodological prescriptions for future research.
Martinez, Virginia; Herencias, Cristina; Jurkevitch, Edouard
This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures. The bio-product targets to be recovered...
Hellwig, SMM; Hazenbos, WLW; van de Winkel, JGJ; Mooi, FR
Bordetella pertussis can attach, invade and survive intracellularly in human macrophages in vitro. To study the significance of this bacterial feature in vivo, we analyzed the presence of viable bacteria in broncho-alveolar lavage (BAL) cells of mice infected with B, pertussis. We found B. pertussis
Bucci, Cecilia; Bakke, Oddmund; Progida, Cinzia
Mutations of genes whose primary function is the regulation of membrane traffic are increasingly being identified as the underlying causes of various important human disorders. Intriguingly, mutations in ubiquitously expressed membrane traffic genes often lead to cell type- or organ-specific disorders. This is particularly true for neuronal diseases, identifying the nervous system as the most sensitive tissue to alterations of membrane traffic. Charcot–Marie–Tooth (CMT) disease is one of the most common inherited peripheral neuropathies. It is also known as hereditary motor and sensory neuropathy (HMSN), which comprises a group of disorders specifically affecting peripheral nerves. This peripheral neuropathy, highly heterogeneous both clinically and genetically, is characterized by a slowly progressive degeneration of the muscle of the foot, lower leg, hand and forearm, accompanied by sensory loss in the toes, fingers and limbs. More than 30 genes have been identified as targets of mutations that cause CMT neuropathy. A number of these genes encode proteins directly or indirectly involved in the regulation of intracellular traffic. Indeed, the list of genes linked to CMT disease includes genes important for vesicle formation, phosphoinositide metabolism, lysosomal degradation, mitochondrial fission and fusion, and also genes encoding endosomal and cytoskeletal proteins. This review focuses on the link between intracellular transport and CMT disease, highlighting the molecular mechanisms that underlie the different forms of this peripheral neuropathy and discussing the pathophysiological impact of membrane transport genetic defects as well as possible future ways to counteract these defects. PMID:22465036
Tago, Damian; Meyer, Damien F
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.
Confocal laser scanning microscopy is the best method to visualize intracellular co-localization of proteins in intact cells. Because of the point scan/pinhole detection system, light contribution from the neighborhood of the scanning spot in the specimen can be eliminated, allowing high Z-axis resolution. Fluorescence detection by sensitive photomultiplier tubes allows the usage of filters with a narrow bandpath, resulting in minimal cross-talk (overlap) between two spectra. This is particularly important in demonstrating co-localization of proteins with multicolor labeling. Here, the methods outlining the detection of transiently expressed tagged proteins and the detection of endogenous proteins are described. Ideally, the intracellular co-localization of two endogenous proteins should be demonstrated. However, when antibodies raised against the protein of interest are unavailable for immunofluorescence or the available cell lines do not express the protein of interest sufficiently enough for immunofluorescence, an alternative method is to transfect cells with expression plasmids that encode tagged proteins and stain the cells with anti-tag antibodies. However, it should be noted that the tagging of proteins of interest or their overexpression could potentially alter the intracellular localization or the function of the target protein.
Bouffard, G G; Rudd, K E; Adhya, S L
A new gene (galM) has been identified as the fourth cistron of the gal operon, encoding enzymes for the metabolism of galactose and lactose in Escherichia coli. Induction of the gal operon either from the gal promoters or from a neighboring prophage lambda promoter expresses the galM gene as well. The new structure of the gal operon from the promoter end is galE-galT-galK-galM in counter-clockwise orientation on the chromosome. Genetic and biochemical analyses have revealed that the galM gene product has mutarotase activity, which converts alpha-aldose to the beta-anomer. Unlike mutarotase from other bacteria in which the enzyme is primarily processed for export and secretion, the mutarotase from E. coli does not appear to be processed and yet is still found in periplasm (and culture media when overexpressed) in significant amounts. Although the interconversion of the sugar anomers occurs spontaneously in pure water in vitro, the in vivo formation of alpha-D-galactopyranose (the substrate for phosphorylation) from beta-D-galactopyranose (generated by beta-galactosidase hydrolysis of lactose) is largely dependent upon the presence of the mutarotase. This shows that efficient lactose metabolism requires mutarotase. These results give credence to the idea that the activity of intracellular water is not high enough to permit a simple extrapolation of observed in vitro reactions to in vivo situations in every case.
Yang, Xinyan; Wu, Liqin; Duan, Xuemei; Cui, Lianzhen; Luo, Jingjing; Li, Gongchu
Lectins exist widely in marine bioresources such as bacteria, algae, invertebrate animals and fishes. Some purified marine lectins have been found to elicit cytotoxicity to cancer cells. However, there are few reports describing the cytotoxic effect of marine lectins on cancer cells through virus-mediated gene delivery. We show here that a replication-deficient adenovirus-carrying gene encoding Haliotis discus discus sialic acid binding lectin (Ad.FLAG-HddSBL) suppressed cancer cell proliferation by inducing apoptosis, as compared to the control virus Ad.FLAG. A down-regulated level of anti-apoptosis factor Bcl-2 was suggested to be responsible for the apoptosis induced by Ad.FLAG-HddSBL infection. Further subcellular localization studies revealed that HddSBL distributed in cell membrane, ER, and the nucleus, but not in mitochondria and Golgi apparatus. In contrast, a previously reported mannose-binding lectin Pinellia pedatisecta agglutinin entered the nucleus as well, but did not distribute in inner membrane systems, suggesting differed intracellular sialylation and mannosylation, which may provide different targets for lectin binding. Further cancer-specific controlling of HddSBL expression and animal studies may help to provide insights into a novel way of anti-cancer marine lectin gene therapy. Lectins may provide a reservoir of anti-cancer genes.
Azad, Kimi; Banerjee, Manidipa; Johnson, John E
Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.
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
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.
Wang, Yuwei; Schaefer, Jeffra K; Mishra, Bhoopesh; Yee, Nathan
The disposal of elemental mercury (Hg(0)) wastes in mining and manufacturing areas has caused serious soil and groundwater contamination issues. Under anoxic conditions, certain anaerobic bacteria can oxidize dissolved elemental mercury and convert the oxidized Hg to neurotoxic methylmercury. In this study, we conducted experiments with the Hg-methylating bacterium Desulfovibrio desulfuricans ND132 to elucidate the role of cellular thiols in anaerobic Hg(0) oxidation. The concentrations of cell-surface and intracellular thiols were measured, and specific fractions of D. desulfuricans ND132 were examined for Hg(0) oxidation activity and analyzed with extended X-ray absorption fine structure (EXAFS) spectroscopy. The experimental data indicate that intracellular thiol concentrations are approximately six times higher than those of the cell wall. Cells reacted with a thiol-blocking reagent were severely impaired in Hg(0) oxidation activity. Spheroplasts lacking cell walls rapidly oxidized Hg(0) to Hg(II), while cell wall fragments exhibited low reactivity toward Hg(0). EXAFS analysis of spheroplast samples revealed that multiple different forms of Hg-thiols are produced by the Hg(0) oxidation reaction and that the local coordination environment of the oxidized Hg changes with reaction time. The results of this study indicate that Hg(0) oxidation in D. desulfuricans ND132 is an intracellular process that occurs by reaction with thiol-containing molecules.
Marleau, Peter; Brubaker, Erik; Kiff, Scott
The various technologies presented herein relate to detecting nuclear material at a large stand-off distance. An imaging system is presented which can detect nuclear material by utilizing time encoded imaging relating to maximum and minimum radiation particle counts rates. The imaging system is integrated with a data acquisition system that can utilize variations in photon pulse shape to discriminate between neutron and gamma-ray interactions. Modulation in the detected neutron count rates as a function of the angular orientation of the detector due to attenuation of neighboring detectors is utilized to reconstruct the neutron source distribution over 360 degrees around the imaging system. Neutrons (e.g., fast neutrons) and/or gamma-rays are incident upon scintillation material in the imager, the photons generated by the scintillation material are converted to electrical energy from which the respective neutrons/gamma rays can be determined and, accordingly, a direction to, and the location of, a radiation source identified.
Labouta, Hagar Ibrahim; Menina, Sara; Kochut, Annika; Gordon, Sarah; Geyer, Rebecca; Dersch, Petra; Lehr, Claus-Michael
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.
McGonigle, John E; Purves, Joanne; Rolff, Jens
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.
Reuben N. S. Rowe
Full Text Available Jay and Given-Wilson have recently introduced the Factorisation (or SF- calculus as a minimal fundamental model of intensional computation. It is a combinatory calculus containing a special combinator, F, which is able to examine the internal structure of its first argument. The calculus is significant in that as well as being combinatorially complete it also exhibits the property of structural completeness, i.e. it is able to represent any function on terms definable using pattern matching on arbitrary normal forms. In particular, it admits a term that can decide the structural equality of any two arbitrary normal forms. Since SF-calculus is combinatorially complete, it is clearly at least as powerful as the more familiar and paradigmatic Turing-powerful computational models of Lambda Calculus and Combinatory Logic. Its relationship to these models in the converse direction is less obvious, however. Jay and Given-Wilson have suggested that SF-calculus is strictly more powerful than the aforementioned models, but a detailed study of the connections between these models is yet to be undertaken. This paper begins to bridge that gap by presenting a faithful encoding of the Factorisation Calculus into the Lambda Calculus preserving both reduction and strong normalisation. The existence of such an encoding is a new result. It also suggests that there is, in some sense, an equivalence between the former model and the latter. We discuss to what extent our result constitutes an equivalence by considering it in the context of some previously defined frameworks for comparing computational power and expressiveness.
Martynova, E U; Kapelinskaia, T V; Schal, C; Mukha, D V
The intracellular localization of the regulatory proteins encoded by the genome of the densovirus of German cockroach was analyzed using western-blotting of nuclear and cytoplasmic extracts of the densovirus-infected passaging cells tissue culture BGE-2. Two of the three regulatory proteins, NS1 and NS3, were shown to possess mainly nuclear localization, while NS2 protein was distributed between the nucleus and cytoplasm. Data obtained provide new information necessary for prediction of the functions of densovirus regulatory proteins. Intracellular localization of NS3 protein was described for the densoviruses for the first time.
Janick, P.A.; Grossman, R.I.; Asakura, T.
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
Hiroyuki Ogata; Patricia Renesto; Stéphane Audic; Catherine Robert; Guillaume Blanc; Pierre-Edouard Fournier; Hugues Parinello; Jean-Michel Claverie; Didier Raoult
We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular alpha-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, sev...
Full Text Available Assembler Encoding is a neuro-evolutionary method in which a neural network is represented in the form of a simple program called Assembler Encoding Program. The task of the program is to create the so-called Network Definition Matrix which maintains all the information necessary to construct the network. To generate Assembler Encoding Programs and the subsequent neural networks evolutionary techniques are used.
The performance of Assembler Encoding strongly depends on operations used in Assembler Encoding Programs. To select the most effective operations, experiments in the optimization and the predator-prey problem were carried out. In the experiments, Assembler Encoding Programs equipped with different types of operations were tested. The results of the tests are presented at the end of the paper.
Benedikt, Jan; Inyushin, Mikhail; Kucheryavykh, Yuriy V.; Rivera, Yomarie; Kucheryavykh, Lilia Y.; Nichols, Colin G.; Eaton, Misty J.; Skatchkov, Serguei N.
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
Poza, M; Prieto-Alcedo, M; Sieiro, C; Villa, T G
The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.
Scott, Victoria C S; Haake, David A; Churchill, Bernard M; Justice, Sheryl S; Kim, Ja-Hong
Patients with persistent lower urinary tract symptoms and negative urine cultures are often difficult to treat. Infection may go undetected in these patients because the concentrations of bacteria in their urine are beneath the threshold of standard urine culture techniques. Empiric treatment may result in temporary relief, followed by recurrent symptoms. Occult and recurrent urinary tract infection may be due to both invasion of the bladder wall by uropathogenic Escherichia coli and the formation of biofilm-like intracellular bacterial communities. This review examines emerging evidence for a role of intracellular bacterial communities in human infection. Copyright © 2015 Elsevier Inc. All rights reserved.
Makarova, K.; Slesarev, A.; Wolf, Y.; Sorokin, A.; Mirkin, B.; Koonin, E.; Pavlov, A.; Pavlova, N.; Karamychev, V.; Polouchine, N.; Shakhova, V.; Grigoriev, I.; Lou, Y.; Rokhsar, D.; Lucas, S.; Huang, K.; Goodstein, D. M.; Hawkins, T.; Plengvidhya, V.; Welker, D.; Hughes, J.; Goh, Y.; Benson, A.; Baldwin, K.; Lee, J. -H.; Diaz-Muniz, I.; Dosti, B.; Smeianov, V; Wechter, W.; Barabote, R.; Lorca, G.; Altermann, E.; Barrangou, R.; Ganesan, B.; Xie, Y.; Rawsthorne, H.; Tamir, D.; Parker, C.; Breidt, F.; Broadbent, J.; Hutkins, R.; O' Sullivan, D.; Steele, J.; Unlu, G.; Saier, M.; Klaenhammer, T.; Richardson, P.; Kozyavkin, S.; Weimer, B.; Mills, D.
Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.
Liu, Xueliang; Lopez, Paola A; Giessen, Tobias W; Giles, Michael; Way, Jeffrey C; Silver, Pamela A
Genetically encoding the synthesis of functional nanomaterials such as magnetic nanoparticles enables sensitive and non-invasive biological sensing and control. Via directed evolution of the natural iron-sequestering ferritin protein, we discovered key mutations that lead to significantly enhanced cellular magnetism, resulting in increased physical attraction of ferritin-expressing cells to magnets and increased contrast for cellular magnetic resonance imaging (MRI). The magnetic mutants further demonstrate increased iron biomineralization measured by a novel fluorescent genetic sensor for intracellular free iron. In addition, we engineered Escherichia coli cells with multiple genomic knockouts to increase cellular accumulation of various metals. Lastly to explore further protein candidates for biomagnetism, we characterized members of the DUF892 family using the iron sensor and magnetic columns, confirming their intracellular iron sequestration that results in increased cellular magnetization.
Cohen, Noga; Pell, Liat; Edelson, Micah G; Ben-Yakov, Aya; Pine, Alex; Dudai, Yadin
We review reports of brain activations that occur immediately prior to the onset or following the offset of to-be-remembered information and can predict subsequent mnemonic success. Memory-predictive pre-encoding processes, occurring from fractions of a second to minutes prior to event onset, are mainly associated with activations in the medial temporal lobe (MTL), amygdala and midbrain, and with enhanced theta oscillations. These activations may be considered as the neural correlates of one or more cognitive operations, including contextual processing, attention, and the engagement of distinct computational modes associated with prior encoding or retrieval. Post-encoding activations that correlate with subsequent memory performance are mainly observed in the MTL, sensory cortices and frontal regions. These activations may reflect binding of elements of the encoded information and initiation of memory consolidation. In all, the findings reviewed here illustrate the importance of brain states in the immediate peri-encoding time windows in determining encoding success. Understanding these brain states and their specific effects on memory may lead to optimization of the encoding of desired memories and mitigation of undesired ones. Copyright © 2014 Elsevier Ltd. All rights reserved.
Ortega, Alvaro D; Quereda, Juan J; Pucciarelli, M Graciela; García-del Portillo, Francisco
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.
Kim, Suk; Kurokawa, Daisuke; Watanabe, Kenta; Makino, Sou-Ichi; Shirahata, Toshikazu; Watarai, Masahisa
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
Brini, Marisa; Calì, Tito; Ottolini, Denis; Carafoli, Ernesto
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.
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...
Tada, Haru; Orii, Hidefumi
Xenopus dazl encoding an RNA-binding protein has been identified as a component of the germ plasm and is involved in the migration and differentiation of the primordial germ cells (PGCs). Here, we investigated the intracellular localization of Dazl in germline cells throughout the lifetime of Xenopus. In early embryogenesis, Dazl was detected initially in the germ plasm and then translocated to a perinuclear region. Then, it was detected within the nucleus in PGCs. Dazl was observed only in the cytoplasm in PGCs when sex differentiation began in the gonads. Dazl was distributed in both the nucleus and cytoplasm of the primary oogonium and spermatogonium, but only in the cytoplasm of the secondary oogonium and spermatogonium. In spermatocytes, Dazl was distributed throughout cytoplasm and localized at the spindles and cytoplasm during meiosis. Then, it was detected as speckles in the nucleus in the round spermatid. The dynamic intracellular localization suggests that Dazl is a multifunctional protein regulating RNA metabolism required for Xenopus germline development.
Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J.A.; Rex Gaskins, H.
Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition
Kolossov, Vladimir L., E-mail: firstname.lastname@example.org [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Beaudoin, Jessica N. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Hanafin, William P. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); DiLiberto, Stephen J. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Kenis, Paul J.A. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801 (United States); Rex Gaskins, H. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Avenue, Urbana, IL 61801 (United States); Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States)
Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.
Niftrik, L.A.M.P. van
Anammox bacteria perform anaerobic ammonium oxidation to dinitrogen gas and belong to the phylum Planctomycetes. Whereas most Prokaryotes consist of one compartment, the cytoplasm bounded by the cytoplasmic membrane and cell wall, the species within this phylum are compartmentalized by intracellular
Kuipers, Oscar P.; Ruyter, Pascalle G.G.A. de; Kleerebezem, Michiel; Vos, Willem M. de
Quorum sensing in lactic acid bacteria (LAB) involves peptides that are directly sensed by membrane-located histidine kinases, after which the signal is transmitted to an intracellular response regulator. This regulator in turn activates transcription of target genes, that commonly include the
Li, Hui; Yu, Chan; Wang, Fei; Chang, Sae Jung; Yao, Jun; Blake, Ruth E.
Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition—including metabolic water produced as a byproduct of metabolism—based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the 18O/16O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ˜30-40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered.
Li, Hui; Yu, Chan; Wang, Fei; Chang, Sae Jung; Yao, Jun; Blake, Ruth E.
Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition—including metabolic water produced as a byproduct of metabolism—based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the 18O/16O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ∼30–40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered. PMID:27170190
Subramanian, Geetha; Barry, Abdoulaye O; Ghigo, Eric; Raoult, Didier; Mediannikov, Oleg
Diplorickettsia massiliensis is an obligate intracellular bacterium from the Coxiellaceae family recently isolated from Ixodes ricinus ticks. The inhibitory effects of antimicrobial agents were assessed by two different methods, immunofluorescence and Gimenez staining assay. Different markers (EEA1, Lamp-1, Cathepsin D, and LysoTracker Red DND99) were used to reveal the nature of the vacuole containing the bacterium. Ciprofloxacin, levofloxacin, and rifampin had MIC values of 2 lg mL(-1). We found that 4 lg mL(-1) of Doxycycline inhibited the growth of D. massiliensis strain. Surprisingly, D. massiliensis was resistant to chloramphenicol up to the concentration of 64 lg mL(-1). We found that penicillin G, ammonium chloride, gentamycin, omeprazole, bafilomycin A1, and chloroquine were not active against D. massiliensis. Studies performed with markers EEA1, Lamp-1, Cathepsin D, and LysoTracker Red DND99 showed that D. massiliensis is localized within an acidic compartment that is not an early phagosome, but a late phagosome or a phagolysosome. Gimenez staining stays a good method that will work with a very low number of bacteria and can be used to determine the MICs of new therapeutic antibiotics precisely. The resistance profile of D. massiliensis was found to be quite unusual for intracellular Gram-negative bacterium with marked resistance to chloramphenicol. Despite of localization in acidic compartment, pH-neutralizing agents do not significantly inhibit intracellular growth of bacterium. The results of these studies prove that antibiotic resistance does not depend on pH of vacuole. This pH-related mechanism seems not to play a contributing role in the overall resistance of D. massiliensis.
Foster, Simon R; Bräuner-Osborne, Hans
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...
Paulo, Cristiana S O; Pires das Neves, Ricardo; Ferreira, Lino S
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.
Full Text Available Encodings or the proof of their absence are the main way to compare process calculi. To analyse the quality of encodings and to rule out trivial or meaningless encodings, they are augmented with quality criteria. There exists a bunch of different criteria and different variants of criteria in order to reason in different settings. This leads to incomparable results. Moreover it is not always clear whether the criteria used to obtain a result in a particular setting do indeed fit to this setting. We show how to formally reason about and compare encodability criteria by mapping them on requirements on a relation between source and target terms that is induced by the encoding function. In particular we analyse the common criteria full abstraction, operational correspondence, divergence reflection, success sensitiveness, and respect of barbs; e.g. we analyse the exact nature of the simulation relation (coupled simulation versus bisimulation that is induced by different variants of operational correspondence. This way we reduce the problem of analysing or comparing encodability criteria to the better understood problem of comparing relations on processes.
Full Text Available Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio = 1/8 (w/v and power density = 15 watt ml(-1], the activity of alkaline phosphomonoesterase (phosphatase in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first plateau of enzyme activity was reached between 60 and 100 s, and a second higher plateau after 300 s. We also found that sonication for 100 s under optimal conditions activated most (about 80% of the alkaline phosphatase that was added to an autoclaved soil, while total bacteria number was not affected. Sonication for 300 s reduced the total bacteria number by three orders of magnitude but had no further effects on enzyme activity. Our results indicate that the first plateau of alkaline phosphatase activity was derived from extracellular enzymes attached to soil particles, and the second plateau to the combination of extracellular and intracellular enzymes after cell lysis. We conclude that our adjusted sonication method may be an alternative to the currently used physiological and chloroform-fumigation methods for differentiating intracellular from extracellular phosphatase activity in soil. Further testing is needed to find out whether this holds for other soil types.
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
Nikitin, D.I.; Tashtemirova, M.A.; Pitryuk, I.A.; Sorokin, V.V.; Oranskaya, M.S.; Nikitin, L.E.
The resistance of seven cultures of eutrophic and oligotrophic bacteria to gamma radiation (at doses up to 360 Gy) was investigated. The bacteria under study were divided into three groups according to their survival ability after irradiation. Methylobacterium organophilum and open-quotes Pedodermatophilus halotoleransclose quotes (LD 50 = 270 Gy) were highly tolerant. By their tolerance, these organisms approached Deinococcus radiodurans. Aquatic ring-shaped (toroidal) bacteria Flectobacillus major and open-quotes Arcocella aquaticaclose quotes (LD 5 = 173 and 210 Gy, respectively) were moderately tolerant. Eutrophic Pseudomonas fluorescens and Escherichia coli (LD 50 = 43 and 38 Gy, respectively) were the most sensitive. X-ray microanalysis showed that in tolerant bacteria the intracellular content of potassium increased and the content of calcium decreased after irradiation. No changes in the element composition of the eutrophic bacterium E. coli were detected. Possible mechanisms of the resistance of oligotrophic bacteria to gamma radiation are discussed
Magnetic resonance imaging (MRI) typically achieves spatial encoding by measuring the projection of a q-dimensional object over q-dimensional spatial bases created by linear spatial encoding magnetic fields (SEMs). Recently, imaging strategies using nonlinear SEMs have demonstrated potential advantages for reconstructing images with higher spatiotemporal resolution and reducing peripheral nerve stimulation. In practice, nonlinear SEMs and linear SEMs can be used jointly to further improve the image reconstruction performance. Here, we propose the multidimensionally encoded (MDE) MRI to map a q-dimensional object onto a p-dimensional encoding space where p > q. MDE MRI is a theoretical framework linking imaging strategies using linear and nonlinear SEMs. Using a system of eight surface SEM coils with an eight-channel radiofrequency coil array, we demonstrate the five-dimensional MDE MRI for a two-dimensional object as a further generalization of PatLoc imaging and O-space imaging. We also present a method of optimizing spatial bases in MDE MRI. Results show that MDE MRI with a higher dimensional encoding space can reconstruct images more efficiently and with a smaller reconstruction error when the k-space sampling distribution and the number of samples are controlled. Copyright © 2012 Wiley Periodicals, Inc.
Full Text Available Bacterial manganese (Mn oxidation plays an important role in the global biogeochemical cycling of Mn and other compounds, and the diversity and prevalence of Mn oxidizers have been well established. Despite many hypotheses of why these bacteria may oxidize Mn, the physiological reasons remain elusive. Intracellular Mn levels were determined for Pseudomonas putida GB-1 grown in the presence or absence of Mn by inductively coupled plasma mass spectrometry (ICP-MS. Mn oxidizing wild type P. putida GB-1 had higher intracellular Mn than non Mn oxidizing mutants grown under the same conditions. P. putida GB-1 had a 5 fold increase in intracellular Mn compared to the non Mn oxidizing mutant P. putida GB-1-007 and a 59 fold increase in intracellular Mn compared to P. putida GB-1 ∆2665 ∆2447. The intracellular Mn is primarily associated with the less than 3 kDa fraction, suggesting it is not bound to protein. Protein oxidation levels in Mn oxidizing and non oxidizing cultures were relatively similar, yet Mn oxidation did increase survival of P. putida GB-1 when oxidatively stressed. This study is the first to link Mn oxidation to Mn homeostasis and oxidative stress protection.
Wei, Pan; Lu, Qiang; Cui, Guimei; Guan, Zhenhong; Yang, Li; Sun, Changjiang; Sun, Wanchun; Peng, Qisheng
Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.
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.
Buisman, H P; Buys, L F; Langermans, J A; van den Broek, P J; van Furth, R
The present study concerns the effects of probenecid on the phagocytosis and intracellular killing of Staphylococcus aureus and Escherichia coli by human monocytes and granulocytes. In both monocytes and granulocytes the inhibitory effect on phagocytosis was very small. Inhibition of intracellular killing of S. aureus by monocytes and granulocytes by probenecid was concentration dependent, being half-maximal at about 2 mM probenecid, and near-maximal at about 5 mM probenecid. The intracellular killing could also be inhibited when probenecid was added when this process was already started. Probenecid also inhibited the intracellular killing of E. coli by granulocytes, but not by monocytes. In the concentration range used, probenecid had no toxic effect on phagocytes or bacteria during the 2 hr of the experiments. PMID:1748482
... Articles | Inside Life Science Home Page Bleach vs. Bacteria By Sharon Reynolds Posted April 2, 2014 Your ... hypochlorous acid to help kill invading microbes, including bacteria. Researchers funded by the National Institutes of Health ...
Full Text Available More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli.
Friederich, Uwe; Billings, Stephen A; Hardie, Roger C; Juusola, Mikko; Coca, Daniel
More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli.
Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob
The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application.
Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grażyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob
The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application. PMID:23305359
Wikraiphat, Chanthiwa; Pudla, Matsayapan; Baral, Pankaj; Kitthawee, Sangvorn; Utaisincharoen, Pongsak
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.
Full Text Available Abstract Background Contamination from fecal bacteria in recreational waters is a major health concern since bacteria capable of causing human disease can be found in animal feces. The Dog Beach area of Ocean Beach in San Diego, California is a beach prone to closures due to high levels of fecal indicator bacteria (FIB. A potential source of these FIB could be the canine feces left behind by owners who do not clean up after their pets. We tested this hypothesis by screening the DNA isolated from canine feces for the bacteriophage-encoded stx gene normally found in the virulent strains of the fecal bacterium Escherichia coli. Results Twenty canine fecal samples were collected, processed for total and bacterial fraction DNA, and screened by PCR for the stx gene. The stx gene was detected in the total and bacterial fraction DNA of one fecal sample. Bacterial isolates were then cultivated from the stx-positive fecal sample. Eighty nine of these canine fecal bacterial isolates were screened by PCR for the stx gene. The stx gene was detected in five of these isolates. Sequencing and phylogenetic analyses of 16S rRNA gene PCR products from the canine fecal bacterial isolates indicated that they were Enterococcus and not E. coli. Conclusions The bacteriophage-encoded stx gene was found in multiple species of bacteria cultivated from canine fecal samples gathered at the shoreline of the Dog Beach area of Ocean Beach in San Diego, California. The canine fecal bacteria carrying the stx gene were not the typical E. coli host and were instead identified through phylogenetic analyses as Enterococcus. This suggests a large degree of horizontal gene transfer of exotoxin genes in recreational waters.
Roux, Aurélien; Cuvelier, Damien; Bassereau, Patricia; Goud, Bruno
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.
Yan, Li; Zhang, Jinfeng; Lee, Chun-Sing; Chen, Xianfeng
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.
Jaque, Daniel; Rosal, Blanca Del; Rodríguez, Emma Martín; Maestro, Laura Martínez; Haro-González, Patricia; Solé, José García
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.
Vaart, Michiel van der
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
Full Text Available Antibiotic-resistant bacteria and genes enter the environment in different ways. The release of these factors into the environment has increased concerns related to public health. The aim of the study was to evaluate the antibiotic resistance genes (ARGs in the environmental resources. In this systematic review, the data were extracted from valid sources of information including ScienceDirect, PubMed, Google Scholar and SID. Evaluation and selection of articles were conducted on the basis of the PRISMA checklist. A total of 39 articles were included in the study, which were chosen from a total of 1249 papers. The inclusion criterion was the identification of genes encoding antibiotic resistance against the eight important groups of antibiotics determined by using the PCR technique in the environmental sources including municipal and hospital wastewater treatment plants, animal and agricultural wastes, effluents from treatment plants, natural waters, sediments, and drinking waters. In this study, 113 genes encoding antibiotic resistance to eight groups of antibiotics (beta-lactams, aminoglycosides, tetracyclines, macrolides, sulfonamides, chloramphenicol, glycopeptides and quinolones were identified in various environments. Antibiotic resistance genes were found in all the investigated environments. The investigation of microorganisms carrying these genes shows that most of the bacteria especially gram-negative bacteria are effective in the acquisition and the dissemination of these pollutants in the environment. Discharging the raw wastewaters and effluents from wastewater treatments acts as major routes in the dissemination of ARGs into environment sources and can pose hazards to public health.
Zurabishvili, S; Mamamtavrishvili, I; Apridonidze, K; Shanidze, L
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.
Margarita O. Shleeva
Full Text Available Earlier we demonstrated that the adenylyl cyclase (AC encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis (Mtb, the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro, the AC-overexpressing pMindRv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo, AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMindRv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo.
Rosenkilde, M M; Waldhoer, M; Lüttichau, H R
expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets....
Liefhebber, Johanna Maaike Pieternella
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
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 ...
... 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 ...
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.
Full Text Available Abstract Background Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. Results We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. Conclusion We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits
Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun
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
Zhang, Zheng; Zhao, Zhen; Margolskee, Robert; Liman, Emily
Bitter, sweet, and umami tastants are detected by G-protein-coupled receptors that signal through a common second-messenger cascade involving gustducin, phospholipase C beta2, and the transient receptor potential M5 (TRPM5) ion channel. The mechanism by which phosphoinositide signaling activates TRPM5 has been studied in heterologous cell types with contradictory results. To resolve this issue and understand the role of TRPM5 in taste signaling, we took advantage of mice in which the TRPM5 promoter drives expression of green fluorescent protein and mice that carry a targeted deletion of the TRPM5 gene to unequivocally identify TRPM5-dependent currents in taste receptor cells. Our results show that brief elevation of intracellular inositol trisphosphate or Ca2+ is sufficient to gate TRPM5-dependent currents in intact taste cells, but only intracellular Ca2+ is able to activate TRPM5-dependent currents in excised patches. Detailed study in excised patches showed that TRPM5 forms a nonselective cation channel that is half-activated by 8 microM Ca2+ and that desensitizes in response to prolonged exposure to intracellular Ca2+. In addition to channels encoded by the TRPM5 gene, we found that taste cells have a second type of Ca2+-activated nonselective cation channel that is less sensitive to intracellular Ca2+. These data constrain proposed models for taste transduction and suggest a link between receptor signaling and membrane potential in taste cells.
Machado, Henrique; Sonnenschein, Eva; Melchiorsen, Jette
- and Gammaproteobacteria collected during the Galathea 3 expedition were sequenced and mined for natural product encoding gene clusters. Results: Independently of genome size, bacteria of all tested genera carried a large number of clusters encoding different potential bioactivities, especially within the Vibrionaceae...... that will facilitate natural product discovery in the future....
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.
Mohn, William W; Wilbrink, Maarten H; Casabon, Israël; Stewart, Gordon R; Liu, Jie; van der Geize, Robert; Eltis, Lindsay D
Bile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9α-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism.
Sherwood, Racquel Kim; Roy, Craig R
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
Punsiri M Colonne
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.
Martens, Kirsten; Bena, Ioana; Droz, Michel; Rácz, Zoltan
Material design at submicron scales would be profoundly affected if the formation of precipitation patterns could be easily controlled. It would allow the direct building of bulk structures, in contrast to traditional techniques which consist of removing material in order to create patterns. Here, we discuss an extension of our recent proposal of using electrical currents to control precipitation bands which emerge in the wake of reaction fronts in A + + B – → C reaction–diffusion processes. Our main result, based on simulating the reaction–diffusion–precipitation equations, is that the dynamics of the charged agents can be guided by an appropriately designed time-dependent electric current so that, in addition to the control of the band spacing, the width of the precipitation bands can also be tuned. This makes straightforward the encoding of information into precipitation patterns and, as an amusing example, we demonstrate the feasibility by showing how to encode a musical rhythm
Full Text Available The majority of H. pylori reside on gastric epithelial cell surfaces and in the overlying mucus, but a small fraction of H. pylori enter host epithelial and immune cells. To explore the role of the nudA invasin in host cell entry, a ΔnudA deletion derivative of strain J99 was constructed and transformants were verified by PCR and by fluorescence in situ hybridization. AGS cells were inoculated with either wild type (WT strain J99 or its ΔnudA mutant to determine the fraction of bacteria that were bound to the cells and inside these cells using the gentamicin protection assay. We observed no significant difference between either the density of H. pylori bound to AGS cell membranes or the density of intracellular H. pylori. To further explore this finding, separate chambers of each culture were fixed in glutaraldehyde for transmission electron microscopy (TEM and immunogold TEM. This addition to the classical gentamicin assay demonstrated that there were significantly more intracellular, and fewer membrane-bound, H. pylori in WT-infected AGS cells than in ΔnudA allele infected cells. Thus, the sum of intracellular and membrane-bound H. pylori was similar in the two groups. Since no other similar TEM study has been performed, it is at present unknown whether our observations can be reproduced by others Taken together however, our observations suggest that the classical gentamicin protection assay is not sufficiently sensitive to analyze H. pylori cell entry and that the addition of TEM to the test demonstrate that nudA plays a role in H. pylori entry into AGS cells in vitro. In addition, deletion of the invasin gene appears to limit H. pylori to the AGS cell surface, where it may be partly protected against gentamicin. In contrast, this specific environment may render H. pylori more vulnerable to host defense and therapeutic intervention, and less prone to trigger normal immune, carcinogenic, and other developmental response pathways.
O'Flaherty, Sarah; Goh, Yong Jun; Klaenhammer, Todd R.
Probiotic bacteria from the Lactobacillus and Bifidobacterium species belong to the Firmicutes and the Actinobacteria phylum, respectively. Lactobacilli are members of the lactic acid bacteria (LAB) group, a broadly defined family of microorganisms that ferment various hexoses into primarily lactic acid. Lactobacilli are typically low G + C gram-positive species which are phylogenetically diverse, with over 100 species documented to date. Bifidobacteria are heterofermentative, high G + C content bacteria with about 30 species of bifidobacteria described to date.
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.
Jose C Garcia-Garcia
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.
Full Text Available Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.
Larissa D Cunha
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.
Cunha, Larissa D; Zamboni, Dario S
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.
Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.
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)
Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar
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.
Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.
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.)
Laura E. Williams
coresident symbionts other than Wolbachia. Although gene order is strictly conserved in four Blochmannia of Camponotus sensu stricto, comparisons with deeply divergent lineages revealed inversions in eight genomic regions, indicating ongoing recombination despite ancestral loss of recA. In sum, the addition of two Blochmannia genomes of divergent host lineages enables reconstruction of early events in evolution of this symbiosis and suggests that Blochmannia lineages may experience distinct, host-associated selective pressures. Understanding how evolutionary forces shape genome reduction in this system may help to clarify forces driving gene loss in other bacteria, including intracellular pathogens.
Shanan, Salah; Bayoumi, Magdi; Saeed, Amir; Sandström, Gunnar; Abd, Hadi
Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 with A. castellanii. The interaction between A. castellanii and V. cholerae strains was studied by means of amoeba cell counts, viable counts of the bacteria in the absence or presence of amoebae, and of the intracellularly growing bacteria, visualised by electron microscopy. These results show that all V. cholerae can grow and survive outside and inside the amoebae, disclosing that V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 all can be considered as facultative intracellular bacteria. PMID:27118300
Full Text Available Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 with A. castellanii. The interaction between A. castellanii and V. cholerae strains was studied by means of amoeba cell counts, viable counts of the bacteria in the absence or presence of amoebae, and of the intracellularly growing bacteria, visualised by electron microscopy. These results show that all V. cholerae can grow and survive outside and inside the amoebae, disclosing that V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 all can be considered as facultative intracellular bacteria.
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  and exogenous tumor-killing proteins 8—10]), is attractive as a
Núñez-Hernández, Cristina; Tierrez, Alberto; Ortega, Alvaro D; Pucciarelli, M Graciela; Godoy, Marta; Eisman, Blanca; Casadesús, Josep; García-del Portillo, Francisco
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.
Núñez-Hernández, Cristina; Tierrez, Alberto; Ortega, Álvaro D.; Pucciarelli, M. Graciela; Godoy, Marta; Eisman, Blanca; Casadesús, Josep
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
Eroglu, Emrah; Rost, Rene; Bischof, Helmut; Blass, Sandra; Schreilechner, Anna; Gottschalk, Benjamin; Depaoli, Maria R; Klec, Christiane; Charoensin, Suphachai; Madreiter-Sokolowski, Corina T; Ramadani, Jeta; Waldeck-Weiermair, Markus; Graier, Wolfgang F; Malli, Roland
Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO• in vivo and in vitro, the real-time monitoring of NO• at the single-cell level is very challenging. The physiological or pathological effects of NO• are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO• are highly desirable. Recently, we expanded the pallet of NO• indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO•) probes (geNOps) that directly respond to cellular NO• fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO• signals in response to two different chemical NO•-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO• levels as compared with the inorganic NO• donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca 2+ indicator fura-2 was performed to visualize the tight regulation of Ca 2+ -dependent NO• formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO• signals in diverse experimental setups.
Balczun, Carsten; Scheid, Patrick L.
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
Stief, P.; Kamp, A.; de Beer, D.
Intracellular nitrate storage allows microorganisms to survive fluctuating nutrient availability and anoxic conditions in aquatic ecosystems. Here we show that diatoms, ubiquitous and highly abundant microalgae, represent major cellular reservoirs of nitrate in an intertidal flat of the German...... Wadden Sea and are potentially involved in anaerobic nitrate respiration. Intracellular nitrate (ICNO3) was present year-round in the sediment and was spatially and temporally correlated with fucoxanthin, the marker photopigment of diatoms. Pyrosequencing of SSU rRNA genes of all domains of life...... confirmed that ICNO3 storage was most likely due to diatoms rather than other known nitrate-storing microorganisms (i.e., large sulfur bacteria and the eukaryotic foraminifers and gromiids). Sedimentary ICNO3 concentrations reached up to 22.3 mu mol dm(-3) at the sediment surface and decreased with sediment...
Van der Henst, Charles; Scrignari, Tiziana; Maclachlan, Catherine; Blokesch, Melanie
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.
Berard, C. A.; Furia, T. J.; Goldberg, E. A.; Greene, R. C.
Encoding mechanism integral to the motor and using the permanent magnets embedded in the rotor eliminates the need for external devices to encode information relating the position and velocity of the rotating member.
Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.
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.
Ruedas-Rama, Maria J., E-mail: email@example.com [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: firstname.lastname@example.org [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)
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.
Verma, S K; Kingsley, K; Irizarry, I; Bergen, M; Kharwar, R N; White, J F
The aim of the present study was to evaluate the effects of the removal of indigenous bacteria from rice seeds on seedling growth and development. Here we report the presence of three indigenous endophytic bacteria in rice seeds that play important roles in modulating seedling development (shoot and root lengths, and formation of root hairs and secondary roots) and defence against pathogens. Seed-associated bacteria were removed using surface sterilization with NaOCl (bleach) followed by antibiotic treatment. When bacteria were absent, growth of seedlings in terms of root hair development and overall seedling size was less than that of seedlings that contained bacteria. Reactive oxygen staining of seedlings showed that endophytic bacteria became intracellular in root parenchyma cells and root hairs. Roots containing endophytic bacteria were seen to stain densely for reactive oxygen, while roots free of bacteria stained lightly for reactive oxygen. Bacteria were isolated and identified as Enterobacter asburiae (VWB1), Pantoea dispersa (VWB2) and Pseudomonas putida (VWB3) by 16S rDNA sequencing. Bacteria were found to produce indole acetic acid (auxins), inhibited the pathogen Fusarium oxysporum and solubilized phosphate. Reinoculation of bacteria onto seedlings derived from surface-disinfected rice and Bermuda grass seeds significantly restored seedling growth and development. Rice seeds harbour indigenous bacterial endophytes that greatly influence seedling growth and development, including root and shoot lengths, root hair formation and disease susceptibility of rice seedlings. This study shows that seeds of rice naturally harbour bacterial endophytes that play key roles in modulation of seedling development. © 2017 The Society for Applied Microbiology.
Kwakman, Paulus H. S.; te Velde, Anje A.; de Boer, Leonie; Speijer, Dave; Vandenbroucke-Grauls, Christina M. J. E.; Zaat, Sebastian A. J.
With the rise in prevalence of antibiotic-resistant bacteria, honey is increasingly valued for its antibacterial activity. To characterize all bactericidal factors in a medical-grade honey, we used a novel approach of successive neutralization of individual honey bactericidal factors. All bacteria
Full Text Available Abstract Background Wax esters are industrially relevant molecules exploited in several applications of oleochemistry and food industry. At the moment, the production processes mostly rely on chemical synthesis from rather expensive starting materials, and therefore solutions are sought from biotechnology. Bacterial wax esters are attractive alternatives, and especially the wax ester metabolism of Acinetobacter sp. has been extensively studied. However, the lack of suitable tools for rapid and simple monitoring of wax ester metabolism in vivo has partly restricted the screening and analyses of potential hosts and optimal conditions. Results Based on sensitive and specific detection of intracellular long-chain aldehydes, specific intermediates of wax ester synthesis, bacterial luciferase (LuxAB was exploited in studying the wax ester metabolism in Acinetobacter baylyi ADP1. Luminescence was detected in the cultivation of the strain producing wax esters, and the changes in signal levels could be linked to corresponding cell growth and wax ester synthesis phases. Conclusions The monitoring system showed correlation between wax ester synthesis pattern and luminescent signal. The system shows potential for real-time screening purposes and studies on bacterial wax esters, revealing new aspects to dynamics and role of wax ester metabolism in bacteria.
Galán, Jorge E; Waksman, Gabriel
Many bacteria have evolved specialized nanomachines with the remarkable ability to inject multiple bacterially encoded effector proteins into eukaryotic or prokaryotic cells. Known as type III, type IV, and type VI secretion systems, these machines play a central role in the pathogenic or symbiotic interactions between multiple bacteria and their eukaryotic hosts, or in the establishment of bacterial communities in a diversity of environments. Here we focus on recent progress elucidating the structure and assembly pathways of these machines. As many of the interactions shaped by these machines are of medical importance, they provide an opportunity to develop novel therapeutic approaches to combat important human diseases. Copyright © 2018 Elsevier Inc. All rights reserved.
Jenner, Dominic; Ducker, Catherine; Clark, Graeme; Prior, Jo; Rowland, Caroline A
The use of in vitro models to understand the interaction of bacteria with host cells is well established. In vitro bacterial infection models are often used to quantify intracellular bacterial load by lysing cell populations and subsequently enumerating the bacteria. Modern established techniques employ the use of fluorescence technologies such as flow cytometry, fluorescent microscopy, and/or confocal microscopy. However, these techniques often lack either the quantification of large data sets (microscopy) or use of gross fluorescence signal which lacks the visual confirmation that can provide additional confidence in data sets. Multispectral imaging flow cytometry (MIFC) is a novel emerging field of technology. This technology captures a bright field and fluorescence image of cells in a flow using a charged coupled device camera. It allows the analysis of tens of thousands of single cell images, making it an extremely powerful technology. Here MIFC was used as an alternative method of analyzing intracellular bacterial infection using Burkholderia thailandensis E555 as a model organism. It has been demonstrated that the data produced using traditional enumeration is comparable to data analyzed using MIFC. It has also been shown that by using MIFC it is possible to generate other data on the dynamics of the infection model rather than viable counts alone. It has been demonstrated that it is possible to inhibit the uptake of bacteria into mammalian cells and identify differences between treated and untreated cell populations. The authors believe this to be the first use of MIFC to analyze a Burkholderia bacterial species during intracellular infection. © 2016 Crown copyright. Published by Wiley Periodicals Inc. on behalf of ISAC. © 2015 International Society for Advancement of Cytometry.
Demon, Dieter; Vande Walle, Lieselotte; Lamkanfi, Mohamed
Caspase-11 contributes to host defense against Gram-negative bacterial pathogens by inducing an inflammatory form of programmed cell death in infected cells. Lipopolysaccharides (LPS) have been identified as the microbial agents that stimulate caspase-11 activation; however, the mechanism of LPS detection has been unknown. In a recent study, Shao and colleagues demonstrate that caspase-11 and its human homologues, caspases -4 and -5, unexpectedly act as direct sensors of cytosolic LPS. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.
Demon, Dieter; Vande Walle, Lieselotte; Lamkanfi, Mohamed
Caspase-11 contributes to host defense against Gram-negative bacterial pathogens by inducing an inflammatory form of programmed cell death in infected cells. Lipopolysaccharides (LPS) have been identified as the microbial agents that stimulate caspase-11 activation; however, the mechanism of LPS detection has been unknown. In a recent study, Shao and colleagues demonstrate that caspase-11 and its human homologues, caspases -4 and -5, unexpectedly act as direct sensors of cytosolic LPS.
associated with shigellosis (otherwise known as bacillary dysentery) include diarrhea, sometimes bloody; fever; stomach cramps; and tenesmus. Possible...host defenses, such as the acidic environment of the stomach , the competing commensal microbiota of the gastrointestinal tract, and the nearly...but are associated with more severe, invasive disease, and can result in complications such as genital ulcers and proctitis (331). Although six
Perrin-Cocon, Laure A.; Chesne, Serge; Pignot-Paintrand, Isabelle; Marche, Patrice N.; Villiers, Christian L. E-mail: email@example.com
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.
Perrin-Cocon, Laure A.; Chesne, Serge; Pignot-Paintrand, Isabelle; Marche, Patrice N.; Villiers, Christian L.
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
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
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.
Moffatt, Pierre; Salois, Patrick; St-Amant, Natalie; Gaumond, Marie-Hélène; Lanctôt, Christian
Terminal differentiation of keratinocytes results in the formation of a cornified layer composed of cross-linked intracellular and extracellular material. Using a signal trap expression screening strategy, we have identified four cDNAs encoding secreted proteins potentially involved in this process. One of the cDNAs is identical to the short isoform of suprabasin, a recently described epidermis-specific protein, which is shown here to contain a functional secretory signal. The second cDNA, sk89, encodes a protein of 493 amino acids, rich in glycine and serine residues. The third cDNA encodes a C-terminal fragment of SK89 (amino acids 410-493). It comprises exons 13 to 18 of the sk89 locus but transcription starts at an isoform-specific exon encoding a distinct secretory signal. The fourth cDNA encodes keratinocyte differentiation-associated protein (KDAP), a precursor protein of 102 amino acids. Subcellular localization by immunofluorescence and detection of the tagged proteins by Western blotting confirmed that the four proteins are secreted. Northern analysis and in situ hybridization revealed that expression of the corresponding genes was restricted to the suprabasal keratinocytes of the epidermis. These genes encoding epidermis-specific secreted products are found in a conserved cluster on human chromosome 19q13.12 and on mouse chromosome 7A3.
Full Text Available Bacteria, which prey on other microorganisms, are commonly found in the environment. While some of these organisms act as solitary hunters, others band together in large consortia before they attack their prey. Anecdotal reports suggest that bacteria practicing such a wolfpack strategy utilize antibiotics as predatory weapons. Consistent with this hypothesis, genome sequencing revealed that these micropredators possess impressive capacities for natural product biosynthesis. Here, we will present the results from recent chemical investigations of this bacterial group, compare the biosynthetic potential with that of non-predatory bacteria and discuss the link between predation and secondary metabolism.
Repin, Rul Aisyah Mat; Mutalib, Sahilah Abdul; Shahimi, Safiyyah; Khalid, Rozida Mohd.; Ayob, Mohd. Khan; Bakar, Mohd. Faizal Abu; Isa, Mohd Noor Mat
In this study, we performed bioinformatics analysis toward genome sequence of Lysinibacillussphaericus (L. sphaericus) to determine gene encoded for gelatinase. L. sphaericus was isolated from soil and gelatinase species-specific bacterium to porcine and bovine gelatin. This bacterium offers the possibility of enzymes production which is specific to both species of meat, respectively. The main focus of this research is to identify the gelatinase encoded gene within the bacteria of L. Sphaericus using bioinformatics analysis of partially sequence genome. From the research study, three candidate gene were identified which was, gelatinase candidate gene 1 (P1), NODE_71_length_93919_cov_158.931839_21 which containing 1563 base pair (bp) in size with 520 amino acids sequence; Secondly, gelatinase candidate gene 2 (P2), NODE_23_length_52851_cov_190.061386_17 which containing 1776 bp in size with 591 amino acids sequence; and Thirdly, gelatinase candidate gene 3 (P3), NODE_106_length_32943_cov_169.147919_8 containing 1701 bp in size with 566 amino acids sequence. Three pairs of oligonucleotide primers were designed and namely as, F1, R1, F2, R2, F3 and R3 were targeted short sequences of cDNA by PCR. The amplicons were reliably results in 1563 bp in size for candidate gene P1 and 1701 bp in size for candidate gene P3. Therefore, the results of bioinformatics analysis of L. Sphaericus resulting in gene encoded gelatinase were identified.
Huang, S.D.; Secor, C.K.; Zweig, R.M.; Ascione, R.
H-producing nonphotosynthetic bacteria are identified and H from sewage treatment plants, H from rumen bacteria, and large-scale production of H through the genetic manipulation of H-producing nonphotosynthetic bacteria are discussed. (Refs. 36).
Malik, Amarila; Radji, Maksum; Kralj, Slavko; Dijkhuizen, Lubbert
Homopolysaccharide (glucan and fructan) synthesis from sucrose by sucrase enzymes in lactic acid bacteria (LAB) has been well studied in the genera Leuconostoc, Streptococcus and Lactobacillus. This study aimed to identify and characterize genes encoding glucansucrase/glucosyltransferase (GTF) and
Lindenburg, Laurens; Merkx, Maarten
Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening. PMID:24991940
Ledermann D, Walter
As in 2009 the scientific world celebrates two hundreds years from the birthday of Charles Darwin and one hundred and fifty from the publication of The Origin of Species, an analysis of his complete work is performed, looking for any mention of bacteria. But it seems that the great naturahst never took knowledge about its existence, something rather improbable in a time when the discovery of bacteria shook the medical world, or he deliberately ignored them, not finding a place for such microscopic beings into his theory of evolution. But the bacteria badly affected his familiar life, killing scarlet fever one of his children and worsening to death the evolution of tuberculosis of his favourite Annie. Darwin himself could suffer the sickness of Chagas, whose etiological agent has a similar level to bacteria in the scale of evolution.
Chhabra, S.R.; Philipp, B.; Eberl, L.
molecules, in different Gram-positive and Gram-negative bacteria they control pathogenicity, secondary metabolite production, biofilm differentiation, DNA transfer and bioluminescence. The development of biosensors for the detection of these signal molecules has greatly facilitated their subsequent chemical...
Full Text Available Phytoplasmas are specialised bacteria that are obligate parasites of plant phloem tissue and insects. These bacteria have resisted all attempts of cell-free cultivation. Genome research is of particular importance to analyse the genetic endowment of such bacteria. Here we review the gene content of the four completely sequenced ‘Candidatus Phytoplasma’ genomes that include those of ‘Ca. P. asteris’ strains OY-M and AY-WB, ‘Ca. P. australiense,’ and ‘Ca. P. mali’. These genomes are characterized by chromosome condensation resulting in sizes below 900 kb and a G + C content of less than 28%. Evolutionary adaption of the phytoplasmas to nutrient-rich environments resulted in losses of genetic modules and increased host dependency highlighted by the transport systems and limited metabolic repertoire. On the other hand, duplication and integration events enlarged the chromosomes and contribute to genome instability. Present differences in the content of membrane and secreted proteins reflect the host adaptation in the phytoplasma strains. General differences are obvious between different phylogenetic subgroups. ‘Ca. P. mali’ is separated from the other strains by its deviating chromosome organization, the genetic repertoire for recombination and excision repair of nucleotides or the loss of the complete energy-yielding part of the glycolysis. Apart from these differences, comparative analysis exemplified that all four phytoplasmas are likely to encode an alternative pathway to generate pyruvate and ATP.
Shi, Deng-Ke; Zhu, Jing; Sun, Ze-Hua; Zhang, Guang; Liu, Rui; Zhang, Tian-Jun; Wang, Sheng-Li; Ren, Ang; Zhao, Ming-Wen
The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50 %, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44 %, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.
Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay
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
Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay
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.
Corti, S; Chevalier, J; Cremieux, A
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
Ruedas-Rama, Maria J; Walters, Jamie D; Orte, Angel; Hall, Elizabeth A H
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.
Mondragón-Terán, Paul; López-Hernández, Luz Berenice; Gutiérrez-Salinas, José; Suárez-Cuenca, Juan Antonio; Luna-Ceballos, Rosa Isela; Erazo Valle-Solís, Aura
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.
Rüdiger, Sten, E-mail: firstname.lastname@example.org
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.
Hitz, Benjamin C; Rowe, Laurence D; Podduturi, Nikhil R; Glick, David I; Baymuradov, Ulugbek K; Malladi, Venkat S; Chan, Esther T; Davidson, Jean M; Gabdank, Idan; Narayana, Aditi K; Onate, Kathrina C; Hilton, Jason; Ho, Marcus C; Lee, Brian T; Miyasato, Stuart R; Dreszer, Timothy R; Sloan, Cricket A; Strattan, J Seth; Tanaka, Forrest Y; Hong, Eurie L; Cherry, J Michael
The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database) and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data) has been released as a separate Python package.
Benjamin C Hitz
Full Text Available The Encyclopedia of DNA elements (ENCODE project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data has been released as a separate Python package.
Baban, Chwanrow K
Anti-cancer therapy faces major challenges, particularly in terms of specificity of treatment. The ideal therapy would eradicate tumor cells selectively with minimum side effects on normal tissue. Gene or cell therapies have emerged as realistic prospects for the treatment of cancer, and involve the delivery of genetic information to a tumor to facilitate the production of therapeutic proteins. However, there is still much to be done before an efficient and safe gene medicine is achieved, primarily developing the means of targeting genes to tumors safely and efficiently. An emerging family of vectors involves bacteria of various genera. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally. Furthermore, invasive species can deliver heterologous genes intra-cellularly for tumor cell expression. Here, we review the use of bacteria as vehicles for gene therapy of cancer, detailing the mechanisms of action and successes at preclinical and clinical levels.
Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max
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...
Sadowsky, Michael J.; Whitman, Richard L.
The Fecal Bacteria offers a balanced, integrated discussion of fecal bacteria and their presence and ecology in the intestinal tract of mammals, in the environment, and in the food supply. This volume covers their use in examining and assessing water quality in order to offer protection from illnesses related to swimming in or ingesting contaminated water, in addition to discussing their use in engineering considerations of water quality, modeling, monitoring, and regulations. Fecal bacteria are additionally used as indicators of contamination of ready-to-eat foods and fresh produce. The intestinal environment, the microbial community structure of the gut microbiota, and the physiology and genomics of this broad group of microorganisms are explored in the book. With contributions from an internationally recognized group of experts, the book integrates medicine, public health, environmental, and microbiological topics in order to provide a unique, holistic understanding of fecal bacteria. Moreover, it shows how the latest basic science and applied research findings are helping to solve problems and develop effective management strategies. For example, readers will discover how the latest tools and molecular approaches have led to our current understanding of fecal bacteria and enabled us to improve human health and water quality. The Fecal Bacteria is recommended for microbiologists, clinicians, animal scientists, engineers, environmental scientists, food safety experts, water quality managers, and students. It will help them better understand fecal bacteria and use their knowledge to protect human and environmental health. They can also apply many of the techniques and molecular tools discussed in this book to the study of a broad range of microorganisms in a variety of habitats.
Challenges for oncology practitioners and researchers include specific treatment and detection of tumours. The ideal anti-cancer therapy would selectively eradicate tumour cells, whilst minimising side effects to normal tissue. Bacteria have emerged as biological gene vectors with natural tumour specificity, capable of homing to tumours and replicating locally to high levels when systemically administered. This property enables targeting of both the primary tumour and secondary metastases. In the case of invasive pathogenic species, this targeting strategy can be used to deliver genes intracellularly for tumour cell expression, while non-invasive species transformed with plasmids suitable for bacterial expression of heterologous genes can secrete therapeutic proteins locally within the tumour environment (cell therapy approach). Many bacterial genera have been demonstrated to localise to and replicate to high levels within tumour tissue when intravenously (IV) administered in rodent models and reporter gene tagging of bacteria has permitted real-time visualisation of this phenomenon. Live imaging of tumour colonising bacteria also presents diagnostic potential for this approach. The nature of tumour selective bacterial colonisation appears to be tumour origin- and bacterial species- independent. While originally a correlation was drawn between anaerobic bacterial colonisation and the hypoxic nature of solid tumours, it is recently becoming apparent that other elements of the unique microenvironment within solid tumours, including aberrant neovasculature and local immune suppression, may be responsible. Here, we consider the pre-clinical data supporting the use of bacteria as a tumour-targeting tool, recent advances in the area, and future work required to develop it into a beneficial clinical tool.
AFRL-RH-WP-TP-2017-0001 Optimal Achievable Encoding for Brain- Machine Interface Eduardo Chichilnisky Leland Stanford Junior...Oct 2016 – 30 Sep 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Optimal Achievable Encoding for Brain- Machine Interface 5b...required. First, we developed novel models of retinal encoding that improve upon the state of the art, by using machine learning methods to
Fikriye Alev Akçay
Full Text Available Metal particles reduced to nano size by nanotechnological methods are confronted in many different fields such as biomedical and physicochemical, pharmaceutical, electric-electronic, automotive and food industries. Nanoparticles can be produced using chemical, physical and biological methods, of which chemical processes are in common use. However, physical and chemical methods are not environmentally friendly and economical because they require the use of high temperature, high pressure and toxic chemicals. For this reason, interest in the production of metal nanoparticles by biological methods, also called green technology, an environmentally friendly and sustainable approach, has increased in recent years. With some plant extracts and intracellular and extracellular secretions of microorganisms, some reduction reactions take place and metal nanoparticles are produced. Bacteria have been actively involved in nanotechnology in recent years due to their diversity in nature, their ease of isolation, and ease of nanoparticle synthesis. In this article, production and application of metal nanoparticles by using bacterial methods have been reviewed.
Zhou, Wei; Zhang, Yanzong; Ding, Xiaohui; Liu, Yan; Shen, Fei; Zhang, Xiaohong; Deng, Shihuai; Xiao, Hong; Yang, Gang; Peng, Hong [Sichuan Agricultural Univ., Chengdu (China). Provincial Key Lab. of Agricultural Environmental Engineering
Magnetotactic bacteria (MTB), which can orient and migrate along a magnetic line of force due to intracellular nanosized magnetosomes, have been a subject of research in the medical field, in dating environmental changes, and in environmental remediation. This paper reviews the recent development of MTB as biosorbents for heavy metals. Ultrastructures and taxis of MTB are investigated. Adsorptions in systems of unitary and binary ions are highlighted, as well as adsorption conditions (temperature, pH value, biomass concentration, and pretreatments). The separation and desorption of MTB in magnetic separators are also discussed. A green method to produce metal nanoparticles is provided, and an energy-efficient way to recover precious metals is put forward during biosorption. (orig.)
Manivasagan, Panchanathan; Kim, Se-Kwon
Extracellular polysaccharides (EPSs) produced by microorganisms are a complex mixture of biopolymers primarily consisting of polysaccharides, as well as proteins, nucleic acids, lipids, and humic substances. Microbial polysaccharides are multifunctional and can be divided into intracellular polysaccharides, structural polysaccharides, and extracellular polysaccharides or exopolysaccharides. Recent advances in biological techniques allow high levels of polysaccharides of interest to be produced in vitro. Biotechnology is a powerful tool to obtain polysaccharides from a variety of marine microorganisms, by controlling the growth conditions in a bioreactor while tailoring the production of biologically active compounds. The aim of this chapter is to give an overview of current knowledge on extracellular polysaccharides producing marine bacteria isolated from marine environment. © 2014 Elsevier Inc. All rights reserved.
Eddy, Sean R
Two clichés of science journalism have now played out around the ENCODE project. ENCODE's publicity first presented a misleading "all the textbooks are wrong" narrative about noncoding human DNA. Now several critiques of ENCODE's narrative have been published, and one was so vitriolic that it fueled "undignified academic squabble" stories that focused on tone more than substance. Neither story line does justice to our actual understanding of genomes, to ENCODE's results, or to the role of big science in biology. Copyright © 2013 Elsevier Ltd. All rights reserved.
Dynapar has developed the Acuro AX71 absolute encoder for use on offshore or land-based oil rig operations. It provides feedback on the operation of automated systems such as draw works, racking systems, rotary tables and top drives. By ensuring that automated systems function properly, this encoder responds to a need by the oil and gas industry to keep workers safe and improve efficiency, particularly for operations in rugged situations. The encoder provides feedback from motor systems to controllers, giving information about position and speed of downhole drill bits. This newly developed encoder is better than commonly used incremental encoders which are not precise in strong electrical noise environments. Rather, the absolute encoder uses a different method of reporting to the controller. A digital signal is transmitted constantly as the device operates. It is less susceptible to noise issues. It is highly accurate, tolerant of noise and is not affected by power outages. However, the absolute encoder is generally more delicate in drilling applications with high ambient temperatures and shock levels. Dynapar addressed this issue by developing compact stainless steel housing that is useful for corrosion resistance in marine applications. The AX71 absolute encoder can withstand up to 100 G of mechanical shock and ambient temperatures of up to 60 degrees C. The encoder is ATEX certified without barriers, and offers the high resolution feedback of 4,000 counts of multiturn rotation and 16,000 counts of position. 1 fig.
Smirnov, Nikolai V.; Latyev, Svjatoslav M.; Naumova, Anastasiia I.
A novel dynamic goniometer for the accuracy of rotary encoders has been developed on the base of the method of comparison with the reference encoder. The set-up of the goniometer considers all constructive and informative characteristics of measured encoders. The novel goniometer construction uses the new compensating method of instrumental errors in automatic working process. The advantages of the dynamic goniometer in combination with an optical rotary encoder at the reduction of the measuring time and a simultaneous increase of the accuracy.
Mussmann, Marc; Hu, Fen Z.; Richter, Michael
Marine sediments are frequently covered by mats of the filamentous Beggiatoa and other large nitrate-storing bacteria that oxidize hydrogen sulfide using either oxygen or nitrate, which they store in intracellular vacuoles. Despite their conspicuous metabolic properties and their biogeochemical...
Frigaard, N.-U.; Bryant, D. A.
The new series "Microbiology Monographs" begins with two volumes on intracellular components in prokaryotes. In this second volume, "Complex Intracellular Structures in Prokaryotes", the components, labelled complex intracellular structures, encompass a multitude of important cellular functions. ...
Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto
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.
Gao, Han; He, Fang; Lin, Xinhua; Wu, Yihui
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.
Claire E. Hills
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.
Bay, Johannes; Kohlhaas, Michael; Maack, Christoph
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.
Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena
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.
Schumacher, Dominik; Helma, Jonas; Schneider, Anselm F. L.; Leonhardt, Heinrich
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
Maturana, Pauline; Graham, Joseph G; Sharma, Uma M; Voth, Daniel E
The intracellular bacterial agent of Q fever, Coxiella burnetii, translocates effector proteins into its host cell cytosol via a Dot/Icm type IV secretion system (T4SS). The T4SS is essential for parasitophorous vacuole formation, intracellular replication, and inhibition of host cell death, but the effectors mediating these events remain largely undefined. Six Dot/Icm substrate-encoding genes were recently discovered on the C. burnetii cryptic QpH1 plasmid, three of which are conserved among all C. burnetii isolates, suggesting that they are critical for conserved pathogen functions. However, the remaining hypothetical proteins encoded by plasmid genes have not been assessed for their potential as T4SS substrates. In the current study, we further defined the T4SS effector repertoire encoded by the C. burnetii QpH1, QpRS, and QpDG plasmids that were originally isolated from acute-disease, chronic-disease, and severely attenuated isolates, respectively. Hypothetical proteins, including those specific to QpRS or QpDG, were screened for translocation using the well-established Legionella pneumophila T4SS secretion model. In total, six novel plasmid-encoded proteins were translocated into macrophage-like cells by the Dot/Icm T4SS. Four newly identified effectors are encoded by genes present only on the QpDG plasmid from severely attenuated Dugway isolates, suggesting that the presence of specific effectors correlates with decreased virulence. These results further support the idea of a critical role for extrachromosomal elements in C. burnetii pathogenesis.
Deveau, Aurelie [French National Insitute for Agricultural Research (INRA); Labbe, Jessy [ORNL
This chapter focuses on the Mycorrhiza Helper Bacteria (MHB), a generic name given to bacteria which stimulate the formation of mycorrhizal symbiosis. By extension, some bacterial strains that positively impact the functioning of mycorrhizal symbiosis are also called MHB. These bacteria have applicative interests, as they indirectly improve the health and growth of tree seedlings. MHB are not restricted to a specific type of ecosystem, but are rather generalist in the way that they associate with both herbaceous and woody mycorrhizal plants from boreal, temperate, arid and tropical ecosystems. However, understanding the molecular mechanisms and their specificities will help us to know more about the ecology of the MHB. The process of acquisition varies between fungal species; while ectomycorrhizal fungi most probably recurrently acquire them from the environment, the association between bacterial endosymbionts and Glomeromycota probably dates back to very ancient times, and has since been vertically transmitted.
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
Meza, Beatriz; de-Bashan, Luz E; Bashan, Yoav
Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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.
... encoder programming shall be protected by a lock or other security measures and be configured so that... for either manual or automatic operation. (2) Inputs. The encoder shall have two inputs, one for audio... initiating the automatic generation of the simultaneous tones shall be protected to prevent accidental...
Gorissen, M.; Eling, P.; Luijtelaar, G. van; Coenen, A.
Benzodiazepines are known to induce amnesic effects. To specify these effects more precisely, 40 healthy volunteers were given 15 mg diazepam or placebo. Effects on a chain of encoding operations were investigated: activation of memory representations, spreading of activation, semantic encoding and
Full Text Available The Arabic Diatessaron Project (henceforth ADP is an international research project in Digital Humanities that aims to collect, digitalise and encode all known manuscripts of the Arabic Diatessaron (henceforth AD, a text that has been relatively neglected in scholarly research. ADP’s final goal is to provide a number of tools that can enable scholars to effectively query, compare and investigate all known variants of the text that will be encoded as far as possible in compliance with the Text Encoding Initiative (TEI guidelines. The paper addresses a number of issues involved in the process of digitalising manuscripts included in the two existing editions (Ciasca 1888 and Marmardji 1935, adding variants in unedited manuscripts, encoding and lemmatising the text. Issues involved in the design of the ADP include presentation of variants, choice of the standard text, applicability of TEI guidelines, automatic translation between different encodings, cross-edition concordances and principles of lemmatisation.
Poirel, Laurent; Jayol, Aurélie; Nordmann, Patrice
SUMMARYPolymyxins are well-established antibiotics that have recently regained significant interest as a consequence of the increasing incidence of infections due to multidrug-resistant Gram-negative bacteria. Colistin and polymyxin B are being seriously reconsidered as last-resort antibiotics in many areas where multidrug resistance is observed in clinical medicine. In parallel, the heavy use of polymyxins in veterinary medicine is currently being reconsidered due to increased reports of polymyxin-resistant bacteria. Susceptibility testing is challenging with polymyxins, and currently available techniques are presented here. Genotypic and phenotypic methods that provide relevant information for diagnostic laboratories are presented. This review also presents recent works in relation to recently identified mechanisms of polymyxin resistance, including chromosomally encoded resistance traits as well as the recently identified plasmid-encoded polymyxin resistance determinant MCR-1. Epidemiological features summarizing the current knowledge in that field are presented. Copyright © 2017 American Society for Microbiology.
Tang, Bor Luen
Intracellular bacterial pathogens drive the formation of host membrane-derived pseudo-organelles that facilitate their replication, survival, or dormancy. The formation and maintenance of these bacteria-containing vacuoles (BCVs) are dependent on the bacteria's ability to usurp the host's intracellular membrane system, in particular dynamic compartments involved in exo-/endocytic membrane traffic and autophagy. Bacteria are typically internalized by phagocytosis, and the compartment matures through endosomal fusion. The bacteria-containing phagosome/endosome often becomes the base for BCV formation. Diverse strategies used by different bacterial pathogens prevent the BCV from being destroyed via the endolysosomal pathway. Furthermore, bacterial survival or proliferation in BCVs could be augmented by host membrane transport processes subverted by secreted bacterial factors, which facilitate the acquisition of membrane sources and nutrients. BCVs may be targeted for destruction by autophagy, and various facultative and obligate intracellular bacteria have evolved ways to evade or even exploit autophagy. Here we review examples of bacterial subversion of host cellular membrane transport and autophagy machinery for a productive invasion.
Full Text Available Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA. All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN. Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists.
Nenert, Rodolphe; Allendorfer, Jane B; Szaflarski, Jerzy P
Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA) with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA). All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions) and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN). Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s) of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists.
Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Antifreeze Proteins of Bacteria. M K Chattopadhyay. General Article Volume 12 Issue 12 December 2007 pp 25-30. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/012/12/0025-0030 ...
Bacteria are thought to be examples of organisms that do not age. ... sues, organs, organ systems, organism, population, species, and .... Humans inevitably grow old through aging. All vertebrates show physical manifestations of aging somewhat similar to humans (other than white hair!). Aging is also seen in plants.
Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Antifreeze Proteins of Bacteria. M K Chattopadhyay. General Article Volume 12 Issue 12 December 2007 pp 25-30. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/012/12/0025-0030. Keywords.
Longenecker, Nevin E.; Oppenheimer, Dan
A study conducted by high school advanced bacteriology students appears to confirm the hypothesis that the incremental administration of antibiotics on several species of bacteria (Escherichia coli, Staphylococcus epidermis, Bacillus sublitus, Bacillus megaterium) will allow for the development of antibiotic-resistant strains. (PEB)
Isolation and characterization of two novel polyhydroxybutyrate (PHB)-producing bacteria. ... subsequently studied using phenotype microarray panels which allowed the testing of the effect of more than 90 different carbon, nitrogen, sulfur and phosphorus sources as well as pH on the growth characteristics of these strains.
Full Text Available Innate immune recognition of bacteria is the first requirement for mounting an effective immune response able to control infection. Over the previous decade, the general paradigm was that extracellular bacteria were only sensed by cell surface-expressed Toll-like receptors (TLRs, whereas cytoplasmic sensors, including members of the Nod-like receptor (NLR family, were specific to pathogens capable of breaching the host cell membrane. It has become apparent, however, that intracellular innate immune molecules, such as the NLRs, play key roles in the sensing of not only intracellular, but also extracellular bacterial pathogens or their components. In this review, we will discuss the various mechanisms used by bacteria to activate NLR signaling in host cells. These mechanisms include bacterial secretion systems, pore-forming toxins and outer membrane vesicles. We will then focus on the influence of NLR activation on the development of adaptive immune responses in different cell types.
Matsumoto, T.; Fukuda, H. [Kobe University, Kobe (Japan). Graduate School of Science and Technology, Division of Molecular Science; Takahashi, S.; Ueda, M.; Tanaka, A. [Kyoto University, Kyoto (Japan). Dept. of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering; Kaieda, M.; Kondo, A. [Kobe University, Kobe (Japan). Faculty of Engineering, Dept. of Chemical Science and Engineering
Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomyces cerevisiae MT8-1. The gene encoding lipase from R. oryzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rPRoROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization. (orig.)
Lewis, Adam J.; Richards, Amanda C.; Mulvey, Matthew A.
Within the mammalian urinary tract uropathogenic bacteria face many challenges, including the shearing flow of urine, numerous antibacterial molecules, the bactericidal effects of phagocytes, and a scarcity of nutrients. These problems may be circumvented in part by the ability of uropathogenic Escherichia coli (UPEC) and several other uropathogens to invade the epithelial cells that line the urinary tract. By entering host cells, uropathogens can gain access to additional nutrients and protection from both host defenses and antibiotic treatments. Translocation through host cells can facilitate bacterial dissemination within the urinary tract, while the establishment of stable intracellular bacterial populations may create reservoirs for relapsing and chronic urinary tract infections (UTIs). Here we review the mechanisms and consequences of host cell invasion by uropathogenic bacteria, with consideration of the defenses that are brought to bear against facultative intracellular pathogens within the urinary tract. The relevance of host cell invasion to the pathogenesis of UTIs in human patients is also assessed, along with some of the emerging treatment options that build upon our growing understanding of the infectious life cycle of UPEC and other uropathogenic bacteria. PMID:28087946
Introduction: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)is increasingly utilized as a rapid technique to identify microorganisms including pathogenic bacteria. However, little attention has been paid to the significant proteomic information encoded in ...
Chu, Qian; Ma, Jiao; Saghatelian, Alan
Molecular biology, genomics and proteomics methods have been utilized to reveal a non-annotated class of endogenous polypeptides (small proteins and peptides) encoded by short open reading frames (sORFs), or small open reading frames (smORFs). We refer to these polypeptides as s(m)ORF-encoded polypeptides or SEPs. The early SEPs were identified via genetic screens, and many of the RNAs that contain s(m)ORFs were originally considered to be non-coding; however, elegant work in bacteria and flies demonstrated that these s(m)ORFs code for functional polypeptides as small as 11-amino acids in length. The discovery of these initial SEPs led to search for these molecules using methods such as ribosome profiling and proteomics, which have revealed the existence of many SEPs, including novel human SEPs. Unlike screens, omics methods do not necessarily link a SEP to a cellular or biological function, but functional genomic and proteomic strategies have demonstrated that at least some of these newly discovered SEPs have biochemical and cellular functions. Here, we provide an overview of these results and discuss the future directions in this emerging field.
Rogério Santos de Oliveira Cruz
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.
Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten
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
Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena
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
Gingl, Ewald; Burger, Anna-M; Barth, Friedrich G
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.
Stein, Olga; Stein, Yechezkiel
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
Mayne, Richard; Adamatzky, Andrew
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.
The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107
Ishikawa, Masahito; Tanaka, Yuya; Suzuki, Risa; Kimura, Kota; Tanaka, Kenya; Kamiya, Kazuhide; Ito, Hidehiro; Kato, Souichiro; Kamachi, Toshiaki; Hori, Katsutoshi; Nakanishi, Shuji
This study aimed to develop a novel method for real-time monitoring of the intracellular redox states in a methanotroph Methylococcus capsulatus, using Peredox as a genetically encoded fluorescent sensor of the NADH:NAD + ratio. As expected, the fluorescence derived from the Peredox-expressing M. capsulatus transformant increased by supplementation of electron donor compounds (methane and formate), while it decreased by specifically inhibiting the methanol oxidation reaction. Electrochemical measurements confirmed that the Peredox fluorescence reliably represents the intracellular redox changes. This study is the first to construct a reliable redox-monitoring method for methanotrophs, which will facilitate to develop more efficient methane-to-methanol bioconversion processes. Copyright © 2017 Elsevier Ltd. All rights reserved.
Vinoth, P; Sankar, P
An in-silico system to encode structure, bonding and properties of coordination complexes is developed. The encoding is achieved through a semantic XML markup frame. Composition of the coordination complexes is captured in terms of central atom and ligands. Structural information of central atom is detailed in terms of electron status of valence electron orbitals. The ligands are encoded with specific reference to the electron environment of ligand centre atoms. Behaviour of ligands to form low or high spin complexes is accomplished by assigning a Ligand Centre Value to every ligand based on the electronic environment of ligand centre atom. Chemical ontologies are used for categorization purpose and to control different hybridization schemes. Complexes formed by the central atoms of transition metal, non-transition elements belonging to s-block, p-block and f-block are encoded with a generic encoding platform. Complexes of homoleptic, heteroleptic and bridged types are also covered by this encoding system. Utility of the encoded system to predict redox electron transfer reaction in the coordination complexes is demonstrated with a simple application. Copyright © 2017 Elsevier Inc. All rights reserved.
Hu, Jiangtao; Wang, Huazhong; Zhao, Lei; Shao, Yu; Wang, Meixia; Osen, Are
Reverse time migration (RTM) is an accurate seismic imaging method for imaging the complex subsurface structure. Traditional common shot RTM suffers from low efficiency due to the large number of single shot gathers, especially for marine seismic data. Phase encoding is commonly used to reduce the computational cost of RTM. Phase encoding in the frequency domain is usually related to time shift in the time domain. Therefore, phase-encoding-based RTM needs time padding to avoid information loss which degrades the efficiency of the time-domain wavefield extrapolator. In this paper, an efficient time-domain RTM scheme based on the amplitude encoding is proposed. This scheme uses the orthogonal cosine basis as the encoding function, which has similar physical meaning to plane wave encoding (i.e. plane-wave components with different surface shooting angles). The proposed scheme can generate a qualified imaging result as well as common shot RTM but with less computational cost. Since this scheme does not need time padding, it is more efficient than the phase encoding schemes and can be conveniently implemented in the time domain. Numerical examples on the Sigsbee2a synthetic dataset demonstrate the feasibility of the proposed method.
Tsunematsu, Noriko; Goto, Mieko; Saiki, Yumiko; Baba, Michiko; Udagawa, Tadashi; Kazumi, Yuko
The bacilli which were isolated from a patient suspected of the mixed infections with Mycobacterium avium and Mycobacterium intracellulare, were analyzed. The genotypes of M. avium in the sedimented fractions of treated sputum and in some colonies isolated from Ogawa medium were compared by the Variable Numbers of Tandem Repeats (VNTR). A woman, aged 57. Mycobacterial species isolated from some colonies by culture in 2004 and 2006 and from the treated sputum in 2006, were determined by DNA sequencing analysis of the 16S rRNA gene. Also, by using VNTR, the genotype of mycobacteria was analyzed. [Results] (1) The colony isolated from Ogawa medium in 2004 was monoclonal M. avium. (2) By VNTR analyses of specimens in 2006, multiple acid-fast bacteria were found in the sputum sediment and in isolated bacteria from Ogawa medium. (3) By analyses of 16S rRNA DNA sequence, M. avium and M. intracellulare were found in the colonies isolated from the sputum sediment and the Ogawa medium in 2006. (4) The same VNTR patterns were obtained in M. avium in 2004 and 2006 when single colony was analyzed. (5) From the showerhead and culvert of the bathroom in the patient's house, M. avium was not detected. By VNTR analyses, it was considered that the mixed infections of M. avium and M. intracellulare had been generated during treatment in this case. Therefore, in the case of suspected complex infection, VNTR analysis would be a useful genotyping method in M. avium complex infection.
Full Text Available We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular alpha-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp and a long (62,829 bp form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin-antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as beta-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria.
Ogata, Hiroyuki; Renesto, Patricia; Audic, Stéphane; Robert, Catherine; Blanc, Guillaume; Fournier, Pierre-Edouard; Parinello, Hugues; Claverie, Jean-Michel; Raoult, Didier
We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular alpha-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin-antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as beta-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria.
Rana, Tanu; Hasan, Rafia J; Nowicki, Stella; Venkatarajan, Mathura S; Singh, Rajbir; Urvil, Petri T; Popov, Vsevolod; Braun, Werner A; Popik, Waldemar; Goodwin, J Shawn; Nowicki, Bogdan J
Escherichia coli-bearing Dr-adhesins (Dr+ E. coli) cause chronic pyelonephritis in pregnant women and animal models. This chronic renal infection correlates with the capacity of bacteria to invade epithelial cells expressing CD55. The mechanism of infection remains unknown. CD55 amino acids in the vicinity of binding pocket-Ser155 for Dr-adhesin were mutated to alanine and subjected to temporal gentamicin-invasion/gentamicin-survival assay in Chinese hamster ovary cells. CD55/microtubule (MT) responses were studied using confocal/electron microscopy, and 3-dimensional structure analysis. Mutant analysis revealed that complement-protective CD55-Ser165 and CD55-Phe154 epitopes control E. coli invasion by coregulating CD55-MT complex expression. Single-point CD55 mutations changed E. coli to either a minimally invasive (Ser165Ala) or a hypervirulent pathogen (Phe154Ala). Thus, single amino acid modifications with no impact on CD55 structure and bacterial attachment can have a profound impact on E. coli virulence. While CD55-Ser165Ala decreased E. coli invasion and led to dormant intracellular persistence, intracellular E. coli in CD55-Phe154Ala developed elongated forms (multiplying within vacuoles), upregulated CD55-MT complexes, acquired CD55 coat, and escaped phagolysosomal fusion. E. coli target complement-protective CD55 epitopes for invasion and exploit CD55-MT complexes to escape phagolysosomal fusion, leading to a nondestructive parasitism that allows bacteria to persist intracellularly.
Zarrabi, N.; Mancini, E.; Tay, J.; Shahand, S.; Sloot, P.M.A.
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
Knoblauch, Jan; Mullendore, Daniel L.; Jensen, Kaare Hartvig
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...
Filipeanu, CM; Brailoiu, E; Henning, RH; Deelman, LE; de Zeeuw, D; Nelemans, SA
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
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 ...
Conover, Matt S; Hadjifrangiskou, Maria; Palermo, Joseph J; Hibbing, Michael E; Dodson, Karen W; Hultgren, Scott J
Uropathogenic Escherichia coli (UPEC) is the primary etiological agent of over 85% of community-acquired urinary tract infections (UTIs). Mouse models of infection have shown that UPEC can invade bladder epithelial cells in a type 1 pilus-dependent mechanism, avoid a TLR4-mediated exocytic process, and escape into the host cell cytoplasm. The internalized UPEC can clonally replicate into biofilm-like intracellular bacterial communities (IBCs) of thousands of bacteria while avoiding many host clearance mechanisms. Importantly, IBCs have been documented in urine from women and children suffering acute UTI. To understand this protected bacterial niche, we elucidated the transcriptional profile of bacteria within IBCs using microarrays. We delineated the upregulation within the IBC of genes involved in iron acquisition, metabolism, and transport. Interestingly, lacZ was highly upregulated, suggesting that bacteria were sensing and/or utilizing a galactoside for metabolism in the IBC. A ΔlacZ strain displayed significantly smaller IBCs than the wild-type strain and was attenuated during competitive infection with a wild-type strain. Similarly, a galK mutant resulted in smaller IBCs and attenuated infection. Further, analysis of the highly upregulated gene yeaR revealed that this gene contributes to oxidative stress resistance and type 1 pilus production. These results suggest that bacteria within the IBC are under oxidative stress and, consistent with previous reports, utilize nonglucose carbon metabolites. Better understanding of the bacterial mechanisms used for IBC development and establishment of infection may give insights into development of novel anti-virulence strategies. Urinary tract infections (UTIs) are one of the most common bacterial infections, impacting mostly women. Every year, millions of UTIs occur in the U.S. with most being caused by uropathogenic E. coli(UPEC). During a UTI, UPEC invade bladder cells and form an intracellular bacterial community
The friendly bacteria within us Commensal bacteria of the intestine: Roles in health and disease B.S. Ramakrishna Professor & Head Gastroenterology & Hepatology Christian Medical College Vellore · Slide 2 · Intestinal bacteria: the hidden organ · Slide 4 · Slide 5 · The normal bacterial flora prevents GI disease · Slide 7.
Short chain fatty acids (SCFA) are main source of energy for colonic epithelial cells · SCFA – role in colonic disease · SCFA prevent mucosal inflammation · Immunoregulation by gut bacteria · Balance of bacterial species in the gut · Immunosensory detection of intestinal bacteria · Pathogenic bacteria release interleukin-8 ...
Radovic-Moreno, Aleksandar Filip
Bacterial infections continue to be a significant concern particularly in healthcare settings and in the developing world. Current challenges include the increasing spread of drug resistant (DR) organisms, the side effects of antibiotic therapy, the negative consequences of clearing the commensal bacterial flora, and difficulties in developing prophylactic vaccines. This thesis was an investigation of the potential of a class of polymeric nanoparticles (NP) to contribute to the management of bacterial infections. More specifically, steps were taken towards using these NPs (1) to achieve greater spatiotemporal control over drug therapy by more targeted antibiotic delivery to bacteria, and (2) to develop a prophylactic vaccine formulation against the common bacterial sexually transmitted disease (STD) caused by Chlamydia trachomatis. In the first part, we synthesized polymeric NPs containing poly(lactic-co-glycolic acid)-block-poly(L-histidine)-block-poly(ethylene glycol) (PLGA-PLH-PEG). We show that these NPs are able to bind to bacteria under model acidic infection conditions and are able to encapsulate and deliver vancomycin to inhibit the growth of Staphylococcus aureus bacteria in vitro. Further work showed that the PLGA-PLH-PEG-based NPs demonstrated the potential for competition for binding bacteria at a site of infection from soluble protein and model phagocytic and tissue-resident cells in a NP composition dependent manner. The NPs demonstrated low toxicity in vitro, were well tolerated by mice in vivo, and circulated in the blood on timescales comparable to control PLGA-PEG NPs. In the second part, we used PLGA-PLH-PEG-based NPs to design a prophylactic vaccine against the obligate intracellular bacterium Chlamydia trachomatis, the most common cause of bacterial STD in the world. Currently, no vaccines against this pathogen are approved for use in humans. We first formulated NPs encapsulating the TLR7 agonist R848 conjugated to poly(lactic acid) (R848-PLA
Pinu, Farhana R; Villas-Boas, Silas G; Aggio, Raphael
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.
Muller, J. A.; Ross, R. P.; Fitzgerald, G. F.; Stanton, C.
Lactic acid bacteria (LAB) have been used for many years as natural biopreservatives in fermented foods. A small group of LAB are also believed to have beneficial health effects on the host, so called probiotic bacteria. Probiotics have emerged from the niche industry from Asia into European and American markets. Functional foods are one of the fastest growing markets today, with estimated growth to 20 billion dollars worldwide by 2010 (GIA, 2008). The increasing demand for probiotics and the new food markets where probiotics are introduced, challenges the industry to produce high quantities of probiotic cultures in a viable and stable form. Dried concentrated probiotic cultures are the most convenient form for incorporation into functional foods, given the ease of storage, handling and transport, especially for shelf-stable functional products. This chapter will discuss various aspects of the challenges associated with the manufacturing of probiotic cultures.
Kontiainen, S; Rinne, E
Bacterial cultures derived from 432 chronic leg ulcers were analysed retrospectively to determine which bacteria are most commonly found in these ulcers. The study covered a 2-year period. Two-thirds of the patients were over 70 years of age. Staphylococcus aureus was found in nearly half of the ulcers studied, Pseudomonas sp. in one-third, pyogenic streptococci and enterococci in every fifth and Proteus sp. in every tenth. The frequency by which pyogenic streptococci were isolated was about 10 to 20 times as high as previously reported. Obligate anaerobic bacteria were also frequently isolated. The sensitivity of the isolates from the second year to antimicrobial agents likely to be chosen if systemic therapy were required is also reported. The results are discussed in relation to previous findings.
In order to ascertain the role of biological activity in ancient sediments, two microbiological studies were carried out. The first was on pleistocenic clay sediments on land, the second on deep oceanic sediments. In the present paper by direct counting the samples is demonstrated the presence of bacteria in a range of 10 5 to 10 7 . Further studies must be carried out to ascertain the activities by in situ incubation methods
Bacteria growing in paper machines can cause several problems. Biofilms detaching from paper machine surfaces may lead to holes and spots in the end product or even break the paper web leading to expensive delays in production. Heat stable endospores will remain viable through the drying section of paper machine, increasing the microbial contamination of paper and board. Of the bacterial species regularly found in the end products, Bacillus cereus is the only one classified as a pathogen. Cer...
Duong, Nancy; Osborne, Suzanne; Bustamante, Víctor H; Tomljenovic, Ana M; Puente, José L; Coombes, Brian K
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.
Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens. PMID:22719929
Catalase/peroxidases (KatGs) are a superfamily of reactive oxygen species (ROS)-degrading enzymes believed to be horizontally acquired by ancient Ascomycota from bacteria. Subsequent gene duplication resulted in two KatG paralogs in ascomycetes: the widely distributed intracellular KatG1 group, and ...
S Opiyo; R Pardy; H Moriyama; E Moriyama
BACKGROUND: Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo2-lipid A, the LPS substructure, involves with nine enzymatic steps. RESULTS: In order to elucidate the evolutionary pathway of Kdo2-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB). Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria. CONCLUSIONS: The nine-enzyme pathway, which was established based on the studies mainly in Escherichia coli K12, appears to be the most derived and optimized form. It is found only in E. coli and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo2-lipid A variants as the likely end products. The Kdo2-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.
Welters, Marij J P; Gouttefangeas, Cécile; Ramwadhdoebe, Tamara H; Letsch, Anne; Ottensmeier, Christian H; Britten, Cedrik M; van der Burg, Sjoerd H
Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables.
Lyttleton, Oliver; Wright, Alexander; Treanor, Darren; Lewis, Paul
The Tissue Microarray Data Exchange Specification (TMA DES) is an XML specification for encoding TMA experiment data. While TMA DES data is encoded in XML, the files that describe its syntax, structure, and semantics are not. The DTD format is used to describe the syntax and structure of TMA DES, and the ISO 11179 format is used to define the semantics of TMA DES. However, XML Schema can be used in place of DTDs, and another XML encoded format, RDF, can be used in place of ISO 11179. Encoding all TMA DES data and metadata in XML would simplify the development and usage of programs which validate and parse TMA DES data. XML Schema has advantages over DTDs such as support for data types, and a more powerful means of specifying constraints on data values. An advantage of RDF encoded in XML over ISO 11179 is that XML defines rules for encoding data, whereas ISO 11179 does not. We created an XML Schema version of the TMA DES DTD. We wrote a program that converted ISO 11179 definitions to RDF encoded in XML, and used it to convert the TMA DES ISO 11179 definitions to RDF. We validated a sample TMA DES XML file that was supplied with the publication that originally specified TMA DES using our XML Schema. We successfully validated the RDF produced by our ISO 11179 converter with the W3C RDF validation service. All TMA DES data could be encoded using XML, which simplifies its processing. XML Schema allows datatypes and valid value ranges to be specified for CDEs, which enables a wider range of error checking to be performed using XML Schemas than could be performed using DTDs.
Full Text Available Background: The Tissue Microarray Data Exchange Specification (TMA DES is an XML specification for encoding TMA experiment data. While TMA DES data is encoded in XML, the files that describe its syntax, structure, and semantics are not. The DTD format is used to describe the syntax and structure of TMA DES, and the ISO 11179 format is used to define the semantics of TMA DES. However, XML Schema can be used in place of DTDs, and another XML encoded format, RDF, can be used in place of ISO 11179. Encoding all TMA DES data and metadata in XML would simplify the development and usage of programs which validate and parse TMA DES data. XML Schema has advantages over DTDs such as support for data types, and a more powerful means of specifying constraints on data values. An advantage of RDF encoded in XML over ISO 11179 is that XML defines rules for encoding data, whereas ISO 11179 does not. Materials and Methods: We created an XML Schema version of the TMA DES DTD. We wrote a program that converted ISO 11179 definitions to RDF encoded in XML, and used it to convert the TMA DES ISO 11179 definitions to RDF. Results: We validated a sample TMA DES XML file that was supplied with the publication that originally specified TMA DES using our XML Schema. We successfully validated the RDF produced by our ISO 11179 converter with the W3C RDF validation service. Conclusions: All TMA DES data could be encoded using XML, which simplifies its processing. XML Schema allows datatypes and valid value ranges to be specified for CDEs, which enables a wider range of error checking to be performed using XML Schemas than could be performed using DTDs.
Kariluoto, Susanna; Edelmann, Minnamari; Herranen, Mirkka; Lampi, Anna-Maija; Shmelev, Anton; Salovaara, Hannu; Korhola, Matti; Piironen, Vieno
Twenty bacteria isolated from three commercial oat bran products were tested for their folate production capability. The bacteria as well as some reference organisms were grown until early stationary phase on a rich medium (YPD), and the amount of total folate in the separated cell mass and the culture medium (supernatant) was determined by microbiological assay. Folate vitamer distribution was determined for eight bacteria including one isolated from rye flakes. For seven bacteria the effect of temperature and pH on folate production was studied in more detail. Relatively large amount of folate was both produced in the cell biomass (up to 20.8microg/g) and released to the culture medium (up to 0.38microg/g) by studied bacteria. The best producers were characterized as Bacillus subtilis ON4, Chryseobacterium sp. NR7, Janthinobacterium sp. RB4, Pantoea agglomerans ON2, and Pseudomonas sp ON8. The level of folate released in culture medium was the highest for B. subtilis ON5, Chryseobacterium sp. NR7, Curtobacterium sp. ON7, Enterococcus durans ON9, Janthinobacterium sp. RB4, Paenibacillus sp. ON10, Propionibacterium sp. RB9, and Staphylococcus kloosii RB7. Marked differences in the distribution of folate vitamers among the bacterial strains were revealed by the HPLC analysis. The main vitamers were tetrahydrofolate, 5,10-methenyltetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate. Increase in the folate content during bacterial growth was accompanied by proportional increase in the 5-methyltetrahydrofolate content and decrease of 5-formyltetrahydrofolate. 10-Formylfolic acid dominated in the culture media of four bacteria, and Janthinobacterium sp. RB4 was also found to excrete 5-methyltetrahydrofolate. Intracellular folate content was higher when the bacteria were grown at 28 degrees C than at 18 degrees C or 37 degrees C and also higher at pH 7 than at pH 5.5. Copyright 2010 Elsevier B.V. All rights reserved.
Michel, R; Burghardt, H; Bergmann, H
The drinking water system of a new hospital building that was highly contaminated with bacteria before opening was investigated too for the prevalence of small free living amoebae. Germ counts resulted in > 100 CFU/ml in 100% of the cold water samples, that showed also growth of P. aeruginosa, whereas E. coli and coliforme bacteria could not be identified. The investigation of 37 water samples for protozoa revealed growth of small freeliving amoebae in 20 samples (54%) belonging to 10 species of the genus Acanthamoeba, Naegleria, Hartmannella, Echinamoeba among others. In addition 2 Ciliate- and 2 Microflagellate-species could be observed. While all Naegleria strains isolated belonged to the N. gruberi-complex two of 16 Acanthamoeba-isolates proved to be pathogenic for laboratory mice. From 7 watersamples positive with P. aeruginosa 5 Acanthamoeba- and 2 Echinamoeba strains could be isolated which revealed intracellular multiplication of P. aeruginosa. Because of their well known resistances against chlorine, the amoebae and their cysts are considered to be vectors for these intracellular bacteria. A complete sanitation of the incriminated drinking water system was accomplished by combined chemical and thermic disinfection measures.
Nifontova, Galina; Zvaigzne, Maria; Baryshnikova, Maria; Korostylev, Evgeny; Ramos-Gomes, Fernanda; Alves, Frauke; Nabiev, Igor; Sukhanova, Alyona
Fabrication of polyelectrolyte microcapsules and their use as carriers of drugs, fluorescent labels, and metal nanoparticles is a promising approach to designing theranostic agents. Semiconductor quantum dots (QDs) are characterized by extremely high brightness and photostability that make them attractive fluorescent labels for visualization of intracellular penetration and delivery of such microcapsules. Here, we describe an approach to design, fabricate, and characterize physico-chemical and functional properties of polyelectrolyte microcapsules encoded with water-solubilized and stabilized with three-functional polyethylene glycol derivatives core/shell QDs. Developed microcapsules were characterized by dynamic light scattering, electrophoretic mobility, scanning electronic microscopy, and fluorescence and confocal microscopy approaches, providing exact data on their size distribution, surface charge, morphological, and optical characteristics. The fluorescence lifetimes of the QD-encoded microcapsules were also measured, and their dependence on time after preparation of the microcapsules was evaluated. The optimal content of QDs used for encoding procedure providing the optimal fluorescence properties of the encoded microcapsules was determined. Finally, the intracellular microcapsule uptake by murine macrophages was demonstrated, thus confirming the possibility of efficient use of developed system for live cell imaging and visualization of microcapsule transportation and delivery within the living cells.
Nifontova, Galina; Zvaigzne, Maria; Baryshnikova, Maria; Korostylev, Evgeny; Ramos-Gomes, Fernanda; Alves, Frauke; Nabiev, Igor; Sukhanova, Alyona
Fabrication of polyelectrolyte microcapsules and their use as carriers of drugs, fluorescent labels, and metal nanoparticles is a promising approach to designing theranostic agents. Semiconductor quantum dots (QDs) are characterized by extremely high brightness and photostability that make them attractive fluorescent labels for visualization of intracellular penetration and delivery of such microcapsules. Here, we describe an approach to design, fabricate, and characterize physico-chemical and functional properties of polyelectrolyte microcapsules encoded with water-solubilized and stabilized with three-functional polyethylene glycol derivatives core/shell QDs. Developed microcapsules were characterized by dynamic light scattering, electrophoretic mobility, scanning electronic microscopy, and fluorescence and confocal microscopy approaches, providing exact data on their size distribution, surface charge, morphological, and optical characteristics. The fluorescence lifetimes of the QD-encoded microcapsules were also measured, and their dependence on time after preparation of the microcapsules was evaluated. The optimal content of QDs used for encoding procedure providing the optimal fluorescence properties of the encoded microcapsules was determined. Finally, the intracellular microcapsule uptake by murine macrophages was demonstrated, thus confirming the possibility of efficient use of developed system for live cell imaging and visualization of microcapsule transportation and delivery within the living cells.
Lan, Ke; Murakami, Masanao; Choudhuri, Tathagata; Kuppers, Daniel A.; Robertson, Erle S.
Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a predominantly latent infection in the infected host. Importantly, during latency, only a small number of viral encoded genes are expressed. This viral gene expression pattern contributes to the establishment of long-term infection as well as the ability of the virus to evade the immune system. Previous studies have been shown that the replication and transcription activator (RTA) encoded by ORF50 activates it downstream genes and initiates viral lytic reactivation through functional interaction with RBP-Jκ, the major downstream effector of the Notch signaling pathway. This indicates that RTA can usurp the conserved Notch signaling pathway and mimic the activities of intracellular Notch1 to modulate gene expression. In this report, we show that the activated intracellular domain of Notch1 (ICN) is aberrantly accumulated in KSHV latently infected pleural effusion lymphoma (PEL) cells. ICN activated the RTA promoter in a dose-dependent manner, and forced expression of ICN in latently infected KSHV-positive cells initiated full blown lytic replication with the production of infectious viral progeny. However, latency-associated nuclear antigen (LANA) which is predominantly expressed during latency can specifically down-modulate ICN-mediated transactivation of RTA and so control KSHV for lytic reactivation. These results demonstrate that LANA can inhibit viral lytic replication by antagonizing ICN function and suggest that LANA is a critical component of the regulatory control mechanism for switching between viral latent and lytic replication by directly interacting with effectors of the conserved cellular Notch1 pathway
Shi, Qi-ping; Cao, Hao-wei; Xu, Rui; Zhang, Dan-dan; Huang, Juan
The transmembrane protein Crumbs (Crb) plays key roles in the establishing and maintaining cell apical-basal polarity in epithelial cells by determining the apical plasma membrane identity. Although its intracellular domain contains only 37 amino acids, it is absolutely essential for its function. In Drosophila, mutations in this intracellular domain result in severe defects in epithelial polarity and abnormal embryonic development. The intracellular domain of Crb shows high homology across species from Drosophila to Mus musculus and Homo sapiens. However, the intracellular domains of the two Crb proteins in C. elegans are rather divergent from those of Drosophila and mammals, raising the question on whether the function of the intracellular domain of the Crb protein is conserved in C. elegans. Using genomic engineering approach, we replaced the intracellular domain of the Drosophila Crb with that of C. elegans Crb2 (CeCrb2), which has extremely low homology with those from the Crb proteins of Drosophila and mammals. Surprisingly, substituting the intracellular domain of Drosophila Crb with that of CeCrb2 did not cause any abnormalities in development of the Drosophila embryo, in terms of expression and localization of Crb and other polarity proteins and apical-basal polarity in embryonic epithelial cells. Our results support the notion that despite their extensive sequence variations, all functionally critical amino acid residues and motifs of the intercellular domain of Crb proteins are fully conserved between Drosophila and C. elegans.
Silverio, Abe L. F.
Trimeric intracellular cation-specific (TRIC) channels are integral to muscle excitation–contraction coupling. TRIC channels provide counter-ionic flux when calcium is rapidly transported from intracellular stores to the cell cytoplasm. Until recently, knowledge of the presence of these proteins was limited to animals. We analyzed the TRIC family and identified a profusion of prokaryotic family members with topologies and motifs similar to those of their eukaryotic counterparts. Prokaryotic members far outnumber eukaryotic members, and although none has been functionally characterized, the evidence suggests that they function as secondary carriers. The presence of fused N- or C-terminal domains of known biochemical functions as well as genomic context analyses provide clues about the functions of these prokaryotic homologs. They are proposed to function in metabolite (e.g., amino acid/ nucleotide) efflux. Phylogenetic analysis revealed that TRIC channel homologs diverged relatively early during evolutionary history and that horizontal gene transfer was frequent in prokaryotes but not in eukaryotes. Topological analyses of TRIC channels revealed that these proteins possess seven putative transmembrane segments (TMSs), which arose by intragenic duplication of a three-TMS polypeptide-encoding genetic element followed by addition of a seventh TMS at the C terminus to give the precursor of all current TRIC family homologs. We propose that this family arose in prokaryotes. PMID:21519847
Xu, Ren; Boudreau, Aaron; Bissell, Mina J
Mammary gland development, functional differentiation, and homeostasis are orchestrated and sustained by a balance of biochemical and biophysical cues from the organ's microenvironment. The three-dimensional microenvironment of the mammary gland, predominantly 'encoded' by a collaboration between the extracellular matrix (ECM), hormones, and growth factors, sends signals from ECM receptors through the cytoskeletal intracellular matrix to nuclear and chromatin structures resulting in gene expression; the ECM in turn is regulated and remodeled by signals from the nucleus. In this chapter, we discuss how coordinated ECM deposition and remodeling is necessary for mammary gland development, how the ECM provides structural and biochemical cues necessary for tissue-specific function, and the role of the cytoskeleton in mediating the extra - to intracellular dialogue occurring between the nucleus and the microenvironment. When operating normally, the cytoskeletal-mediated dynamic and reciprocal integration of tissue architecture and function directs mammary gland development, tissue polarity, and ultimately, tissue-specific gene expression. Cancer occurs when these dynamic interactions go awry for an extended time.
Mathematical models of virus dynamics have not previously acknowledged spatial resolution at the intracellular level despite substantial arguments that favor the consideration of intracellular spatial dependence. The replication of the hepatitis C virus (HCV) viral RNA (vRNA) occurs within special replication complexes formed from membranes derived from endoplasmatic reticulum (ER). These regions, termed membranous webs, are generated primarily through specific interactions between nonstructural virus-encoded proteins (NSPs) and host cellular factors. The NSPs are responsible for the replication of the vRNA and their movement is restricted to the ER surface. Therefore, in this study we developed fully spatio-temporal resolved models of the vRNA replication cycle of HCV. Our simulations are performed upon realistic reconstructed cell structures-namely the ER surface and the membranous webs-based on data derived from immunostained cells replicating HCV vRNA. We visualized 3D simulations that reproduced dynamics resulting from interplay of the different components of our models (vRNA, NSPs, and a host factor), and we present an evaluation of the concentrations for the components within different regions of the cell. Thus far, our model is restricted to an internal portion of a hepatocyte and is qualitative more than quantitative. For a quantitative adaption to complete cells, various additional parameters will have to be determined through further in vitro cell biology experiments, which can be stimulated by the results deccribed in the present study.
Lee, Hyo Jung; Park, Jihoon; Lee, Joo-Young; Kim, Pil
Overexpression of the genes encoding phosphoeneolpyruvate carboxykinase (pckA) and NAD-dependent malic enzyme (maeA) facilitates higher intracellular ATP and NAD(P)H concentrations, respectively, under aerobic conditions in Escherichia coli. To verify a hypothesis that higher intracellular energy reserves might contribute to H2 fermentation, wild-type E. coli strains overexpressing pckA and maeA were cultured under anaerobic conditions in a glucose minimal medium. Overexpression of pckA and maeA enabled E. coli to produce 3- times and 4-times greater H2 (193 and 284 nmol, respectively) than the wild type (66 nmol H2). The pckA and maeA genes were further overexpressed in a hydrogenase-3-enhanced E. coli strain. The hydrogenase-3-enhanced strain (W3110+fhlA) produced 322 nmol H2, whereas the ATP-enhanced strain (W3110+fhlA+pckA) produced 50% increased H2 (443 nmol). Total H2 in the NAD(P)H-enhanced strain (W3110+fhlA+maeA) was similar to that in the control strain at 319 nmol H2. Possible explanations for the contribution of the increased cellular energy reserves to the enhanced hydrogen fermentation observed are discussed based on the viewpoint of metabolic engineering strategy.
Martin, Gale L
This article proposes that visual encoding learning improves reading fluency by widening the span over which letters are recognized from a fixated text image so that fewer fixations are needed to cover a text line. Encoder is a connectionist model that learns to convert images like the fixated text images human readers encode into the corresponding letter sequences. The computational theory of classification learning predicts that fixated text-image size makes this learning difficult but that reducing image variability and biasing learning should help. Encoder confirms these predictions. It fails to learn as image size increases but achieves humanlike visual encoding accuracy when image variability is reduced by regularities in fixation positions and letter sequences and when learning is biased to discover mapping functions based on the sequential, componential structure of text. After training, Encoder exhibits many humanlike text familiarity effects. ((c) 2004 APA, all rights reserved)
Reva, Oleg; Tümmler, Burkhard
Long genes should be rare in archaea and eubacteria because of the demanding costs of time and resources for protein production. The search in 580 sequenced prokaryotic genomes, however, revealed 0.2% of all genes to be longer than 5 kb (absolute number: 3732 genes). Eighty giant bacterial genes of more than 20 kb in length were identified in 47 taxa that belong to the phyla Thermotogae (1), Chlorobi (3), Planctomycetes (1), Cyanobacteria (2), Firmicutes (7), Actinobacteria (9), Proteobacteria (23) or Euryarchaeota (1) (number of taxa in brackets). Giant genes are strain-specific, differ in their tetranucleotide usage from the bulk genome and occur preferentially in non-pathogenic environmental bacteria. The two longest bacterial genes known to date were detected in the green sulfur bacterium Chlorobium chlorochromatii CaD3 encoding proteins of 36 806 and 20 647 amino acids, being surpassed in length only by the human titin coding sequence. More than 90% of bacterial giant genes either encode a surface protein or a polyketide/non-ribosomal peptide synthetase. Most surface proteins are acidic, threonine-rich, lack cystein and harbour multiple amino acid repeats. Giant proteins increase bacterial fitness by the production of either weapons towards or shields against animate competitors or hostile environments.
Nichols, P. D.; Henson, J. M.; Guckert, J. B.; Nivens, D. E.; White, D. C.
Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis of freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at approximately 1740 cm-1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggest that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled the composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum arabic (amide I/carbohydrate C-O approximately 1150 cm-1) and bacteria-poly-beta-hydroxybutyrate (amide I/carbonyl approximately 1740 cm-1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR spectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at approximately 1440 and 1090 cm-1 was seen in FT-IR spectra of the V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactions within adherent microbial consortia.
Villadsen, Ingrid Stampe
This section contains the following single lecture notes: Eukaryotic Cell Biology. Kingdom Fungi. Cell Division. Meiosis and Recombination. Genetics of Yeast. Organisation of the Chromosome. Organization and genetics of the mitochondrial Geneme. Regulatio of Gene Expression. Intracellular Compart...
Full Text Available Magnesium (Mg is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ, that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg.
Stamatoyannopoulos, John A; Snyder, Michael; Hardison, Ross; Ren, Bing; Gingeras, Thomas; Gilbert, David M; Groudine, Mark; Bender, Michael; Kaul, Rajinder; Canfield, Theresa; Giste, Erica; Johnson, Audra; Zhang, Mia; Balasundaram, Gayathri; Byron, Rachel; Roach, Vaughan; Sabo, Peter J; Sandstrom, Richard; Stehling, A Sandra; Thurman, Robert E; Weissman, Sherman M; Cayting, Philip; Hariharan, Manoj; Lian, Jin; Cheng, Yong; Landt, Stephen G; Ma, Zhihai; Wold, Barbara J; Dekker, Job; Crawford, Gregory E; Keller, Cheryl A; Wu, Weisheng; Morrissey, Christopher; Kumar, Swathi A; Mishra, Tejaswini; Jain, Deepti; Byrska-Bishop, Marta; Blankenberg, Daniel; Lajoie, Bryan R; Jain, Gaurav; Sanyal, Amartya; Chen, Kaun-Bei; Denas, Olgert; Taylor, James; Blobel, Gerd A; Weiss, Mitchell J; Pimkin, Max; Deng, Wulan; Marinov, Georgi K; Williams, Brian A; Fisher-Aylor, Katherine I; Desalvo, Gilberto; Kiralusha, Anthony; Trout, Diane; Amrhein, Henry; Mortazavi, Ali; Edsall, Lee; McCleary, David; Kuan, Samantha; Shen, Yin; Yue, Feng; Ye, Zhen; Davis, Carrie A; Zaleski, Chris; Jha, Sonali; Xue, Chenghai; Dobin, Alex; Lin, Wei; Fastuca, Meagan; Wang, Huaien; Guigo, Roderic; Djebali, Sarah; Lagarde, Julien; Ryba, Tyrone; Sasaki, Takayo; Malladi, Venkat S; Cline, Melissa S; Kirkup, Vanessa M; Learned, Katrina; Rosenbloom, Kate R; Kent, W James; Feingold, Elise A; Good, Peter J; Pazin, Michael; Lowdon, Rebecca F; Adams, Leslie B
To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.
Zhihua, Li; Xuetao, Hu; Jiyong, Shi; Xiaobo, Zou; Xiaowei, Huang; Xucheng, Zhou; Tahir, Haroon Elrasheid; Holmes, Mel; Povey, Malcolm
A simple and rapid bacteria counting method based on polyaniline (PANI)/bacteria thin film was proposed. Since the negative effects of immobilized bacteria on the deposition of PANI on glass carbon electrode (GCE), PANI/bacteria thin films containing decreased amount of PANI would be obtained when increasing the bacteria concentration. The prepared PANI/bacteria film was characterized with cyclic voltammetry (CV) technique to provide quantitative index for the determination of the bacteria count, and electrochemical impedance spectroscopy (EIS) was also performed to further investigate the difference in the PANI/bacteria films. Good linear relationship of the peak currents of the CVs and the log total count of bacteria (Bacillus subtilis) could be established using the equation Y=-30.413X+272.560 (R(2)=0.982) over the range of 5.3×10(4) to 5.3×10(8)CFUmL(-1), which also showed acceptable stability, reproducibility and switchable ability. The proposed method was feasible for simple and rapid counting of bacteria. Copyright © 2016 Elsevier B.V. All rights reserved.
Full Text Available Abstract Background Reuterin produced from glycerol by Lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. It has been postulated that reuterin could play a role in the probiotic effects of Lb. reuteri. Reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. Moreover reuterin's mode of action has not yet been elucidated. Glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin. The aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols (LMW-SH such as glutathione, hydrogen peroxide and/or reuterin sensitivity. Reuterin was produced by Lb. reuteri SD2112 in pure glycerol solution, purified and used to test the minimal inhibitory (MIC and minimal bactericidal concentrations (MBC. Hydrogen peroxide sensitivity and intracellular LMW-SH concentration were also analysed. Results Our data showed that most tested intestinal bacteria showed MIC below that for a sensitive indicator Escherichia coli (7.5–15 mM. Lactobacilli and Clostridium clostridioforme were more resistant with MIC ranging from 15 to 50 mM. No correlation between bacterial intracellular concentrations of LMW-SH, including glutathione, and reuterin or hydrogen peroxide sensitivities were found. Conclusion Our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of LMW-SH was not directly linked to their capacity to resist reuterin or hydrogen peroxide. This suggests that detoxification by LMW-SH such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance
Goessweiner-Mohr, Nikolaus; Arends, Karsten; Keller, Walter; Grohmann, Elisabeth
Conjugative transfer is the most important means of spreading antibiotic resistance and virulence factors among bacteria. The key vehicles of this horizontal gene transfer are a group of mobile genetic elements, termed conjugative plasmids. Conjugative plasmids contain as minimum instrumentation an origin of transfer (oriT), DNA-processing factors (a relaxase and accessory proteins), as well as proteins that constitute the trans-envelope transport channel, the so-called mating pair formation (Mpf) proteins. All these protein factors are encoded by one or more transfer (tra) operons that together form the DNA transport machinery, the Gram-positive type IV secretion system. However, multicellular Gram-positive bacteria belonging to the streptomycetes appear to have evolved another mechanism for conjugative plasmid spread reminiscent of the machinery involved in bacterial cell division and sporulation, which transports double-stranded DNA from donor to recipient cells. Here, we focus on the protein key players involved in the plasmid spread through the two different modes and present a new secondary structure homology-based classification system for type IV secretion protein families. Moreover, we discuss the relevance of conjugative plasmid transfer in the environment and summarize novel techniques to visualize and quantify conjugative transfer in situ.
Matt S. Conover
Full Text Available Uropathogenic Escherichia coli (UPEC is the primary etiological agent of over 85% of community-acquired urinary tract infections (UTIs. Mouse models of infection have shown that UPEC can invade bladder epithelial cells in a type 1 pilus-dependent mechanism, avoid a TLR4-mediated exocytic process, and escape into the host cell cytoplasm. The internalized UPEC can clonally replicate into biofilm-like intracellular bacterial communities (IBCs of thousands of bacteria while avoiding many host clearance mechanisms. Importantly, IBCs have been documented in urine from women and children suffering acute UTI. To understand this protected bacterial niche, we elucidated the transcriptional profile of bacteria within IBCs using microarrays. We delineated the upregulation within the IBC of genes involved in iron acquisition, metabolism, and transport. Interestingly, lacZ was highly upregulated, suggesting that bacteria were sensing and/or utilizing a galactoside for metabolism in the IBC. A ΔlacZ strain displayed significantly smaller IBCs than the wild-type strain and was attenuated during competitive infection with a wild-type strain. Similarly, a galK mutant resulted in smaller IBCs and attenuated infection. Further, analysis of the highly upregulated gene yeaR revealed that this gene contributes to oxidative stress resistance and type 1 pilus production. These results suggest that bacteria within the IBC are under oxidative stress and, consistent with previous reports, utilize nonglucose carbon metabolites. Better understanding of the bacterial mechanisms used for IBC development and establishment of infection may give insights into development of novel anti-virulence strategies.
Mertens, Katja; Samuel, James E
Survival of intracellular pathogenic bacteria depends on the ability to resist host-mediated degradation and to generate a replicative niche within the host. Usually, after internalization by professional phagocytic cells, the bacteria containing vacuole or phagosome traffics through the endocytic pathway, progressively acidifies and develops into a degradative mature phagolysosome. In this environment bacteria are exposed to a wide variety of anti-microbial agents, such as defensins, proteases, and reactive oxygen species (ROS) and reactive nitrogen species (RNS). Most parasitizing bacteria have evolved strategies to interfere with this maturation process and to direct the development of an environment that supports survival and replication. C. burnetii also follows this paradigm, but directs the biogenesis of a unique parasitophorous vacuole (PV), which resembles, yet is distinct from a terminal phagolysosome. Within the environment of the PV, C. burnetii is exposed to varying levels of ROS and RNS, which represent the primary defense mechanism of the host cell against this invading microorganism. Major mediators for ROS and RNS are superoxide (O (2) (-) ) and nitric oxide (NO(*)), generated by the cellular NADPH oxidase (phox) and inducible nitric oxide synthase (iNOS), respectively. C. burnetii employs several strategies to evade oxidative stress; on the host side (i) delaying phagolysosome fusion and (ii) inhibiting cellular NADPH oxidase. On the bacterial side, maintaining genome stability by (iii) evolving a preference for a low iron environment, (iv) expressing a minimal and likely crucial set of DNA repair genes and (v) detoxifying the PV by ROS and RNS degrading enzymes. Overall defense mechanisms in C. burnetii against oxidative and nitrosative stress and the regulation thereof are not fully defined and our knowledge is mainly based on genome sequence information. Comparison with E. coli as a model bacterium reveals that defense strategies of C. burnetii
M. Sloan Siegrist
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.
Full Text Available Phytoremediation is an emerging technology that uses plants in order to cleanup pollutants including xenobiotics and heavy metals from soil, water and air. Inoculation of plants with plant growth promoting endophytic and rhizospheric bacteria can enhance efficiency of phytoremediation. Genomic analysis of four plant-associated strains belonging to the Stenotrophomonas maltophilia species revealed the presence of genes encoding proteins involved in plant growth promotion, biocontrol of phytopathogens, biodegradation of xenobiotics, heavy metals resistance and plant-bacteria-environment interaction. The results of this analysis suggest great potential of bacteria belonging to Stenotrophomonas maltophilia species in enhancing phytoremediation efficiency.
Moleres, Javier; Sinha, Sunita; Fernández-Calvet, Ariadna; Porsch, Eric A.; St. Geme, Joseph W.; Nislow, Corey; Redfield, Rosemary J.; Garmendia, Junkal
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
Li, Zhongyuan; Zhang, Dahu; He, Yuanqiao; Ding, Zhiyong; Mao, Fei; Luo, Tao; Zhang, Xiaoping
A worldwide decline in the quality of human semen is currently occurring. In mammals, sperm are produced from diploid stem-cell spermatogonia by spermatogenesis in testes and become mature in epididymis. Nevertheless, these biological processes can be affected by Gram-negative bacterial infection mediated by lipopolysaccharide (LPS), the major endotoxin of Gram-negative bacteria. It is well known that LPS can disturb spermatogenesis and affect sperm maturation and quality in vivo. However, the effect of LPS on the ejaculated mature sperm in vitro remains unclear. Thus, this study aimed to assess the in vitro toxicity of LPS on human sperm function and to elucidate the underlying mechanism. Human sperm were incubated with LPS (0.1-100 μg/ml) for 1-12 h in vitro and, subsequently, sperm viability, motility and capacitation, and the acrosome reaction were examined. LPS dose-dependently inhibited total and progressive motility and the ability to move through a viscous medium of the sperm but did not affect sperm viability, capacitation, and the acrosome reaction. To explore the underlying mechanism of LPS's actions, we examined the effects of LPS on the intracellular concentrations of cyclic adenosine monophosphate (cAMP) and calcium ([Ca(2+)]i) and protein-tyrosine phosphorylation of human sperm, which are key regulators of human sperm function. LPS decreased intracellular cAMP dose-dependently but had no effect on [Ca(2+)]i and protein-tyrosine phosphorylation of human sperm. These findings suggest that LPS inhibits human sperm motility by decreasing intracellular cAMP.
D'Alvise, Paul; Magdenoska, Olivera; Melchiorsen, Jette
species Ruegeria mobilis are associated with intracellular concentrations of the signal compound cyclic dimeric guanosinmonophosphate (c-di-GMP), which in bacteria regulates transitions between motile and sessile life stages. Genes for diguanylate cyclases and phosphodiesterases, which are involved in c...... formation and production of the potent antibiotic tropodithietic acid (TDA). An introduced phosphodiesterase gene decreased c-di-GMP and reduced biofilm formation and TDA production. tdaC, a key gene for TDA biosynthesis, was expressed only in attached or biofilm-forming cells, and expression was induced...
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
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
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
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...
Cellular compartmentalization and intracellular transport mechanisms are important to establish and maintain the spatial organisation of proteins and organelles needed to ensure proper cellular functioning. Especially in polarized cells like neurons, the proper distribution of proteins into the
Varian, Bernard J.; Goureshetti, Sravya; Poutahidis, Theofilos; Lakritz, Jessica R.; Levkovich, Tatiana; Kwok, Caitlin; Teliousis, Konstantinos; Ibrahim, Yassin M.; Mirabal, Sheyla; Erdman, Susan E.
Muscle wasting, known as cachexia, is a debilitating condition associated with chronic inflammation such as during cancer. Beneficial microbes have been shown to optimize systemic inflammatory tone during good health; however, interactions between microbes and host immunity in the context of cachexia are incompletely understood. Here we use mouse models to test roles for bacteria in muscle wasting syndromes. We find that feeding of a human commensal microbe, Lactobacillus reuteri, to mice is sufficient to lower systemic indices of inflammation and inhibit cachexia. Further, the microbial muscle-building phenomenon extends to normal aging as wild type animals exhibited increased growth hormone levels and up-regulation of transcription factor Forkhead Box N1 [FoxN1] associated with thymus gland retention and longevity. Interestingly, mice with a defective FoxN1 gene (athymic nude) fail to inhibit sarcopenia after L. reuteri therapy, indicating a FoxN1-mediated mechanism. In conclusion, symbiotic bacteria may serve to stimulate FoxN1 and thymic functions that regulate inflammation, offering possible alternatives for cachexia prevention and novel insights into roles for microbiota in mammalian ontogeny and phylogeny. PMID:26933816
Suravajhala, Srinivasa Sandeep; Saini, Deepak; Nott, Prabhu
Luminescence in Vibrio fischeri is a model for quorum-sensing-gene-regulation in bacteria. We study luminescence response of V. fischeri to both internal and external cues at the single cell and population level. Experiments with ES114, a wild-type strain, and ainS mutant show that luminescence induction in cultures is not always proportional to cell-density and there is always a basal level of luminescence. At any given concentration of the exogenously added signals, C6-HSL and C8-HSL, luminescence per cell reaches a maximum during the exponential phase and decreases thereafter. We hypothesize that (1) C6-HSL production and LuxR activity are not proportional to cell-density, and (2) there is a shift in equilibrium from C6-HSL to C8-HSL during the later stages of growth of the culture. RT-PCR analysis of luxI and luxR shows that the expression of these genes is maximum corresponding to the highest level of luminescence. The shift in equilibrium is shown by studying competitive binding of C6-HSL and C8-HSL to LuxR. We argue that luminescence is a unicellular behaviour, and an intensive property like per cell luminescence is more important than gross luminescence of the population in understanding response of bacteria to chemical signalling. Funding from the Department of Science and Technology, India is acknowledged.
Bennett, Neil J; Ashiru, Omodele; Morgan, Fiona J E; Pang, Yin; Okecha, Georgina; Eagle, Rob A; Trowsdale, John; Sissons, J G Patrick; Wills, Mark R
Human CMV (HCMV) encodes multiple genes that control NK cell activation and cytotoxicity. Some of these HCMV-encoded gene products modulate NK cell activity as ligands expressed at the cell surface that engage inhibitory NK cell receptors, whereas others prevent the infected cell from upregulating ligands that bind to activating NK cell receptors. A major activating NKR is the homodimeric NKG2D receptor, which has eight distinct natural ligands in humans. It was shown that HCMV is able to prevent the surface expression of five of these ligands (MIC A/B and ULBP1, 2, and 6). In this article, we show that the HCMV gene product UL142 can prevent cell surface expression of ULBP3 during infection. We further show that UL142 interacts with ULBP3 and mediates its intracellular retention in a compartment that colocalizes with markers of the cis-Golgi complex. In doing so, UL142 prevents ULBP3 trafficking to the surface and protects transfected cells from NK-mediated cytotoxicity. This is the first description of a viral gene able to mediate downregulation of ULBP3.
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
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.
Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David
Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: email@example.com.
Ordway, Diane; Viveiros, Miguel; Leandro, Clara; Bettencourt, Rosário; Almeida, Josefina; Martins, Marta; Kristiansen, Jette E.; Molnar, Joseph; Amaral, Leonard
The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant Mycobacterium tuberculosis. These compounds have not been used as anti-M. tuberculosis agents because their in vitro activities take place at concentrations which are beyond those that are clinically achievable. In addition, chronic administration of CPZ produces frequent severe side effects. Because CPZ has been shown to enhance the killing of intracellular M. tuberculosis at concentrations in the medium that are clinically relevant, we have investigated whether TZ, a phenothiazine whose negative side effects are less frequent and serious than those associated with CPZ, kills M. tuberculosis organisms that have been phagocytosed by human macrophages, which have nominal killing activities against these bacteria. Both CPZ and TZ killed intracellular antibiotic-sensitive and -resistant M. tuberculosis organisms when they were used at concentrations in the medium well below those present in the plasma of patients treated with these agents. These concentrations in vitro were not toxic to the macrophage, nor did they affect in vitro cellular immune processes. TZ thus appears to be a serious candidate for the management of a freshly diagnosed infection of pulmonary tuberculosis or as an adjunct to conventional antituberculosis therapy if the patient originates from an area known to have a high prevalence of multidrug-resistant M. tuberculosis isolates. Nevertheless, we must await the outcomes of clinical trials to determine whether TZ itself may be safely and effectively used as an antituberculosis agent. PMID:12604522
De Mello, Walmor C
HighlightsIntracellular renin disrupts chemical communication in the heartAngiotensinogen enhances the effect of reninIntracellular enalaprilat reduces significantly the effect of reninIntracellular renin increases the inward calcium currentHarmful versus beneficial effect during myocardial infarction The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; (1) under normal conditions, Lucifer Yellow flows from cell to cell through gap junctions; (2) the intracellular dialysis of renin (100 nM) disrupts chemical communication - an effect enhanced by simultaneous administration of angiotensinogen (100 nM); (3) enalaprilat (10(-9) M) administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; (4) aliskiren (10(-8) M) inhibited the effect of renin on chemical communication; (5) the possible role of intracellular renin independently of angiotensin II (Ang II) was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; (6) the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed; (7) the present results indicate that intracellular renin due to internalization or in situ synthesis causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.
Miskolci, Veronika; Hodgson, Louis; Cox, Dianne; Vancurova, Ivana
Most cytokines are stored in the cytoplasm until their release into the extracellular environment; however, some cytokines have been reported to localize in the nucleus. Traditional whole cell extract preparation does not provide information about the intracellular localization of cytokines. Here, we describe how to prepare cytoplasmic and nuclear extracts that can be analyzed by immunoblotting. While in this chapter we use this method to analyze intracellular localization of interleukin-8 (I...
Wang, Tingting; Cheng, Lijun; Zhang, Wenhao; Xu, Xiuhong; Meng, Qingxin; Sun, Xuewei; Liu, Huajing; Li, Hongtao; Sun, Yu
Composting is widely used to transform waste into valuable agricultural organic fertilizer. Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the global nitrogen cycle, but their role in composting remains poorly understood. In the present study, the community structure, diversity, and abundance of anammox bacteria were analyzed using cloning and sequencing methods by targeting the 16S rRNA gene and the hydrazine oxidase gene ( hzo ) in samples isolated from compost produced from cow manure and rice straw. A total of 25 operational taxonomic units were classified based on 16S rRNA gene clone libraries, and 14 operational taxonomic units were classified based on hzo gene clone libraries. The phylogenetic tree analysis of the 16S rRNA gene and deduced HZO protein sequences from the corresponding encoding genes indicated that the majority of the obtained clones were related to the known anammox bacteria Candidatus "Brocadia," Candidatus "Kuenenia," and Candidatus "Scalindua." The abundances of anammox bacteria were determined by quantitative PCR, and between 2.13 × 10 5 and 1.15 × 10 6 16S rRNA gene copies per gram of compost were found. This study provides the first demonstration of the existence of anammox bacteria with limited diversity in cow manure composting.
Li, Yi; Lei, Xueqian; Zhu, Hong; Zhang, Huajun; Guan, Chengwei; Chen, Zhangran; Zheng, Wei; Fu, Lijun; Zheng, Tianling
Chitinase producing bacteria can involve extensively in nutrient cycling and energy flow in the aquatic environment through degradation and utilization of chitin. It is well known that diatoms cells are encased by box-like frustules composed of chitin. Thus the chitin containing of diatoms shall be a natural target of chitinase producing bacteria, however, the interaction between these two organismic groups has not been studied thus far. Therefore, in this study, the algicidal mechanism of one chitinase producing bacterium (strain LY03) on Thalassiosira pseudonana was investigated. The algicidal range and algicidal mode of strain LY03 were first studied, and then bacterial viability, chemotactic ability and direct interaction characteristic between bacteria and diatom were also confirmed. Finally, the characteristic of the intracellular algicidal substance was identified and the algicidal mechanism was determined whereby algicidal bacterial cells showed chemotaxis to algal cells, fastened themselves on algal cells with their flagella, and then produced chitinase to degrade algal cell walls, and eventually caused algal lysis and death. It is the first time to investigate the interaction between chitinase producing bacteria and diatoms, and this novel special interaction mode was confirmed in this study, which will be helpful in protection and utilization of diatoms resources.
Pei, Gang; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel
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.
Kovtunovych, G.; Lar, O.; Kovalchuk, M.; Negrutska, V.; Rogutski, I.; Kozyrovska, N.; Kordyum, V.
The ability to grow plants in space self-perpetuating gardens is actual for providing an advanced life support system for humans during extended missions. Nevertheless, studies of molecular mechanisms of plant-bacteria interactions in the flight experiments are still in their infancy. Space factors affect cell microenvironment and signal transduction through membranes, and it may result in induction in bacteria of some genes regulated by the systems, sensing environmental signals. In space a risk of genetic rearrangements is increased, and some changes in bacterial DNA expected. As a consequence, bacteria may exhibit novel characters, e.g., pathogenicity. During the previous our experience we have determined an increase of internal colonisation of the rice roots with bacteria in space flight. It is important to characterise molecular-genetic plant-bacteria interactions influenced with physical factors. Genes coding for bacterial pectinases provide a suitable model for studies of well integrated objectives, concerning plant-bacteria interactions. From examining a mode of expression of pectinases encoding genes of a plant-associated bacterium and nucleotide polymorphism within pectate lyase- and polygalacturonase encoding genes in microcosm experiments in earth and space flight we get new knowledges about understanding some physical factors as the environmental signals and the possible risk of changes in interactions of bacteria with the plant.
Ramachandran, Gayetri; Miguel-Arribas, Andrés; Abia, David; Singh, Praveen K; Crespo, Isidro; Gago-Córdoba, César; Hao, Jian An; Luque-Ortega, Juan Roman; Alfonso, Carlos; Wu, Ling J; Boer, D Roeland; Meijer, Wilfried J J
Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.
Kowalchuk, G.A.; Stienstra, A.W.; Heilig, G.H.J.; Stephen, J.R.; Woldendorp, J.W.
The community structure of beta-subclass Proteobacteria ammonia-oxidizing bacteria was determined in semi-natural chalk grassland soils at different stages of secondary succession. Both culture-mediated (most probable number; MPN) and direct nucleic acid-based approaches targeting genes encoding 16S
Ubiquitous isethionate (2-hydroxyethanesulfonate) is dissimilated by diverse bacteria. Growth of Cupriavidus necator H16 with isethionate was observed, as was inducible membrane-bound isethionate dehydrogenase (IseJ) and inducible transcription of the genes predicted to encode IseJ and a transporter (IseU). Biodiversity in isethionate transport genes was observed and investigated by transcription experiments.
Wright, Steven M; McDougall, Mary Preston
Encoding of spatial information in magnetic resonance imaging is conventionally accomplished by using magnetic field gradients. During gradient encoding, the position in k-space is determined by a time-integral of the gradient field, resulting in a limitation in imaging speed due to either gradient power or secondary effects such as peripheral nerve stimulation. Partial encoding of spatial information through the sensitivity patterns of an array of coils, known as parallel imaging, is widely used to accelerate the imaging, and is complementary to gradient encoding. This paper describes the one-dimensional limit of parallel imaging in which all spatial localization in one dimension is performed through encoding by the radiofrequency (RF) coil. Using a one-dimensional array of long and narrow parallel elements to localize the image information in one direction, an entire image is obtained from a single line of k-space, avoiding rapid or repeated manipulation of gradients. The technique, called single echo acquisition (SEA) imaging, is described, along with the need for a phase compensation gradient pulse to counteract the phase variation contained in the RF coil pattern which would otherwise cause signal cancellation in each imaging voxel. Image reconstruction and resolution enhancement methods compatible with the speed of the technique are discussed. MR movies at frame rates of 125 frames per second are demonstrated, illustrating the ability to monitor the evolution of transverse magnetization to steady state during an MR experiment as well as demonstrating the ability to image rapid motion. Because this technique, like all RF encoding approaches, relies on the inherent spatially varying pattern of the coil and is not a time-integral, it should enable new applications for MRI that were previously inaccessible due to speed constraints, and should be of interest as an approach to extending the limits of detection in MR imaging.
The interaction of antimicrobial peptides with the membrane and intracellular targets of Staphylococcus aureus investigated by ATP leakage, DNA-binding analysis, and the expression of a LexA-controlled gene, recA
Gottschalk, Sanne; Thomsen, Line Elnif
The analysis of how antimicrobial peptides (AMPs) interact with bacterial membranes and intracellular targets is important for our understanding of how these molecules affect bacteria. Increased knowledge may aid the design of AMPs that work on their target bacterium without inducing bacterial re...
Risgaard-Petersen, Nils; Kristiansen, Michael; Frederiksen, Rasmus
In marine sediments cathodic oxygen reduction at the sediment surface can be coupled to anodic sulfide oxidation in deeper anoxic layers through electrical currents mediated by filamentous, multicellular bacteria of the Desulfobulbaceae family, the so-called cable bacteria. Until now, cable...... bacteria have only been reported from marine environments. In this study, we demonstrate that cable bacteria also occur in freshwater sediments. In a first step, homogenized sediment collected from the freshwater stream Giber Å, Denmark, was incubated in the laboratory. After 2 weeks, pH signatures...... marine cable bacteria, with the genus Desulfobulbus as the closest cultured lineage. The results of the present study indicate that electric currents mediated by cable bacteria could be important for the biogeochemistry in many more environments than anticipated thus far and suggest a common evolutionary...
Fink, Lisbeth Nielsen
Certain lactic acid bacteria (LAB) are part of the commensal intestinal flora and considered beneficial for health, as they compete with pathogens for adhesion sites in the intestine and ferment otherwise indigestible compounds. Another important property of these so-called probiotic bacteria...... with bacteria, and the cytokine pattern induced by specific bacteria resembled the pattern induced in MoDC, except for TNF-alpha and IL-6, which were induced in response to different bacteria in blood DC/monocytes and monocyte-derived DC. Autologous NK cells produced IFN-gamma when cultured with blood DC......, monocytes and monocyte-derived DC and IL-12-inducing bacteria, whereas only DC induced IFN-gamma production in allogeneic T cells. In vitro-generated DC is a commonly used model of tissue DC, but they differ in certain aspects from intestinal DC, which are in direct contact with the intestinal microbiota...
Living matter is a non-equilibrium system in which many components work in parallel to perpetuate themselves through a fluctuating environment. Physiological states or functionalities revealed by a particular environment are called phenotypes. Transitions between phenotypes may occur either spontaneously or via interaction with the environment. Even in the same environment, genetically identical bacteria can exhibit different phenotypes of a continuous or discrete nature. In this thesis, we pursued three lines of investigation into discrete phenotypic heterogeneity in bacterial populations: the quantitative characterization of the so-called bacterial persistence, a theoretical model of phenotypic switching based on those measurements, and the design of artificial genetic networks which implement this model. Persistence is the phenotype of a subpopulation of bacteria with a reduced sensitivity to antibiotics. We developed a microfluidic apparatus, which allowed us to monitor the growth rates of individual cells while applying repeated cycles of antibiotic treatments. We were able to identify distinct phenotypes (normal and persistent) and characterize the stochastic transitions between them. We also found that phenotypic heterogeneity was present prior to any environmental cue such as antibiotic exposure. Motivated by the experiments with persisters, we formulated a theoretical model describing the dynamic behavior of several discrete phenotypes in a periodically varying environment. This theoretical framework allowed us to quantitatively predict the fitness of dynamic populations and to compare survival strategies according to environmental time-symmetries. These calculations suggested that persistence is a strategy used by bacterial populations to adapt to fluctuating environments. Knowledge of the phenotypic transition rates for persistence may provide statistical information about the typical environments of bacteria. We also describe a design of artificial
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.
Khaled M. Soradi
Full Text Available Double patterning photolithography (DPL is considered one of the best solutions used for enabling 32 nm/22 nm technology. In this paper, we propose a new technique for double patterning post decomposition conflict resolution. The algorithm is based on lines positions encoding followed by code pattern matching. Experimental results show that the usage of encoded patterns decreases the time needed for pattern matching and increases the matching accuracy. The overall manual problem solution time is reduced to about 1%.
Michael S Humphrys
Full Text Available We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness. Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.
Hol, Felix J H; Hubert, Bert; Dekker, Cees; Keymer, Juan E
During antibiotic treatment, antibiotic concentration gradients develop. Little is know regarding the effects of antibiotic gradients on populations of nonresistant bacteria. Using a microfluidic device, we show that high-density motile Escherichia coli populations composed of nonresistant bacteria can, unexpectedly, colonize environments where a lethal concentration of the antibiotic kanamycin is present. Colonizing bacteria establish an adaptively resistant population, which remains viable for over 24 h while exposed to the antibiotic. Quantitative analysis of multiple colonization events shows that collectively swimming bacteria need to exceed a critical population density in order to successfully colonize the antibiotic landscape. After colonization, bacteria are not dormant but show both growth and swimming motility under antibiotic stress. Our results highlight the importance of motility and population density in facilitating adaptive resistance, and indicate that adaptive resistance may be a first step to the emergence of genetically encoded resistance in landscapes of antibiotic gradients.
Staicu, Lucian; Oremland, Ronald S.; Tobe, Ryuta; Mihara, Hisaaki
Bacteria and selenium (Se) are closely interlinked as the element serves both essential nutrient requirements and energy generation functions. However, Se can also behave as a powerful toxicant for bacterial homeostasis. Conversely, bacteria play a tremendous role in the cycling of Se between different environmental compartments, and bacterial metabolism has been shown to participate to all valence state transformations undergone by Se in nature. Bacteria possess an extensive molecular repertoire for Se metabolism. At the end of the 1980s, a novel mode of anaerobic respiration based on Se oxyanions was experimentally documented for the first time. Following this discovery, specific enzymes capable of reducing Se oxyanions and harvesting energy were found in a number of anaerobic bacteria. The genes involved in the expression of these enzymes have later been identified and cloned. This iterative approach undertaken outside-in led to the understanding of the molecular mechanisms of Se transformations in bacteria. Based on the extensive knowledge accumulated over the years, we now have a full(er) view from the inside out, from DNA-encoding genes to enzymes and thermodynamics. Bacterial transformations of Se for assimilatory purposes have been the object of numerous studies predating the investigation of Se respiration. Remarkable contributions related to the understating of the molecular picture underlying seleno-amino acid biosynthesis are reviewed herein. Under certain circumstances, Se is a toxicant for bacterial metabolism and bacteria have evolved strategies to counteract this toxicity, most notably by the formation of elemental Se (nano)particles. Several biotechnological applications, such as the production of functional materials and the biofortification of crop species using Se-utilizing bacteria, are presented in this chapter.
Saks, Elzbieta; Jankiewicz, Urszula
Chitinolytic bacteria play an important role in degradation of chitin, one of the most abundant biopolymers in nature. These microorganisms synthesize specific enzymes, that catalyze hydrolysis of beta-1,4-glycosidic bonds in low-digestible chitin polymers, turning it into low-molecular, easy to digest compounds. During last decades many bacterial chitinolytic enzymes have been studied and characterized, mainly for their potential applications in agriculture, industry and medicine. Several chitinase classifications have been proposed, either on the base of substrate specificity or amino acid sequence similarities. X-ray crystallography and NMR spectroscopy techniques enabled the determination of three dimensional structure of some chitinases, what was helpful in explaining their catalytic mechanism. Development of biotechnology and molecular biology enables a deep research in regulation and cloning of bacterial chitinase genes.
Full Text Available The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.
Li, Sixing; Huang, Tony Jun; Ma, Fen; Zeng, Xiangqun; Bachman, Hunter; Cameron, Craig E
Bacterial separation from human blood samples can help with the identification of pathogenic bacteria for sepsis diagnosis. In this work, we report an acoustofluidic device for label-free bacterial separation from human blood samples. In particular, we exploit the acoustic radiation force generated from a tilted-angle standing surface acoustic wave (taSSAW) field to separate Escherichia coli from human blood cells based on their size difference. Flow cytometry analysis of the E. coli separated from red blood cells shows a purity of more than 96%. Moreover, the label-free electrochemical detection of the separated E. coli displays reduced non-specific signals due to the removal of blood cells. Our acoustofluidic bacterial separation platform has advantages such as label-free separation, high biocompatibility, flexibility, low cost, miniaturization, automation, and ease of in-line integration. The platform can be incorporated with an on-chip sensor to realize a point-of-care sepsis diagnostic device. (paper)
Li, Sixing; Ma, Fen; Bachman, Hunter; Cameron, Craig E.; Zeng, Xiangqun; Huang, Tony Jun
Bacterial separation from human blood samples can help with the identification of pathogenic bacteria for sepsis diagnosis. In this work, we report an acoustofluidic device for label-free bacterial separation from human blood samples. In particular, we exploit the acoustic radiation force generated from a tilted-angle standing surface acoustic wave (taSSAW) field to separate Escherichia coli from human blood cells based on their size difference. Flow cytometry analysis of the E. coli separated from red blood cells shows a purity of more than 96%. Moreover, the label-free electrochemical detection of the separated E. coli displays reduced non-specific signals due to the removal of blood cells. Our acoustofluidic bacterial separation platform has advantages such as label-free separation, high biocompatibility, flexibility, low cost, miniaturization, automation, and ease of in-line integration. The platform can be incorporated with an on-chip sensor to realize a point-of-care sepsis diagnostic device.
Full Text Available Abstract A successful infection of the human intestine by enteropathogenic bacteria depends on the ability of bacteria to attach and colonize the intestinal epithelium and, in some cases, to invade the host cell, survive intracellularly and disseminate from cell to cell. To accomplish these processes bacteria have evolved an arsenal of molecules that are mostly secreted by dedicated type III secretion systems, and that interact with the host, subverting normal cellular functions. Here we overview the most important molecular strategies developed by enteropathogenic Escherichia coli, Salmonella enterica, Shigella flexneri, and Yersinia enterocolitica to cause enteric infections. Despite having evolved different effectors, these four microorganisms share common host cellular targets.
Yuen, KY; Lau, SKP; Woo, PCY; Lau, ATK; To, APC
Although DNA encoding antibiotic resistance has been discovered in antibiotic preparations, its significance for the development of antibiotic resistance in bacteria is unknown. No phylogenetic evidence was obtained for recent horizontal transfer of antibiotic resistance genes from antibiotic-producing organisms to bacteria from human or animal sources.
Full Text Available Understanding single-neuron computations and encoding performed by spike-generation mechanisms of cortical neurons is one of the central challenges for cell electrophysiology and computational neuroscience. An established paradigm to study spike encoding in controlled conditions in vitro uses intracellular injection of a mixture of signals with fluctuating currents that mimic in vivo-like background activity. However this technique has two serious limitations: it uses current injection, while synaptic activation leads to changes of conductance, and current injection is technically most feasible in the soma, while the vast majority of synaptic inputs are located on the dendrites. Recent progress in optogenetics provides an opportunity to circumvent these limitations. Transgenic expression of light-activated ionic channels, such as Channelrhodopsin2 (ChR2, allows induction of controlled conductance changes even in thin distant dendrites. Here we show that photostimulation provides a useful extension of the tools to study neuronal encoding, but it has its own limitations. Optically induced fluctuating currents have a low cutoff (~70 Hz, thus limiting the dynamic range of frequency response of cortical neurons. This leads to severe underestimation of the ability of neurons to phase-lock their firing to high frequency components of the input. This limitation could be worked around by using short (2 ms light stimuli which produce membrane potential responses resembling EPSPs by their fast onset and prolonged decay kinetics. We show that combining application of short light stimuli to different parts of dendritic tree for mimicking distant EPSCs with somatic injection of fluctuating current that mimics fluctuations of membrane potential in vivo, allowed us to study fast encoding of artificial EPSPs photoinduced at different distances from the soma. We conclude that dendritic photostimulation of ChR2 with short light pulses provides a powerful tool to
Ioannidis, Panagiotis; Dunning Hotopp, Julie C; Sapountzis, Panagiotis
as their patterns of sequence evolution will aid studies of cell replication and cell density, as well as the potential genetic manipulation of these widespread intracellular bacteria. RESULTS: Using features that have been previously experimentally verified in the alpha-Proteobacterium Caulobacter crescentus...... bacteria while fundamental characteristics like presence of DnaA and IHF binding sites as well as the boundary genes are more widely conserved. The relative paucity of CtrA binding sites in the ori regions, as well as the absence of key enzymes associated with DNA replication in the respective genomes...
Urbani, Jacopo; Dutta, Sourav; Gurajada, Sairam; Weikum, Gerhard
Many Web applications require efficient querying of large Knowledge Graphs (KGs). We propose KOGNAC, a dictionary-encoding algorithm designed to improve SPARQL querying with a judicious combination of statistical and semantic techniques. In KOGNAC, frequent terms are detected with a frequency
Purpose: To investigate the distribution of plasmid-encoded extended spectrum beta-lacatamases (ESBLs) in Lahore, Pakistan using different phenotypic and molecular methods. Methods: Escherichia coli and Klebsiella spp were obtained over a period of nineteen months (June 2007 to December 2008). Both were tested ...
Cloning, characterization and expression of OsFMO(t) in rice encoding a flavin monooxygenase. Jicai Yi, Lanna Liu, Youpei Cao, Jiazuo Li and Mantong Mei. J. Genet. 92, 471–480. Figure 1. Examples of PCR analysis of the presence of the genes for HPT and GUS in transgenic plants. Genomic DNA of putative.
de Vries, Walter; Berkhout, Ben
RNA silencing or RNAi interference (RNAi) serves as an innate antiviral mechanism in plants, fungi and animals. Human viruses, like plant viruses, encode suppressor proteins or RNAs that block or modulate the RNAi pathway. This review summarizes the mechanisms by which pathogenic human viruses
Nieuwenstein, Mark; Kromm, Maria
Studies on visual long-term memory have shown that we have a tremendous capacity for remembering pictures of objects, even at a highly detailed level. What remains unclear, however, is whether encoding objects at such a detailed level comes at any cost. In the current study, we examined how the
Raw, domestic sewage of Kuwait City contained about 106 ml−1 colony forming units of Enterobacter hormaechei subsp. oharae (56.6%), Klebsiella spp. (36%), and Escherichia coli (7.4%), as characterized by their 16S rRNA-gene sequences. The isolated coliforms grew successfully on a mineral medium with crude oil vapor as a sole source of carbon and energy. Those strains also grew, albeit to different degrees, on individual n-alkanes with carbon chains between C9 and C36 and on the individual aromatic hydrocarbons, toluene, naphthalene, phenanthrene, and biphenyl as sole sources of carbon and energy. These results imply that coliforms, like other hydrocarbonoclastic microorganisms, oxidize hydrocarbons to the corresponding alcohols and then to aldehydes and fatty acids which are biodegraded by β-oxidation to acetyl CoA. The latter is a well-known key intermediate in cell material and energy production. E. coli cells grown in the presence of n-hexadecane (but not in its absence) exhibited typical intracellular hydrocarbon inclusions, as revealed by transmission electron microscopy. Raw sewage samples amended with crude oil, n-hexadecane, or phenanthrene lost these hydrocarbons gradually with time. Meanwhile, the numbers of total and individual coliforms, particularly Enterobacter, increased. It was concluded that coliform bacteria in domestic sewage, probably in other environmental materials too, are effective hydrocarbon-biodegrading microorganisms. PMID:29082238
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
Nielsen, Jesper Sejrup; Lei, Lisbeth Kristensen; Ebersbach, Tine
Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species...... studied thus far. Here, we provide evidence for Hfq-dependent translational repression in the Gram-positive human pathogen Listeria monocytogenes, which is known to encode at least 50 sRNAs. We show that the Hfq-binding sRNA LhrA controls the translation and degradation of its target mRNA by an antisense...... mechanism, and that Hfq facilitates the binding of LhrA to its target. The work presented here provides the first experimental evidence for Hfq-dependent riboregulation in a Gram-positive bacterium. Our findings indicate that modulation of translation by trans-encoded sRNAs may occur by both Hfq...
Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki
We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159–165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD+ ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses. PMID
Bacterial viruses (i.e. phages) are ubiquitous intracellular parasites of bacteria, that along with protist grazers account for majority of bacterial mortality in nature. Phages impose strong selection for bacterial phage-resistance, which is often coupled with fitness costs on bacterial traits such as growth ability, virulence or motility. Traditionally phage-host interactions have been studied with two species systems in the laboratory, neglecting the complex web of interactions presen...
Husník, Filip; Nikoh, N.; Koga, R.; Ross, L.; Duncan, R.P.; Fuije, M.; Tanaka, M.; Satoh, N.; Bachtrog, D.; Wilson, A.C.C.; von Dohlen, C.D.; Fukatsu, T.; McCutcheon, J.P.
Roč. 153, č. 7 (2013), s. 1567-1578 ISSN 0092-8674 Grant - others:GA ČR(CZ) GAP505/10/1401; GA ČR(CZ) GA13-01878S Program:GA Institutional support: RVO:60077344 Keywords : intracellular bacteria * beta-proteobacteria * reduced genomes * host cell * evolution * endosymbionts * Wolbachia Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 33.116, year: 2013
Libby, Elizabeth A.; Roggiani, Manuela; Goulian, Mark
It has long been hypothesized that subcellular positioning of chromosomal loci in bacteria may be influenced by gene function and expression state. Here we provide direct evidence that membrane protein expression affects the position of chromosomal loci in Escherichia coli. For two different membrane proteins, we observed a dramatic shift of their genetic loci toward the membrane upon induction. In related systems in which a cytoplasmic protein was produced, or translation was eliminated by mutating the start codon, a shift was not observed. Antibiotics that block transcription and translation similarly prevented locus repositioning toward the membrane. We also found that repositioning is relatively rapid and can be detected at positions that are a considerable distance on the chromosome from the gene encoding the membrane protein (>90 kb). Given that membrane protein-encoding genes are distributed throughout the chromosome, their expression may be an important mechanism for maintaining the bacterial chromosome in an expanded and dynamic state. PMID:22529375
Gedik, H.; Voss, T.A.; Voss, A.
Money is one of the most frequently passed items in the world. The aim of this study was to ascertain the survival status of bacteria including Staphylococcus aureus, Escherichia coli, and Vancomycin- Resistant Enterococci (VRE) on banknotes from different countries and the transmission of bacteria
Walmor eDe Mello
Full Text Available The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; 1 under normal conditions, Lucifer Yellow flows from cell-to-cell through gap junctions; 2 the intracellular dialysis of renin (100nM disrupts chemical communication-an effect enhanced by simultaneous administration of angiotensinogen (100nM; 3 enalaprilat (10-9M administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; 4 aliskiren (10-8M inhibited the effect of renin on chemical communication;5 the possible role of intracellular renin independently of angiotensin II (Ang II was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; 6 the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed;7 the present results indicate that intracellular renin due to internalization or in situ synthesis, causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.
Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.
Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca 2+ on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery
Kono, Nozomu; Arai, Hiroyuki
Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E. © 2014 The Authors. Traffic published by John Wiley & Sons Ltd.
Fuller Brian G
Full Text Available Abstract Gradients are used in a number of biological systems to transmit spatial information over a range of distances. The best studied are morphogen gradients where information is transmitted over many cell lengths. Smaller mitotic gradients reflect the need to organize several distinct events along the length of the mitotic spindle. The intracellular gradients that characterize mitosis are emerging as important regulatory paradigms. Intracellular gradients utilize intrinsic auto-regulatory feedback loops and diffusion to establish stable regions of activity within the mitotic cytosol. We review three recently described intracellular mitotic gradients. The Ran GTP gradient with its elaborate cascade of nuclear transport receptors and cargoes is the best characterized, yet the dynamics underlying the robust gradient of Ran-GTP have received little attention. Gradients of phosphorylation have been observed on Aurora B kinase substrates both before and after anaphase onset. In both instances the phosphorylation gradient appears to result from a soluble gradient of Aurora B kinase activity. Regulatory properties that support gradient formation are highlighted. Intracellular activity gradients that regulate localized mitotic events bare several hallmarks of self-organizing biologic systems that designate spatial information during pattern formation. Intracellular pattern formation represents a new paradigm in mitotic regulation.
Bjerg, Jesper Tataru; Damgaard, Lars Riis; Holm, Simon Agner
Cable bacteria are filamentous bacteria that electrically couple sulfide oxidation and oxygen reduction at centimeter distances, and observations in sediment environments have suggested that they are motile. By time-lapse microscopy, we found that cable bacteria used gliding motility on surfaces...... with a highly variable speed of 0.50.3 ms1 (meanstandard deviation) and time between reversals of 155108 s. They frequently moved forward in loops, and formation of twisted loops revealed helical rotation of the filaments. Cable bacteria responded to chemical gradients in their environment, and around the oxic......-anoxic interface, they curled and piled up, with straight parts connecting back to the source of sulfide. Thus, it appears that motility serves the cable bacteria in establishing and keeping optimal connections between their distant electron donor and acceptors in a dynamic sediment environment....
Chongsiriwatana, Nathaniel P; Lin, Jennifer S; Kapoor, Rinki; Wetzler, Modi; Rea, Jennifer A C; Didwania, Maruti K; Contag, Christopher H; Barron, Annelise E
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.
Wedde, Marianne; Weise, Christoph; Nuck, Rolf; Altincicek, Boran; Vilcinskas, Andreas
The insect metalloproteinase inhibitor (IMPI) from the greater wax moth, Galleria mellonella, represents the first and to date only specific inhibitor of microbial metalloproteinases reported from animals. Here, we report on the characterization including carbohydrate analysis of two recombinant constructs encoded by impi cDNA either upstream or downstream of the furin cleavage site identified. rIMPI-1, corresponding to native IMPI purified from hemolymph, is encoded by the N-terminal part of the impi sequence, whereas rIMPI-2 is encoded by its C-terminal part. rIMPI-1 is glycosylated at N48 with GlcNAc2Man3, showing fucosylation to different extents. Similarly, rIMPI-2 is glycosylated at N149 with GlcNAc2Man3, but is fully fucosylated. rIMPI-1 represents a promising template for the design of second-generation antibiotics owing to its specific activity against thermolysin-like metalloproteinases produced by human pathogenic bacteria such as Vibrio vulnificus. In contrast, rIMPI-2 does not inhibit bacterial metalloproteinases, but is moderately active against recombinant human matrix metalloproteinases (MMPs). Both microbial metalloproteinases and MMPs induce expression of the impi gene when injected into G. mellonella larvae. These findings provide evidence that the impi gene encodes two distinct inhibitors, one inhibiting microbial metalloproteinases and contributing to innate immunity, the other putatively mediating regulation of endogenous MMPs during metamorphosis.
Rita C. Ruiz
Full Text Available Plasmid encoded toxin (Pet is a serine protease originally described in enteroaggregative Escherichia coli (EAEC prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.
Manuel E. Del Cogliano
Full Text Available Shiga toxin (Stx is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non-alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: (1 direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, (2 cationic properties are necessary but not sufficient for bacteriophage inactivation, and (3 inactivation by cationic peptides could be sequence (or structure specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.
FREEMAN, B A; CIRCO, R
Freeman, Bob A. (University of Chicago, Chicago, Ill.) and Richard Circo. Effect of tetracyclines on the intracellular amino acids of molds. J. Bacteriol. 86:38-44. 1963.-The tetracycline antibiotics were shown to alter the amino acid metabolism of molds whose growth is not markedly affected. Eight molds were grown in the presence of these antiobiotics; four exhibited a general reduction in the concentration of the intracellular amino acids, except for glutamic acid and alanine. In most of these four cultures, the tetracyclines also caused the complete disappearance of arginine, lysine, proline, phenylalanine, and tyrosine from the intracellular amino acid pool. The significance of these observations and the usefulness of the method in the study of the mechanisms of antibiotic action are discussed.
Huang, Beijing K.; Sikes, Hadley D.
Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS) in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies. PMID:25460730
Van Damme, Ilse; d'Ydewalle, Géry
Recent studies with the Deese/Roediger-McDermott (Deese 1959; Roediger & McDermott, 1995) paradigm have revealed that amnesic patients do not only show impaired veridical memory, but also diminished false memory for semantically related lure words. Due to the typically used explicit retrieval instructions, however, this finding may reflect problems at encoding, at recollection, or both. Therefore, the present experiments examined implicit as well as explicit false memory in patients suffering from Korsakoff's syndrome and controls. In Experiment 1, encoding instructions either focused on remembering individual list words, or on discovering semantic relationships among the words. In Experiment 2, different presentation durations were used. Results emphasize the distinction between automatic and intentional retrieval: Korsakoff patients' veridical and false memory scores were diminished when explicit recollection was required, but not when memory was tested implicitly. Encoding manipulations only significantly affected veridical memory: Priming was reduced with thematic encoding, and explicit retrieval was facilitated when given more study time.
Van Grinsven Bart Robert, Nicolaas; Cleij, Thomas
A method for characterizing bacteria includes passing a liquid containing an analyte comprising a first bacteria and a second bacteria over and in contact with a polymer material on a substrate. The polymer material is formulated to bind to the first bacteria, and the first bacteria binds to the
Tvermoes, Brooke E.; Bird, Gary S.; Freedman, Jonathan H.
Background Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca2+]i) and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal. Methodology/Principal Finding In the present report, the effects of cadmium on [Ca2+]i under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60), which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca2+]i mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression. Conclusions/Significance These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription. PMID:21694771
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
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.
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
Boutet, Isabelle; Collin, Charles; Faubert, Jocelyn
Configural relations and a critical band of spatial frequencies (SFs) in the middle range are particularly important for face recognition. We report the results of four experiments in which the relationship between these two types of information was examined. In Experiments 1, 2A, and 2B, the face inversion effect (FIE) was used to probe configural face encoding. Recognition of upright and inverted faces and nonface objects was measured in four conditions: a no-filter condition and three SF conditions (low, medium, and high frequency). We found significant FIEs of comparable magnitudes for all frequency conditions. In Experiment 3, discrimination of faces on the basis of either configural or featural modifications was measured under the same four conditions. Although the ability to discriminate configural modifications was superior in the medium-frequency condition, so was the ability to discriminate featural modifications. We conclude that the band of SF that is critical for face recognition does not contribute preferentially to configural encoding.
Krath, Britta N.; Hove-Jensen, Bjarne
The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...
Wong,; Pak C. , Wong; Kwong K. , Foote; Harlan, P [Richland, WA
Current technologies allow the generation of artificial DNA molecules and/or the ability to alter the DNA sequences of existing DNA molecules. With a careful coding scheme and arrangement, it is possible to encode important information as an artificial DNA strand and store it in a living host safely and permanently. This inventive technology can be used to identify origins and protect R&D investments. It can also be used in environmental research to track generations of organisms and observe the ecological impact of pollutants. Today, there are microorganisms that can survive under extreme conditions. As well, it is advantageous to consider multicellular organisms as hosts for stored information. These living organisms can provide as memory housing and protection for stored data or information. The present invention provides well for data storage in a living organism wherein at least one DNA sequence is encoded to represent data and incorporated into a living organism.
Full Text Available The benchmark for computation is typically given as Turing computability; the ability for a computation to be performed by a Turing Machine. Many languages exploit (indirect encodings of Turing Machines to demonstrate their ability to support arbitrary computation. However, these encodings are usually by simulating the entire Turing Machine within the language, or by encoding a language that does an encoding or simulation itself. This second category is typical for process calculi that show an encoding of lambda-calculus (often with restrictions that in turn simulates a Turing Machine. Such approaches lead to indirect encodings of Turing Machines that are complex, unclear, and only weakly equivalent after computation. This paper presents an approach to encoding Turing Machines into intensional process calculi that is faithful, reduction preserving, and structurally equivalent. The encoding is demonstrated in a simple asymmetric concurrent pattern calculus before generalised to simplify infinite terms, and to show encodings into Concurrent Pattern Calculus and Psi Calculi.
Fuentes, Juan Luis; Garbayo, Inés; Cuaresma, María; Montero, Zaida; González-Del-Valle, Manuel; Vílchez, Carlos
A greater insight on the control of the interactions between microalgae and other microorganisms, particularly bacteria, should be useful for enhancing the efficiency of microalgal biomass production and associated valuable compounds. Little attention has been paid to the controlled utilization of microalgae-bacteria consortia. However, the studies of microalgal-bacterial interactions have revealed a significant impact of the mutualistic or parasitic relationships on algal growth. The algal growth, for instance, has been shown to be enhanced by growth promoting factors produced by bacteria, such as indole-3-acetic acid. Vitamin B12 produced by bacteria in algal cultures and bacterial siderophores are also known to be involved in promoting faster microalgal growth. More interestingly, enhancement in the intracellular levels of carbohydrates, lipids and pigments of microalgae coupled with algal growth stimulation has also been reported. In this sense, massive algal production might occur in the presence of bacteria, and microalgae-bacteria interactions can be beneficial to the massive production of microalgae and algal products. This manuscript reviews the recent knowledge on the impact of the microalgae-bacteria interactions on the production of microalgae and accumulation of valuable compounds, with an emphasis on algal species having application in aquaculture.
Kornfeld, A.; Schiewe, J.; Dykes, J.
Our sonic environment is the matter of subject in multiple domains which developed individual means of its description. As a result, it lacks an established visual language through which knowledge can be connected and insights shared. We provide a visual communication framework for the systematic and coherent documentation of sound in large-scale environments. This consists of visual encodings and mappings of acoustic parameters into distinct graphic variables that present plausible solutions...
Nielsen, John; Janda, Kim D.
The recent application of "combinatorial libraries" to supplement existing drug screening processes might simplify and accelerate the search for new lead compounds or drugs. Recently, a scheme for encoded combinatorial chemistry was put forward to surmount a number of the limitations possessed by...... by existing methodologies. Here we detail the synthesis of several matrices and the necessary chemistry to implement the conceptual scheme. In addition, we disclose how this novel technology permits a controlled ′dendritic" display of the chemical libraries....
Amin, Shady A; Parker, Micaela S; Armbrust, E Virginia
Diatoms and bacteria have cooccurred in common habitats for hundreds of millions of years, thus fostering specific associations and interactions with global biogeochemical consequences. Diatoms are responsible for one-fifth of the photosynthesis on Earth, while bacteria remineralize a large portion of this fixed carbon in the oceans. Through their coexistence, diatoms and bacteria cycle nutrients between oxidized and reduced states, impacting bioavailability and ultimately feeding higher trophic levels. Here we present an overview of how diatoms and bacteria interact and the implications of these interactions. We emphasize that heterotrophic bacteria in the oceans that are consistently associated with diatoms are confined to two phyla. These consistent bacterial associations result from encounter mechanisms that occur within a microscale environment surrounding a diatom cell. We review signaling mechanisms that occur in this microenvironment to pave the way for specific interactions. Finally, we discuss known interactions between diatoms and bacteria and exciting new directions and research opportunities in this field. Throughout the review, we emphasize new technological advances that will help in the discovery of new interactions. Deciphering the languages of diatoms and bacteria and how they interact will inform our understanding of the role these organisms have in shaping the ocean and how these interactions may change in future oceans.
Amin, Shady A.; Parker, Micaela S.
Summary: Diatoms and bacteria have cooccurred in common habitats for hundreds of millions of years, thus fostering specific associations and interactions with global biogeochemical consequences. Diatoms are responsible for one-fifth of the photosynthesis on Earth, while bacteria remineralize a large portion of this fixed carbon in the oceans. Through their coexistence, diatoms and bacteria cycle nutrients between oxidized and reduced states, impacting bioavailability and ultimately feeding higher trophic levels. Here we present an overview of how diatoms and bacteria interact and the implications of these interactions. We emphasize that heterotrophic bacteria in the oceans that are consistently associated with diatoms are confined to two phyla. These consistent bacterial associations result from encounter mechanisms that occur within a microscale environment surrounding a diatom cell. We review signaling mechanisms that occur in this microenvironment to pave the way for specific interactions. Finally, we discuss known interactions between diatoms and bacteria and exciting new directions and research opportunities in this field. Throughout the review, we emphasize new technological advances that will help in the discovery of new interactions. Deciphering the languages of diatoms and bacteria and how they interact will inform our understanding of the role these organisms have in shaping the ocean and how these interactions may change in future oceans. PMID:22933565
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
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.
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.
Pamela A. Mosier-Boss
Full Text Available Surface enhanced Raman spectroscopy (SERS has been widely used for chemical detection. Moreover, the inherent richness of the spectral data has made SERS attractive for use in detecting biological materials, including bacteria. This review discusses methods that have been used to obtain SERS spectra of bacteria. The kinds of SERS substrates employed to obtain SERS spectra are discussed as well as how bacteria interact with silver and gold nanoparticles. The roll of capping agents on Ag/Au NPs in obtaining SERS spectra is examined as well as the interpretation of the spectral data.
Full Text Available Brucella spp. is a species of facultative intracellular Gram-negative bacteria that induces abortion and causes sterility in domesticated mammals and chronic undulant fever in humans. Important determinants of Brucella's virulence and potential for chronic infection include the ability to circumvent the host cell's internal surveillance system and the capability to proliferate within dedicated and non-dedicated phagocytes. Hence, identifying genes necessary for intracellular survival may hold the key to understanding Brucella infection. In the present study, microarray analysis reveals that 7.82% (244/3334 of all Brucella abortus genes were up-regulated and 5.4% (180/3334 were down-regulated in RAW264.7 cells, compared to free-living cells in TSB. qRT-PCR verification further confirmed a >5-fold up-regulation for fourteen genes. Functional analysis classified araC, ddp, and eryD as to partake in information storage and processing, alp, flgF and virB9 to be involved in cellular processes, hpcd and aldh to play a role in metabolism, mfs and nikC to be involved in both cellular processes and metabolism, and four hypothetical genes (bruAb1_1814, bruAb1_0475, bruAb1_1926, and bruAb1_0292 had unknown functions. Furthermore, we constructed a B. abortus 2308 mutant Δddp where the ddp gene is deleted in order to evaluate the role of ddp in intracellular survival. Infection assay indicated significantly higher adherence and invasion abilities of the Δddp mutant, however it does not survive well in RAW264.7 cells. Brucella may survive in hostile intracellular environment by modulating gene expression.
Maresca, Julia A; Gomez Maqueo Chew, Aline; Ponsatí, Marta Ros
Bacteriochlorophylls (BChls) c and d, two of the major light-harvesting pigments in photosynthetic green sulfur bacteria, differ only by the presence of a methyl group at the C-20 methine bridge position in BChl c. A gene potentially encoding the C-20 methyltransferase, bchU, was identified...
Martínez, B.; Sillanpää, J.; Smit, E.; Korhonen, T.K.; Pouwels, P.H.
The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393T. The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting
Sakamoto, K; Konings, WN
For brewing industry, beer spoilage bacteria have been problematic for centuries. They include some lactic acid bacteria such as Lactobacillus brevis, Lactobacillus lindneri and Pediococcus damnosus, and some Gram-negative bacteria such as Pectinatus cerevisiiphilus, Pectinatus frisingensis and
Købmann, Brian Jensen; Solem, Christian; Pedersen, M.B.
of the genes encoding F-1-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F-1-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase...... threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions...
Dieudonné, Alexandre; Daniel, Thomas L; Sane, Sanjay P
Antennal mechanosensors play a key role in control and stability of insect flight. In addition to the well-established role of antennae as airflow detectors, recent studies have indicated that the sensing of antennal vibrations by Johnston's organs also provides a mechanosensory feedback relevant for flight stabilization. However, few studies have addressed how the individual units, or scolopidia, of the Johnston's organs encode these antennal vibrations and communicate it to the brain. Here, we characterize the encoding properties of individual scolopidia from the Johnston's organs in the hawk moth, Manduca sexta, through intracellular neurophysiological recordings from axons of the scolopidial neurons. We stimulated the flagellum-pedicel joint using a custom setup that delivered mechanical stimuli of various (step, sinusoidal, frequency and amplitude sweeps) waveforms. Single units of the Johnston's organs typically displayed phaso-tonic responses to step stimuli with short (3-5 ms) latencies. Their phase-locked response to sinusoidal stimuli in the 0.1-100 Hz frequency range showed high fidelity (vector strengths>0.9). The neurons were able to encode different phases of the stimulus motion and were also extremely sensitive to small amplitude (<0.05 deg) deflections with some indication of directional tuning. In many cases, the firing frequency of the neurons varied linearly as a function of the stimulus frequency at wingbeat and double wingbeat frequencies, which may be relevant to their role in flight stabilization. Iontophoretic fills of these neurons with fluorescent dyes showed that they all projected in the antennal mechanosensory and motor center (AMMC) area of the brain. Taken together, these results showcase the speed and high sensitivity of scolopidia of the Johnston's organs, and hence their ability to encode fine antennal vibrations. © 2014. Published by The Company of Biologists Ltd.
Full Text Available "nBackground: Current treatment of hemophilia which is one of the most common bleeding disorders, involves replacement therapy using concentrates of FVIII and FIX .However, these concentrates have been associated with viral infections and thromboembolic complications and development of antibodies. "nThe use of recombinant human factor VII (rhFVII is effective for the treatment of patients with hemophilia A or B, who develop antibodies ( referred as inhibitors against replacement therapy , because it induces coagulation independent of FVIII and FIX. However, its short half-life and high cost have limited its use. One potential solution to this problem may be the use of FVIIa gene transfer, which would attain continuing therapeutic levels of expression from a single injection. The aim of this study was to engineer a novel hFVII (human FVII gene containing a cleavage site for the intracellular protease and furin, by PCR mutagenesis "nMethods: The sequence encoding light and heavy chains of hFVII, were amplified by using hFVII/pTZ57R and specific primers, separately. The PCR products were cloned in pTZ57R vector. "nResults and discussion: Cloning was confirmed by restriction analysis or PCR amplification using specific primers and plasmid universal primers. Mutagenesis of sequence encoding light and heavy chain was confirmed by restriction enzyme. "nConclusion: In the present study, it was provided recombinant plasmids based on mutant form of DNA encoding light and heavy chains. Joining mutant form of DNA encoding light chain with mutant heavy chain led to a new variant of hFVII. This variant can be activated by furin and an increase in the proportion of activated form of FVII. This mutant form of hFVII may be used for gene therapy of hemophilia.
Juenemann, Katrin; Wiemhoefer, Anne; Reits, Eric A.
Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington's disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion
The goal of this thesis was to investigate the combination of ultrasound and microbubbles (USMB) for intracellular delivery of (model) drugs in vitro. We have focused on clinically approved drugs, i.e. cisplatin, and microbubbles, i.e. SonoVue™, to facilitate clinical translation. In addition, model
Hansen, Kasper Bø; Bräuner-Osborne, Hans
Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...
In this study, we examined the intracellular localization of the product of Na+/H+ antiporter gene (MzNHX1) cloned from Malus zumi. Analysis using yeast cells expressing a fusion protein of MzNHX1 and green fluorescent protein confirmed the localization of MzNHX1 on the tonoplast.
Novak, I; Hug, M; Greger, R
In order to study the mechanism of H+ and HCO3- transport in a HCO3- secreting epithelium, pancreatic ducts, we have measured the intracellular pH (pHi) in this tissue using the pH sensitive probe BCECF. We found that exposures of ducts to solutions containing acetate/acetic acid or NH4+/NH3 buff...
therapeutic and vaccine development. Keywords - gene therapy, vaccine, bioinspired, biotherapeutic I. INTRODUCTION The efficacy of many protein and DNA...DNA, RNA and proteins . While these therapeutics have tremendous potential, effectively formulating and delivering them has also been a widely...intracellular trafficking that is inspired by biological polymers, i.e. proteins , that are involved in controlling vesicular trafficking pathways. For
Sep 5, 2007 ... binding lectin of Escherichia coli (Kawasaki et al., 1989) and Salmonella (Ihara et al., 1991). However some reports could not find any effect of mannose binding lectin on complement activation upon extracellular infec- tion of Staphylococcus aureus (Cunion et al., 2001). In intracellular infections, there is ...
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 ...
There is increasing interest in using live-cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of multip...
Full Text Available Voltage sensing domains of Kv channels control ionic conductance through coupling of the movement of charged residues in the S4 segment to conformational changes at the cytoplasmic region of the pore domain, that allow K+ ions to flow. Conformational transitions within the voltage sensing domain caused by changes in the applied voltage across the membrane field are coupled to the conducting pore region and the gating of ionic conductance. However, several other factors not directly linked to the voltage dependent movement of charged residues within the voltage sensor impact the dynamics of the voltage sensor, such as inactivation, ionic conductance, intracellular ion identity and block of the channel by intracellular ligands. The effect of intracellular ions on voltage sensor dynamics is of importance in the interpretation of gating current measurements and the physiology of pore/voltage sensor coupling. There is a significant amount of variability in the reported kinetics of voltage sensor deactivation kinetics of Kv channels attributed to different mechanisms such as open state stabilization, immobilization and relaxation processes of the voltage sensor. Here we separate these factors and focus on the causal role that intracellular ions can play in allosterically modulating the dynamics of Kv voltage sensor deactivation kinetics. These considerations are of critical importance in understanding the molecular determinants of the complete channel gating cycle from activation to deactivation.
Kok, JW; NikolovaKarakashian, M; Klappe, K; Alexander, C; Merrill, AH
This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DN-Cer), an intermediate in de novo sphingolipid biosynthesis, When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl-DH-Cer (C-6-NBD-DH-Cer) was incubated with HT29, NRK, BHK,
Kuzu, Omer F; Gowda, Raghavendra; Noory, Mohammad A; Robertson, Gavin P
Demand for cholesterol is high in certain cancers making them potentially sensitive to therapeutic strategies targeting cellular cholesterol homoeostasis. A potential approach involves disruption of intracellular cholesterol transport, which occurs in Niemann-Pick disease as a result of acid sphingomyelinase (ASM) deficiency. Hence, a class of lysosomotropic compounds that were identified as functional ASM inhibitors (FIASMAs) might exhibit chemotherapeutic activity by disrupting cancer cell cholesterol homoeostasis. Here, the chemotherapeutic utility of ASM inhibition was investigated. The effect of FIASMAs on intracellular cholesterol levels, cholesterol homoeostasis, cellular endocytosis and signalling cascades were investigated. The in vivo efficacy of ASM inhibition was demonstrated using melanoma xenografts and a nanoparticle formulation was developed to overcome dose-limiting CNS-associated side effects of certain FIASMAs. Functional ASM inhibitors inhibited intracellular cholesterol transport leading to disruption of autophagic flux, cellular endocytosis and receptor tyrosine kinase signalling. Consequently, major oncogenic signalling cascades on which cancer cells were reliant for survival were inhibited. Two tested ASM inhibitors, perphenazine and fluphenazine that are also clinically used as antipsychotics, were effective in inhibiting xenografted tumour growth. Nanoliposomal encapsulation of the perphenazine enhanced its chemotherapeutic efficacy while decreasing CNS-associated side effects. This study suggests that disruption of intracellular cholesterol transport by targeting ASM could be utilised as a potential chemotherapeutic approach for treating cancer.
Carlsson, Carl Michael; Bengtson, Per; Cucak, Helena
these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3 bound glycoform increased in cancer, suggesting...
Apr 18, 2017 ... if the histidine residue is protonated. Lontok et al., in their chimeric S protein studies used C terminal 11 amino acids of SARS-S protein attached to the plasma membrane re- porter protein VSV-G to show KXHXX motif is an intra- cellular localization signal for SARS, and the intracellular distribution closely ...
Full Text Available The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, ultramicrotomy, and scanning transmission X-ray microscopy (STXM. Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.
Fischer, Natalie; Mak, Tim N.; Shinohara, Debika Biswal; Sfanos, Karen S.; Meyer, Thomas F.
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
Method: The intracellular fibrinolytic protease produced by Ganoderma lucidum VK12 was isolated from the mycelia grown in MCDBF broth ... The inhibitory effect of different metal ions and commercial protease inhibitors on enzyme activity was studied. ... sodium hydroxide and 2.9 %w/v sodium carbonate in glass-distilled ...
Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is localized in the ER or ERGIC compartment and OC43 spike protein is predominantly localized in thelysosome. Differential localization can be explained by signal sequence. The sequence alignment ...
Three new studies have identified intestinal bacteria that appear to influence the response to checkpoint inhibitors. This Cancer Currents blog post explains how the researchers think their findings could be used to improve patients’ responses to these immunotherapy drugs.
Ramaiah, N.; Rodrigues, V.; Alwares, E.; Rodrigues, C.; Baksh, R.; Jayan, S.; Mohandass, C.
indiscriminate, deliber- ate, accidental, or regular/routine disposals), higher will be the number of coliforms in environmental samples. Further, microbiologists rely on the principle that higher the incidence of sewage indicator bacteria in any environment...
Lactic acid bacteria (LAB) is a heterotrophic Gram-positive bacteria which under goes lactic acid fermentations and leads to production of lactic acid as an end product. LAB includes Lactobacillus, Leuconostoc, Pediococcus, Lactococcus and Streptococcus which are grouped together in the family lactobacillaceae. LAB shows numerous antimicrobial activities due to production of antibacterial and antifungal compounds such as organic acids, bacteriocins, diacetyl, hydrogen peroxide and reutrin. LA...
Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.
Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....
Full Text Available Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-λ genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-λ genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-λ genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-λ. We also found that IFN-λ genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-λ2/λ3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-λ2. Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues.
Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M
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.
Zutz, Christoph; Chiang, Yi Ming; Faehnrich, Bettina; Bacher, Markus; Hellinger, Roland; Kluger, Bernhard; Wagner, Martin; Strauss, Joseph; Rychli, Kathrin
Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives. Copyright © 2017 Elsevier GmbH. All rights reserved.
Taylor, F J
Wallin (1927) first published the notion that the fusion of bacteria with host cells was the principal source of genetic novelty for speciation. He suggested that mitochondria are transitional elements in this process. While the significance that he attributed to symbiosis now seem excessive, he was one of the first authors to be aware of the evolutionary potential of symbiotic events and his view of mitochondria may not seem strange to many cell biologist today. The most significant evolutionary development which has been attributed to intracellular symbiosis is the origin of eukaryotic cellular organization. The current status of the 'serial endosymbiosis hypothesis' is briefly review. The case for the symbiotic origin of the chloroplast, based principally on 16 S RNA oligonucleotide cataloguing, is very strong. Mitochondrial origins are more obscure but also appear to be symbiotic due to recent 18 S cataloguing from wheat embryos. The probablility of the multiple origin of some eukaryotic organelles is also examined, the processes in question being the acquisition of distinct stocks of chloroplasts from disparate photosynthetic prokaryotes and the secondary donation of organelles from degenerate eukaryotic endosymbionts to their hosts, with specific reference to the dinoflagellates Peridinium balticum, Kryptoperidinium foliaceum and the ciliate Mesodinium rubrum. It is concluded that the evolutionary potential of intracellular symbiosis ('cytobiosis': a term introduced in this paper) is great, with the best established influence being on the origin of eukaryotic chloroplasts. Together with the potential effects of viral vectors, symbiosis serves as a supplementary speciation mechanism capable of producing directed evolutionary changes. It is likely that these processes will explain some of the apparent anomalies in evolutionary rates and direction which are not readily explicable by the conventional synthetic theory of evolution.
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.
Stief, Peter; Kamp, Anja; de Beer, Dirk
Intracellular nitrate storage allows microorganisms to survive fluctuating nutrient availability and anoxic conditions in aquatic ecosystems. Here we show that diatoms, ubiquitous and highly abundant microalgae, represent major cellular reservoirs of nitrate in an intertidal flat of the German Wadden Sea and are potentially involved in anaerobic nitrate respiration. Intracellular nitrate (ICNO3) was present year-round in the sediment and was spatially and temporally correlated with fucoxanthin, the marker photopigment of diatoms. Pyrosequencing of SSU rRNA genes of all domains of life confirmed that ICNO3 storage was most likely due to diatoms rather than other known nitrate-storing microorganisms (i.e., large sulfur bacteria and the eukaryotic foraminifers and gromiids). Sedimentary ICNO3 concentrations reached up to 22.3 µmol dm(-3) at the sediment surface and decreased with sediment depth to negligible concentrations below 5 cm. Similarly, the ICNO3/fucoxanthin ratio and porewater nitrate (PWNO3) concentrations decreased with sediment depth, suggesting that ICNO3 of diatoms is in equilibrium with PWNO3, but is enriched relative to PWNO3 by 2-3 orders of magnitude. Cell-volume-specific ICNO3 concentrations in a diatom mat covering the sediment surface during spring were estimated at 9.3-46.7 mmol L(-1). Retrieval of 18S rRNA gene sequences related to known nitrate-storing and nitrate-ammonifying diatom species suggested that diatoms in dark and anoxic sediment layers might be involved in anaerobic nitrate respiration. Due to the widespread dominance of diatoms in microphytobenthos, the total nitrate pool in coastal marine sediments may generally be at least two times larger than derived from porewater measurements and partially be recycled to ammonium.
Stief, Peter; Kamp, Anja; de Beer, Dirk
Intracellular nitrate storage allows microorganisms to survive fluctuating nutrient availability and anoxic conditions in aquatic ecosystems. Here we show that diatoms, ubiquitous and highly abundant microalgae, represent major cellular reservoirs of nitrate in an intertidal flat of the German Wadden Sea and are potentially involved in anaerobic nitrate respiration. Intracellular nitrate (ICNO3) was present year-round in the sediment and was spatially and temporally correlated with fucoxanthin, the marker photopigment of diatoms. Pyrosequencing of SSU rRNA genes of all domains of life confirmed that ICNO3 storage was most likely due to diatoms rather than other known nitrate-storing microorganisms (i.e., large sulfur bacteria and the eukaryotic foraminifers and gromiids). Sedimentary ICNO3 concentrations reached up to 22.3 µmol dm-3 at the sediment surface and decreased with sediment depth to negligible concentrations below 5 cm. Similarly, the ICNO3/fucoxanthin ratio and porewater nitrate (PWNO3) concentrations decreased with sediment depth, suggesting that ICNO3 of diatoms is in equilibrium with PWNO3, but is enriched relative to PWNO3 by 2-3 orders of magnitude. Cell-volume-specific ICNO3 concentrations in a diatom mat covering the sediment surface during spring were estimated at 9.3-46.7 mmol L-1. Retrieval of 18S rRNA gene sequences related to known nitrate-storing and nitrate-ammonifying diatom species suggested that diatoms in dark and anoxic sediment layers might be involved in anaerobic nitrate respiration. Due to the widespread dominance of diatoms in microphytobenthos, the total nitrate pool in coastal marine sediments may generally be at least two times larger than derived from porewater measurements and partially be recycled to ammonium. PMID:24023845
Van'kova, A. A.; Ivanov, P. I.; Emtsev, V. T.
Filtrating (ultramicroscopic) forms (FF) of bacteria were studied in a soddy-podzolic soil and the root zone of alfalfa plants as part of populations of the most widespread physiological groups of soil bacteria. FF were obtained by filtering soil solutions through membrane filters with a pore diameter of 0.22 μm. It was established that the greater part of the bacteria in the soil and in the root zone of the plants has an ultramicroscopic size: the average diameter of the cells is 0.3 μm, and their length is 0.6 μm, which is significantly less than the cell size of banal bacteria. The number of FF varies within a wide range depending on the physicochemical conditions of the habitat. The FF number's dynamics in the soil is of a seasonal nature; i.e., the number of bacteria found increases in the summer and fall and decreases in the winter-spring period. In the rhizosphere of the alfalfa, over the vegetation period, the number of FF and their fraction in the total mass of the bacteria increase. A reverse tendency is observed in the rhizoplane. The morphological particularities (identified by an electron microscopy) and the nature of the FF indicate their physiological activity.
Landt, Stephen G.; Marinov, Georgi K.; Kundaje, Anshul; Kheradpour, Pouya; Pauli, Florencia; Batzoglou, Serafim; Bernstein, Bradley E.; Bickel, Peter; Brown, James B.; Cayting, Philip; Chen, Yiwen; DeSalvo, Gilberto; Epstein, Charles; Fisher-Aylor, Katherine I.; Euskirchen, Ghia; Gerstein, Mark; Gertz, Jason; Hartemink, Alexander J.; Hoffman, Michael M.; Iyer, Vishwanath R.; Jung, Youngsook L.; Karmakar, Subhradip; Kellis, Manolis; Kharchenko, Peter V.; Li, Qunhua; Liu, Tao; Liu, X. Shirley; Ma, Lijia; Milosavljevic, Aleksandar; Myers, Richard M.; Park, Peter J.; Pazin, Michael J.; Perry, Marc D.; Raha, Debasish; Reddy, Timothy E.; Rozowsky, Joel; Shoresh, Noam; Sidow, Arend; Slattery, Matthew; Stamatoyannopoulos, John A.; Tolstorukov, Michael Y.; White, Kevin P.; Xi, Simon; Farnham, Peggy J.; Lieb, Jason D.; Wold, Barbara J.; Snyder, Michael
Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals. PMID:22955991
Goss, Foster R; Zhou, Li; Plasek, Joseph M; Broverman, Carol; Robinson, George; Middleton, Blackford; Rocha, Roberto A
Allergy documentation and exchange are vital to ensuring patient safety. This study aims to analyze and compare various existing standard terminologies for representing allergy information. Five terminologies were identified, including the Systemized Nomenclature of Medical Clinical Terms (SNOMED CT), National Drug File-Reference Terminology (NDF-RT), Medication Dictionary for Regulatory Activities (MedDRA), Unique Ingredient Identifier (UNII), and RxNorm. A qualitative analysis was conducted to compare desirable characteristics of each terminology, including content coverage, concept orientation, formal definitions, multiple granularities, vocabulary structure, subset capability, and maintainability. A quantitative analysis was also performed to compare the content coverage of each terminology for (1) common food, drug, and environmental allergens and (2) descriptive concepts for common drug allergies, adverse reactions (AR), and no known allergies. Our qualitative results show that SNOMED CT fulfilled the greatest number of desirable characteristics, followed by NDF-RT, RxNorm, UNII, and MedDRA. Our quantitative results demonstrate that RxNorm had the highest concept coverage for representing drug allergens, followed by UNII, SNOMED CT, NDF-RT, and MedDRA. For food and environmental allergens, UNII demonstrated the highest concept coverage, followed by SNOMED CT. For representing descriptive allergy concepts and adverse reactions, SNOMED CT and NDF-RT showed the highest coverage. Only SNOMED CT was capable of representing unique concepts for encoding no known allergies. The proper terminology for encoding a patient's allergy is complex, as multiple elements need to be captured to form a fully structured clinical finding. Our results suggest that while gaps still exist, a combination of SNOMED CT and RxNorm can satisfy most criteria for encoding common allergies and provide sufficient content coverage.
The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (approximately 1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.
Christon, Philip R.; Meyer, Wallace W.
Optoelectronic system measures relative angular position of shaft or other device to be turned, also measures absolute angular position after device turned through small angle. Relative angular position measured with fine resolution by optoelectronically counting finely- and uniformly-spaced light and dark areas on encoder disk as disk turns past position-sensing device. Also includes track containing coarsely- and nonuniformly-spaced light and dark areas, angular widths varying in proportion to absolute angular position. This second track provides gating and indexing signal.
Full Text Available Cellular immunity to Mycobacterium tuberculosis (Mtb requires a coordinated response between the innate and adaptive arms of the immune system, resulting in a type 1 cytokine response, which is associated with control of infection. The contribution of innate lymphocytes to immunity against Mtb remains controversial. We established an in vitro system to study this question. Interferon-gamma is produced when splenocytes from uninfected mice are cultured with Mtb-infected macrophages, and, under these conditions, bacterial replication is suppressed. This innate control of bacterial replication is dependent on CD1d-restricted invariant NKT (iNKT cells, and their activation requires CD1d expression by infected macrophages as well as IL-12 and IL-18. We show that iNKT cells, even in limiting quantities, are sufficient to restrict Mtb replication. To determine whether iNKT cells contribute to host defense against tuberculosis in vivo, we adoptively transferred iNKT cells into mice. Primary splenic iNKT cells obtained from uninfected mice significantly reduce the bacterial burden in the lungs of mice infected with virulent Mtb by the aerosol route. Thus, iNKT cells have a direct bactericidal effect, even in the absence of synthetic ligands such as alpha-galactosylceramide. Our finding that iNKT cells protect mice against aerosol Mtb infection is the first evidence that CD1d-restricted NKT cells mediate protection against Mtb in vivo.
Lu, Rui; Folimonov, Alexey; Shintaku, Michael; Li, Wan-Xiang; Falk, Bryce W.; Dawson, William O.; Ding, Shou-Wei
Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP- or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. RNA interference | citrus tristeza virus | virus synergy | antiviral immunity
Li, Liping; Tetu, Sasha G; Paulsen, Ian T; Hassan, Karl A
The core genomes of most bacterial species include a large number of genes encoding putative efflux pumps. The functional roles of most of these pumps are unknown, however, they are often under tight regulatory control and expressed in response to their substrates. Therefore, one way to identify pumps that function in antimicrobial resistance is to examine the transcriptional responses of efflux pump genes to antimicrobial shock. By conducting complete transcriptomic experiments following antimicrobial shock treatments, it may be possible to identify novel drug efflux pumps encoded in bacterial genomes. In this chapter we describe a complete workflow for conducting transcriptomic analyses by RNA sequencing, to determine transcriptional changes in bacteria responding to antimicrobials.
This paper describes a simple method for making dual beam encoded extended fractional Fourier transform (EFRT) security holograms. The hologram possesses different stages of encoding so that security features are concealed and remain invisible to the counterfeiter. These concealed and encoded anticounterfeit ...
Abstract. This paper describes a simple method for making dual beam encoded ex- tended fractional Fourier transform (EFRT) security holograms. The hologram possesses different stages of encoding so that security features are concealed and remain invisible to the counterfeiter. These concealed and encoded ...
Shields, Conor J
OBJECTIVE: This study sought to determine whether hypertonic saline (HTS) infusion modulates the host response to bacterial challenge. METHODS: Sepsis was induced in 30 Balb-C mice by intraperitoneal injection of Escherichia coli (5 x 107 organisms per animal). In 10 mice, resuscitation was performed at 0 and 24 hours with a 4 mL\\/kg bolus of HTS (7.5% NaCl), 10 animals received 4 mL\\/kg of normal saline (0.9% NaCl), and the remaining animals received 30 mL\\/kg of normal saline. Samples of blood, spleen, and lung were cultured at 8 and 36 hours. Polymorphonucleocytes were incubated in isotonic or hypertonic medium before culture with E. coli. Phagocytosis was assessed by flow cytometry, whereas intracellular bacterial killing was measured after inhibition of phagocytosis with cytochalasin B. Intracellular formation of free radicals was assessed by the molecular probe CM-H(2)DCFDA. Mitogen-activated protein (MAP) kinase p38 and ERK-1 phosphorylation, and nuclear factor kappa B (NFkappaB) activation were determined. Data are represented as means (SEM), and an analysis of variance test was performed to gauge statistical significance. RESULTS: Significantly reduced bacterial culture was observed in the animals resuscitated with HTS when compared with their NS counterparts, in blood (51.8 +\\/- 4.3 vs. 82.0 +\\/- 3.3 and 78.4 +\\/- 4.8, P = 0.005), lung (40.0 +\\/- 4.1 vs. 93.2 +\\/- 2.1 and 80.9 +\\/- 4.7, P = 0.002), and spleen (56.4 +\\/- 3.8 vs. 85.4 +\\/- 4.2 and 90.1 +\\/- 5.9, P = 0.05). Intracellular killing of bacteria increased markedly (P = 0.026) and superoxide generation was enhanced upon exposure to HTS (775.78 +\\/- 23.6 vs. 696.57 +\\/- 42.2, P = 0.017) despite inhibition of MAP kinase and NFkappaB activation. CONCLUSIONS: HTS significantly enhances intracellular killing of bacteria while attenuating receptor-mediated activation of proinflammatory cascades.
Hosni M Hassan
Full Text Available In the ancient anaerobic environment, ferrous iron (Fe2+ was one of the first metal cofactors. Oxygenation of the ancient world challenged bacteria to acquire the insoluble ferric iron (Fe3+ and later to defend against reactive oxygen species (ROS generated by the Fenton chemistry. To acquire Fe3+, bacteria produce low-molecular weight compounds, known as siderophores, which have extremely high affinity for Fe3+. However, during infection the host restricts iron from pathogens by producing iron- and siderophore-chelating proteins, by exporting iron from intracellular pathogen-containing compartments, and by limiting absorption of dietary iron. Ferric Uptake Regulator (Fur is a transcription factor which utilizes Fe2+ as a corepressor and represses siderophore synthesis in pathogens. Fur, directly or indirectly, controls expression of enzymes that protect against ROS damage. Thus, the challenges of iron homeostasis and defense against ROS are addressed via Fur. Although the role of Fur as a repressor is well documented, emerging evidence demonstrates that Fur can function as an activator. Fur activation can occur through three distinct mechanisms 1 indirectly via small RNAs, 2 binding at cis regulatory elements that enhance recruitment of the RNA polymerase holoenzyme (RNAP, and 3 functioning as an antirepressor by removing or blocking DNA binding of a repressor of transcription. In addition, Fur homologs control defense against peroxide stress (PerR and control uptake of other metals such as zinc (Zur and manganese (Mur in pathogenic bacteria. Fur family members are important for virulence within bacterial pathogens since mutants of fur, perR, or zur exhibit reduced virulence within numerous animal and plant models of infection. This review focuses on the breadth of Fur regulation in pathogenic bacteria.
Troxell, Bryan; Hassan, Hosni M
In the ancient anaerobic environment, ferrous iron (Fe(2+)) was one of the first metal cofactors. Oxygenation of the ancient world challenged bacteria to acquire the insoluble ferric iron (Fe(3+)) and later to defend against reactive oxygen species (ROS) generated by the Fenton chemistry. To acquire Fe(3+), bacteria produce low-molecular weight compounds, known as siderophores, which have extremely high affinity for Fe(3+). However, during infection the host restricts iron from pathogens by producing iron- and siderophore-chelating proteins, by exporting iron from intracellular pathogen-containing compartments, and by limiting absorption of dietary iron. Ferric Uptake Regulator (Fur) is a transcription factor which utilizes Fe(2+) as a corepressor and represses siderophore synthesis in pathogens. Fur, directly or indirectly, controls expression of enzymes that protect against ROS damage. Thus, the challenges of iron homeostasis and defense against ROS are addressed via Fur. Although the role of Fur as a repressor is well-documented, emerging evidence demonstrates that Fur can function as an activator. Fur activation can occur through three distinct mechanisms (1) indirectly via small RNAs, (2) binding at cis regulatory elements that enhance recruitment of the RNA polymerase holoenzyme (RNAP), and (3) functioning as an antirepressor by removing or blocking DNA binding of a repressor of transcription. In addition, Fur homologs control defense against peroxide stress (PerR) and control uptake of other metals such as zinc (Zur) and manganese (Mur) in pathogenic bacteria. Fur family members are important for virulence within bacterial pathogens since mutants of fur, perR, or zur exhibit reduced virulence within numerous animal and plant models of infection. This review focuses on the breadth of Fur regulation in pathogenic bacteria.
Lin, Wei [Institute of Geology and Geophysics; Bazylinski, Dennis A [Ames Laboratory; Xiao, Tian [Chinese Academy of Sciences; Wu, Long-Fei [v; Pan, Yongxin [Institute of Geology and Geophysics
Magnetotactic bacteria (MTB) are unique in their ability to synthesize intracellular nano-sized minerals of magnetite and/or greigite magnetosomes for magnetic orientation. Thus, they provide an excellent model system to investigate mechanisms of biomineralization. MTB play important roles in bulk sedimentary magnetism and have numerous versatile applications in paleoenvironmental reconstructions, and biotechnological and biomedical fields. Significant progress has been made in recent years in describing the composition of MTB communities and distribution through innovative cultivation-dependent and -independent techniques. In this review, the most recent contributions to the field of diversity and biogeography of MTB are summarized and reviewed. Emphasis is on the novel insights into various factors/processes potentially affecting MTB community distribution. An understanding of the present-day biogeography of MTB, and the ruling parameters of their spatial distribution, will eventually help us predict MTB community shifts with environmental changes and assess their roles in global iron cycling.
Southam, Gordon; Beveridge, Terrance J.
A laboratory simulation was developed to provide mechanistic information about placer (nugget) gold development in the natural environment. To initiate the simulation, ionic gold was immobilized to a high capacity by Bacillus subtilis 168 (116.2 μg/mg dry weight bacteria) as fine-grained intracellular colloids (5-50 nm). During the low-temperature diagenesis experiment (60°C), the release of organics due to bacterial autolysis coincided with the in vitro formation of hexagonal-octahedral gold crystals (20 μm). This octahedral gold was observed to aggregate, forming fine-grained placer gold (50 μm). In addition to achieving a fundamental understanding into secondary gold deposition, a significant economic benefit could be realized by employing this environmentally safe procedure to concentrate widely dispersed gold in placer deposits to facilitate mining by conventional methodologies.
Lim, Jeong-A; Shin, Hakdong; Heu, Sunggi; Ryu, Sangryeol
Concerns over drug-resistant bacteria have stimulated interest in developing alternative methods to control bacterial infections. Endolysin, a phage-encoded enzyme that breaks down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle, is reported to be effective for the control of bacterial pathogenic bacteria. Bioinformatic analysis of the SPN9CC bacteriophage genome revealed a gene that encodes an endolysin with a domain structure similar to those of the endolysins produced by the P1 and P22 coliphages. The SPN9CC endolysin was purified with a C-terminal oligo-histidine tag. The endolysin was relatively stable and active over a broad temperature range (from 24°C to 65°C). It showed maximal activity at 50°C, and its optimum pH range was from pH 7.5 to 8.5. The SPN9CC endolysin showed antimicrobial activity against only gram-negative bacteria and functioned by cutting the glycosidic bond of peptidoglycan. Interestingly, the SPN9CC endolysin could lyse intact gram-negative bacteria in the absence of EDTA as an outer membrane permeabilizer. The exogenous lytic activity of the SPN9CC endolysin makes it a potential therapeutic agent against gram-negative bacteria.
Magnetotactic bacteria (mtb) migrate along geomagnetic field lines, i.e., they behave like self-propelled magnetic compass needles. Mtb make single-magnetic-domain crystals of magnetite (Fe 3 O4) and greigite (Fe 3 S4) in intracellular structures called magnetosomes. The magnetosomes are arranged in linear chains that comprise permanent magnetic dipoles with remanent moments approaching the saturation moment, causing the mtb to be oriented in the geomagnetic field as they swim. This allows them to keep their heading and efficiently migrate to, and remain in, a preferred, microaerobic, aquatic habitat. The mtb have solved the difficult problem of designing a permanent magnet that is sufficiently robust to cause the cell to be oriented in the geomagnetic field at ambient temperature, yet fit inside a micron-sized object, and be assembled in situ from potentially toxic materials scavenged from the environment. I will describe some recent advances in mtb genetics that illuminate the process by which they make and arrange their magnetosomes.
Luo, Meng; Geng, Haiyan
We investigated the psychological mechanism of survival processing advantage from the perspective of false memory in two experiments. Using a DRM paradigm in combination with analysis based on signal detection theory, we were able to separately examine participants' utilization of verbatim representation and gist representation. Specifically, in Experiment 1, participants rated semantically related words in a survival scenario for a survival condition but rated pleasantness of words in the same DRM lists for a non-survival control condition. The results showed that participants demonstrated more gist processing in the survival condition than in the pleasantness condition; however, the degree of item-specific processing in the two encoding conditions did not significantly differ. In Experiment 2, the control task was changed to a category rating task, in which participants were asked to make category ratings of words in the category lists. We found that the survival condition involved more item-specific processing than did the category condition, but we found no significant difference between the two encoding conditions at the level of gist processing. Overall, our study demonstrates that survival processing can simultaneously promote gist and item-specific representations. When the control tasks only promoted either item-specific representation or gist representation, memory advantages of survival processing occurred.
Kerner, T.; Conkling, C. R.; Oerter, B.
The V123 Synchronous Encoder Module transmits events to distributed trigger modules and embedded decoders around the RHIC rings where they are used to provide beam instrumentation triggers [1,2,3]. The RHIC beam synchronous event link hardware is mainly comprised of three VMEbus board designs, the central input modules (V201), and encoder modules (V123), and the distributed trigger modules (V124). Two beam synchronous links, one for each ring, are distributed via fiberoptic and fanned out via twisted wire pair cables. The V123 synchronizes with the RF system clock derived from the beam bucket frequency and a revolution fiducial pulse. The RF system clock is used to create the beam synchronous event link carrier and events are synchronized with the rotation fiducial. A low jitter RF clock is later recovered from this carrier by phase lock loops in the trigger modules. Prioritized hardware and software triggers fill up to 15 beam event code transmission slots per revolution while tracking the ramping RF acceleration frequency and storage frequency. The revolution fiducial event is always the first event transmitted which is used to synchronize the firing of the abort kicker and to locate the first bucket for decoders distributed about the ring
The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga. © Woo et al.
Rogan, Mark P
Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease?
Bateman, Stacey L.; Seed, Patrick C.
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
Fejzo, Marlena Schoenberg; Myhre, Ronny; Colodro-Conde, Lucía; MacGibbon, Kimber W; Sinsheimer, Janet S; Reddy, M V Prasad Linga; Pajukanta, Päivi; Nyholt, Dale R; Wright, Margaret J; Martin, Nicholas G; Engel, Stephanie M; Medland, Sarah E; Magnus, Per; Mullin, Patrick M
Hyperemesis Gravidarum (HG), severe nausea/vomiting in pregnancy (NVP), can cause poor maternal/fetal outcomes. Genetic predisposition suggests the genetic component is essential in discovering an etiology. We performed whole-exome sequencing of 5 families followed by analysis of variants in 584 cases/431 controls. Variants in RYR2 segregated with disease in 2 families. The novel variant L3277R was not found in any case/control. The rare variant, G1886S was more common in cases (p = 0.046) and extreme cases (p = 0.023). Replication of G1886S using Norwegian/Australian data was supportive. Common variants rs790899 and rs1891246 were significantly associated with HG and weight loss. Copy-number analysis revealed a deletion in a patient. RYR2 encodes an intracellular calcium release channel involved in vomiting, cyclic-vomiting syndrome, and is a thyroid hormone target gene. Additionally, RYR2 is a downstream drug target of Inderal, used to treat HG and CVS. Thus, herein we provide genetic evidence for a pathway and therapy for HG. Copyright Â© 2016 Elsevier Ireland Ltd. All rights reserved.
Dong, Aiwu; Zhu, Yan; Yu, Yu; Cao, Kaiming; Sun, Chongrong; Shen, Wen-Hui
The nucleosome assembly protein 1 (NAP1) is considered to be a conserved histone chaperone, facilitating the assembly of nucleosomes in all eukaryotes. However, studies in yeast and animal cells also indicated that NAP1 proteins have diverse functions likely independent of nucleosome-assembly activity. Here, we describe the isolation and characterization of cDNAs encoding NAP1-like proteins from the monocotyledon rice ( Oryza sativa L.) and the dicotyledon tobacco ( Nicotiana tabacum L.). Northern-blot analysis demonstrated that the two rice NAP1-like genes are predominantly expressed in stem tissues such as root and shoot apical meristems as well as in young flowers. During the cell cycle, all four tobacco NAP1-like genes are highly expressed, with one of them showing a slightly increased expression at the G1/S transition. These results are consistent with a role for plant NAP1-like proteins in cell division. In vitro binding assays revealed that different NAP1-like proteins bind, with distinct relative binding strengths, to different classes of histone. Intracellular localization analyses showed that some NAP1-like proteins could be targeted into the nucleus whereas others are exclusively cytoplasm-localized. It is thus likely that different plant NAP1-like proteins have distinct functions in vivo. Plant NAP1-like proteins were observed to concentrate around the metaphase plate and in the phragmoplast, suggesting a role in mitotic events and cytokinesis.
Yu, Xingang; Abdullahi, Auwalu Yusuf; Wu, Sheng; Pan, Weida; Shi, Xianli; Hu, Wei; Tan, Liping; Li, Kangxin; Wang, Zhen; Li, Guoqing
To study prokaryotic expression and subcellular localization of α -13 giardin in Giardia lamblia trophozoites, α -13 giardin gene was amplified and cloned into prokaryotic expression vector pET-28a(+). The positive recombinant plasmid was transformed into E. coli BL21(DE3) for expression by using IPTG and autoinduction expression system (ZYM-5052). The target protein was validated by SDS-PAGE and Western blotting and purified by Ni-NTA Resin. Rabbits were immunized with purified fusion proteins for preparation of polyclonal antibody; then the intracellular location of α -13 giardin was determined by fluorescence immunoassay. The results showed that the length of α -13 giardin gene was 1038 bp, encoding a polypeptide of 345 amino acids. The expressed product was a fusion protein with about 40 kDa largely present in soluble form. The target protein accounted for 21.0% of total proteins after being induced with IPTG, while it accounted for 28.8% with ZYM-5052. The anti- α 13-giardin polyclonal antibody possessed good antigenic specificity as well as excellent binding activity with recombinant α -13 giardin. Immunofluorescence assays revealed that α -13 giardin was localized in the cytoplasm of G. lamblia trophozoite, suggesting that it is a cytoplasm-associated protein. The present study may lay a foundation for further functional research on α -13 giardin of G. lamblia .
Lee, S-K; Lee, K-E; Jeong, T-S; Hwang, Y-H; Kim, S; Hu, J C-C; Simmer, J P; Kim, J-W
Mutations in a family with sequence similarity 83 member H (FAM83H) cause autosomal-dominant hypocalcification amelogenesis imperfecta (ADH CAI). All FAM83H ADHCAI-causing mutations terminate translation or shift the reading frame within the specific exon 5 segment that encodes from Ser(287) to Glu(694). Mutations near Glu(694) cause a milder, more localized phenotype. We identified disease-causing FAM83H mutations in two families with ADHCAI: family 1 (g.3115C>T, c.1993 C>T, p.Q665X) and family 2 (g.3151C>T, c.2029 C>T, p.Q677X). We also tested the hypothesis that truncation mutations alter the intracellular localization of FAM83H. Wild-type FAM83H and p.E694X mutant FAM83H fused to green fluorescent protein (GFP) localized in the cytoplasm of HEK293T cells, but the mutant FAM83H proteins (p.R325X, p.W460X, and p.Q677X) fused to GFP localized mainly in the nucleus with slight expression in the cytoplasm. We conclude that nuclear targeting of the truncated FAM83H protein contributes to the severe, generalized enamel phenotype.
Bateman, Stacey L; Seed, Patrick C
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.
Reinoso, Claudia A.; Auger, Christopher; Appanna, Vasu D.; Vásquez, Claudio C.
Highlights: ► Tellurite-exposed E. coli exhibits decreased α-KG dehydrogenase activity. ► Cells lacking α-KGDH genes are more sensitive to ROS than isogenic, wt E. coli. ► KG accumulation may serve to face tellurite-mediated oxidative damage in E. coli. -- Abstract: The tellurium oxyanion tellurite is toxic to most organisms because of its ability to generate oxidative stress. However, the detailed mechanism(s) how this toxicant interferes with cellular processes have yet to be fully understood. As part of our effort to decipher the molecular interactions of tellurite with living systems, we have evaluated the global metabolism of α-ketoglutarate a known antioxidant in Escherichia coli. Tellurite-exposed cells displayed reduced activity of the KG dehydrogenase complex (KGDHc), resulting in increased intracellular KG content. This complex’s reduced activity seems to be due to decreased transcription in the stressed cells of sucA, a gene that encodes the E1 component of KGDHc. Furthermore, it was demonstrated that the increase in total reactive oxygen species and superoxide observed upon tellurite exposure was more evident in wild type cells than in E. coli with impaired KGDHc activity. These results indicate that KG may be playing a pivotal role in combating tellurite-mediated oxidative damage.
Mavromatis, K.; Kuyler Doyle, C.; Lykidis, A.; Ivanova, N.; Francino, P.; Chain, P.; Shin, M.; Malfatti, S.; Larimer, F.; Copeland,A.; Detter, J.C.; Land, M.; Richardson, P.M.; Yu, X.J.; Walker, D.H.; McBride, J.W.; Kyrpides, N.C.
Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, a-proteobacterium is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, and 17 putative pseudogenes, and a substantial proportion of non-coding sequence (27 percent). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences, and a unique serine-threonine bias associated with the potential for O-glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly G:C tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Proteins associated with pathogen-host interactions were identified including a small group of proteins (12) with tandem repeats and another with eukaryotic-like ankyrin domains (7).
Parisi, Maria-Giovanna; Li, Hui; Jouvet, Lionel B P; Dyrynda, Elisabeth A; Parrinello, Nicolo; Cammarata, Matteo; Roch, Philippe
Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12h post-injection for Micrococcus lysodeikticus, 24h for Vibrio splendidus and more than 48 h for Vibrio anguillarum. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with -66% 9h post-injection of living bacteria and -56% 3h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (+90% at 12h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides.
Li, T. T.; Qu, A.; Yuan, X. N.; Tan, F. X.; Li, X. W.; Wang, T.; Zhang, L. H.
Moderate halophilic bacteria are of halophilic bacteria whose suitable growth of NaCl is 5-10%. When the moderate halophilic bacteria response to high osmotic stress, the intracellular will synthesize small organic molecule compatible solutes. Ectoine, which is the major synthetic osmotic compatible solutes for moderate halophilic bacteria, can help microbial enzymes, nucleic acids and the whole cell resist to hypertonic, high temperature, freezing and other inverse environment. In order to increase the Ectoine production of Moderate halophilic bacteria Halomonas sp. H02, the Ectoine fermentation medium component was optimized by Plackett-Burman (PB) and Response Surface Methodology (RSM) based on the principle of non-complete equilibrium The results of PB experiments showed that the three main influencing factors of Moderate halophilic bacteria Halomonas sp. H02 synthesis Ectoine culture medium were C5H8NNaO4 concentration, NaCl concentration and initial pH. According to the center point of the steepest climbing experiment, the central combination design experiment was used to show that the model is consistent with the actual situation. The optimum combination of three influencing factors were C5H8NNaO4 41 g/L, NaCl 87.2 g/L and initial pH 5.9, and the predicted amount of Ectoine was 1835.8 mg/L, increased by 41.6%.
A Al-Mariri; R Akel; AQ Abbady
Brucella species are facultative intracellular gram-negative bacteria that can multiply within phagocytic and non-phagocytic cells of humans or animals as end hosts. B. melitensis causes abortion in pregnant animals and undulant fever in humans. A 41 kDa surface protein (sp41) is associated with bacterial adherence and invasion of HeLa cells. The role of this protein a is important for the interaction with host cells. Previously, the putative periplasmic binding protein p39 had been described...
Glaasker, E; Konings, W.N; Poolman, B.
Intracellular pH in bacteria can be measured efficiently between internal pH values of 6.5 and 8.5 with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and-6]-carboxyfluorescein (BCECF). A new fluorescent pH probe with a lower pK(a)(app) than BCECF was synthesized from fluorescein
Knudsen, J; Faergeman, N J; Skøtt, H
Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates. We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2. Both proteins contain 86 amino acid residues and are identical except for four conservative substitut...... resulted in a significant expansion of the intracellular acyl-CoA pool. Finally, Southern-blotting analysis of the two genes encoding ACBP types 1 and 2 in S. carlsbergensis strongly indicated that this species is a hybrid between S. cerevisiae and Saccharomyces monacensis....
Salerian, Alen J
"Human body may produce bacteria" proposes that human body may produce bacteria and represent an independent source of infections contrary to the current paradigm of infectious disorders proposed by Louis Pasteur in 1880. The following observations are consistent with this hypothesis: A. Bidirectional transformations of both living and nonliving things have been commonly observed in nature. B. Complex multicellular organisms harbor the necessary properties to produce bacteria (water, nitrogen and oxygen). C. Physical laws suggest any previously observed phenomenon or action will occur again (life began on earth; a non living thing). D. Animal muscle cells may generate energy (fermentation). E. Sterilized food products (i.e. boiled eggs), may produce bacteria and fungus under special conditions and without any exposure to foreign living cells. "Human body may produce bacteria" may challenge the current medical paradigm that views human infectious disorders as the exclusive causative byproducts of invading foreign cells. It may also introduce new avenues to treat infectious disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.