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Sample records for chlamydial outer membrane

  1. Humoral immune responses in koalas (Phascolarctos cinereus) either naturally infected with Chlamydia pecorum or following administration of a recombinant chlamydial major outer membrane protein vaccine.

    Science.gov (United States)

    Khan, Shahneaz Ali; Polkinghorne, Adam; Waugh, Courtney; Hanger, Jon; Loader, Jo; Beagley, Kenneth; Timms, Peter

    2016-02-03

    The development of a vaccine is a key strategy to combat the widespread and debilitating effects of chlamydial infection in koalas. One such vaccine in development uses recombinant chlamydial major outer membrane protein (rMOMP) as an antigen and has shown promising results in several koala trials. Previous chlamydial vaccine studies, primarily in the mouse model, suggest that both cell-mediated and antibody responses will be required for adequate protection. Recently, the important protective role of antibodies has been highlighted. In our current study, we conducted a detailed analysis of the antibody-mediated immune response in koalas that are either (a) naturally-infected, and/or (b) had received an rMOMP vaccine. Firstly, we observed that naturally-infected koalas had very low levels of Chlamydia pecorum-specific neutralising antibodies. A strong correlation between low IgG total titers/neutralising antibody levels, and higher C. pecorum infection load was also observed in these naturally-infected animals. In vaccinated koalas, we showed that the vaccine was able to boost the humoral immune response by inducing strong levels of C. pecorum-specific neutralising antibodies. A detailed characterisation of the MOMP epitope response was also performed in naturally-infected and vaccinated koalas using a PepScan epitope approach. This analysis identified unique sets of MOMP epitope antibodies between naturally-infected non-protected and diseased koalas, versus vaccinated koalas, with the latter group of animals producing a unique set of specific epitope-directed antibodies that we demonstrated were responsible for the in vitro neutralisation activity. Together, these results show the importance of antibodies in chlamydial infection and immunity following vaccination in the koala.

  2. Recombinant outer membrane vesicles carrying Chlamydia muridarum HtrA induce antibodies that neutralize chlamydial infection in vitro

    OpenAIRE

    Bartolini, Erika; Ianni, Elvira; Frigimelica, Elisabetta; Petracca, Roberto; Galli, Giuliano; Berlanda Scorza, Francesco; Norais, Nathalie; Laera, Donatello; Giusti, Fabiola; Pierleoni, Andrea; Donati, Manuela; Cevenini, Roberto; Finco, Oretta; Grandi, Guido; Grifantini, Renata

    2013-01-01

    Background: Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent v...

  3. Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens.

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    Karin Aistleitner

    Full Text Available The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydiaouter membrane proteins, PomS (pc1489 and PomT (pc1077, are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts.

  4. Liposome delivery of Chlamydia muridarum major outer membrane protein primes a Th1 response that protects against genital chlamydial infection in a mouse model

    DEFF Research Database (Denmark)

    Hansen, Jon; Jensen, Klaus Thorleif; Follmann, Frank;

    2008-01-01

    ) received a genital inoculation of 1.5 x 10(5) inclusion-forming units of C. muridarum. The role played by CD4(+) T cells in MOMP/CAF01-raised immunity was investigated by depleting CD4(+) T cells in vaccinated mice, and antigen conformation dependence was evaluated by vaccination with recombinant MOMP....... RESULTS: Mice vaccinated with MOMP/alum displayed a strong anti-MOMP humoral response with high IgG1 titers, low levels of IFN-gamma and tumor necrosis factor (TNF)-alpha, and only a slight reduction in chlamydial load. Mice vaccinated with MOMP/CAF01 displayed high titers of IgG2b, IFN-gamma, and TNF...

  5. The leptospiral outer membrane.

    Science.gov (United States)

    Haake, David A; Zückert, Wolfram R

    2015-01-01

    The outer membrane (OM) is the front line of leptospiral interactions with their environment and the mammalian host. Unlike most invasive spirochetes, pathogenic leptospires must be able to survive in both free-living and host-adapted states. As organisms move from one set of environmental conditions to another, the OM must cope with a series of conflicting challenges. For example, the OM must be porous enough to allow nutrient uptake, yet robust enough to defend the cell against noxious substances. In the host, the OM presents a surface decorated with adhesins and receptors for attaching to, and acquiring, desirable host molecules such as the complement regulator, Factor H.Factor H. On the other hand, the OM must enable leptospires to evade detection by the host's immune system on their way from sites of invasion through the bloodstream to the protected niche of the proximal tubule. The picture that is emerging of the leptospiral OM is that, while it shares many of the characteristics of the OMs of spirochetes and Gram-negative bacteria, it is also unique and different in ways that make it of general interest to microbiologists. For example, unlike most other pathogenic spirochetes, the leptospiral OM is rich in lipopolysaccharide (LPS). Leptospiral LPS is similar to that of Gram-negative bacteria but has a number of unique structural features that may explain why it is not recognized by the LPS-specific Toll-like receptor 4 of humans. As in other spirochetes, lipoproteins are major components of the leptospiral OM, though their roles are poorly understood. The functions of transmembrane outer membrane proteins (OMPs) in many cases are better understood, thanks to homologies with their Gram-negative counterparts and the emergence of improved genetic techniques. This chapter will review recent discoveries involving the leptospiral OM and its role in leptospiral physiology and pathogenesis.

  6. Identification of Chlamydia trachomatis outer membrane complex proteins by differential proteomics.

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    Liu, Xiaoyun; Afrane, Mary; Clemmer, David E; Zhong, Guangming; Nelson, David E

    2010-06-01

    The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.

  7. Small RNAs controlling outer membrane porins

    DEFF Research Database (Denmark)

    Valentin-Hansen, Poul; Johansen, Jesper; Rasmussen, Anders A

    2007-01-01

    Gene regulation by small non-coding RNAs has been recognized as an important post-transcriptional regulatory mechanism for several years. In Gram-negative bacteria such as Escherichia coli and Salmonella, these RNAs control stress response and translation of outer membrane proteins and therefore...... are key regulators of environmental stress. Recent work has revealed an intimate interplay between small RNA regulation of outer membrane proteins and the stress-induced sigmaE-signalling system, which has an essential role in the maintenance of the integrity of the outer membrane....

  8. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

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    McClafferty Heather

    2005-01-01

    Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

  9. Loss of elongation factor P disrupts bacterial outer membrane integrity

    DEFF Research Database (Denmark)

    Zou, S Betty; Hersch, Steven J; Roy, Hervé;

    2012-01-01

    increased uptake of the hydrophobic dye 1-N-phenylnaphthylamine (NPN). Analysis of the membrane proteomes of wild-type and efp mutant Salmonella strains reveals few changes, including the prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant...... overexpression of an outer membrane porin....

  10. BACTERIAL OUTER MEMBRANE VESICLES AND VACCINE APPLICATIONS

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    Reinaldo eAcevedo

    2014-03-01

    Full Text Available Vaccines based on outer membrane vesicles (OMV were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of self meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA, serogroup W (dOMVW and serogroup X (dOMVX were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC, Bordetella pertussis (dOMVBP, Mycobacterium smegmatis (dOMVSM and BCG (dOMVBCG. The immunogenicity of the OMV have been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice have shown their protective potential. dOMVB has been evaluated with non-self neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates.

  11. Assembly of outer-membrane proteins in bacteria and mitochondria.

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    Tommassen, Jan

    2010-09-01

    The cell envelope of Gram-negative bacteria consists of two membranes separated by the periplasm. In contrast with most integral membrane proteins, which span the membrane in the form of hydrophobic alpha-helices, integral outer-membrane proteins (OMPs) form beta-barrels. Similar beta-barrel proteins are found in the outer membranes of mitochondria and chloroplasts, probably reflecting the endosymbiont origin of these eukaryotic cell organelles. How these beta-barrel proteins are assembled into the outer membrane has remained enigmatic for a long time. In recent years, much progress has been reached in this field by the identification of the components of the OMP assembly machinery. The central component of this machinery, called Omp85 or BamA, is an essential and highly conserved bacterial protein that recognizes a signature sequence at the C terminus of its substrate OMPs. A homologue of this protein is also found in mitochondria, where it is required for the assembly of beta-barrel proteins into the outer membrane as well. Although accessory components of the machineries are different between bacteria and mitochondria, a mitochondrial beta-barrel OMP can be assembled into the bacterial outer membrane and, vice versa, bacterial OMPs expressed in yeast are assembled into the mitochondrial outer membrane. These observations indicate that the basic mechanism of OMP assembly is evolutionarily highly conserved.

  12. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

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    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.

  13. Structural basis for lipopolysaccharide insertion in the bacterial outer membrane.

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    Qiao, Shuai; Luo, Qingshan; Zhao, Yan; Zhang, Xuejun Cai; Huang, Yihua

    2014-07-03

    One of the fundamental properties of biological membranes is the asymmetric distribution of membrane lipids. In Gram-negative bacteria, the outer leaflet of the outer membrane is composed predominantly of lipopolysaccharides (LPS). The export of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane, through the periplasm to the surface. Of the seven Lpt proteins, the LptD-LptE complex is responsible for inserting LPS into the external leaflet of the outer membrane. Here we report the crystal structure of the ∼110-kilodalton membrane protein complex LptD-LptE from Shigella flexneri at 2.4 Å resolution. The structure reveals an unprecedented two-protein plug-and-barrel architecture with LptE embedded into a 26-stranded β-barrel formed by LptD. Importantly, the secondary structures of the first two β-strands are distorted by two proline residues, weakening their interactions with neighbouring β-strands and creating a potential portal on the barrel wall that could allow lateral diffusion of LPS into the outer membrane. The crystal structure of the LptD-LptE complex opens the door to new antibiotic strategies targeting the bacterial outer membrane.

  14. Dissecting Escherichia coli outer membrane biogenesis using differential proteomics.

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    Alessandra M Martorana

    Full Text Available The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality.

  15. Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts

    Science.gov (United States)

    1985-01-01

    We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes. PMID:3988805

  16. A progenitor of the outer membrane LamB trimer.

    OpenAIRE

    Stader, J.; Silhavy, T J

    1988-01-01

    During its localization to the outer membrane, LamB possesses distinctive biochemical properties as it passes through the cytoplasmic membrane. Because LamB entered this dynamic state with an attached signal sequence and leaves after cleavage, we call this export-related form of LamB the early-translocation form (et-LamB).

  17. An efficient depyrogenation method for recombinant bacterial outer membrane lipoproteins.

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    Basto, Afonso P; Morais, Joana; Marcelino, Eduardo; Leitão, Alexandre; Santos, Dulce M

    2014-06-01

    Bacterial outer membrane lipoproteins are anchored in the outer membrane lipid layer in close association with lipopolysaccharides (LPS) and with other hydrophobic membrane proteins, making their purification technically challenging. We have previously shown that a thorough delipidation of outer membrane preparations from the Escherichia coli expression host is an important step to eliminate contaminant proteins when purifying recombinant antigens expressed in fusion with the Pseudomonas aeruginosa OprI lipoprotein. Here we report the cloning and expression of three antigens in fusion with OprI (ovalbumin, eGFP and BbPDI) and our efforts to deal with the variable LPS contamination levels observed in different batches of purified lipoproteins. The use of polymyxin B columns or endotoxin removal polycationic magnetic beads for depyrogenation of purified lipoproteins resulted in high protein losses and the use of Triton X-114 or sodium deoxycholate during the course of affinity chromatography showed to be ineffective to reduce LPS contamination. Instead, performing a hot phenol/water LPS extraction from outer membrane preparations prior to metal affinity chromatography allowed the purification of the recombinant fusion lipoproteins with LPS contents below 0.02EU/μg of protein. The purified recombinant lipoproteins retain their capacity to stimulate bone marrow-derived dendritic cells allowing for the study of their immunomodulatory properties through TLR2/1. This is a simple and easy to scale up method that can also be considered for the purification of other outer membrane lipoproteins.

  18. Simulations of outer membrane channels and their permeability.

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    Pothula, Karunakar R; Solano, Carlos J F; Kleinekathöfer, Ulrich

    2016-07-01

    Channels in the outer membrane of Gram-negative bacteria provide essential pathways for the controlled and unidirectional transport of ions, nutrients and metabolites into the cell. At the same time the outer membrane serves as a physical barrier for the penetration of noxious substances such as antibiotics into the bacteria. Most antibiotics have to pass through these membrane channels to either reach cytoplasmic bound targets or to further cross the hydrophobic inner membrane. Considering the pharmaceutical significance of antibiotics, understanding the functional role and mechanism of these channels is of fundamental importance in developing strategies to design new drugs with enhanced permeation abilities. Due to the biological complexity of membrane channels and experimental limitations, computer simulations have proven to be a powerful tool to investigate the structure, dynamics and interactions of membrane channels. Considerable progress has been made in computer simulations of membrane channels during the last decade. The goal of this review is to provide an overview of the computational techniques and their roles in modeling the transport across outer membrane channels. A special emphasis is put on all-atom molecular dynamics simulations employed to better understand the transport of molecules. Moreover, recent molecular simulations of ion, substrate and antibiotics translocation through membrane pores are briefly summarized. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

  19. Substrate specificity within a family of outer membrane carboxylate channels.

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    Elif Eren

    2012-01-01

    Full Text Available Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

  20. E. coli outer membrane and interactions with OmpLA.

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    Wu, Emilia L; Fleming, Patrick J; Yeom, Min Sun; Widmalm, Göran; Klauda, Jeffery B; Fleming, Karen G; Im, Wonpil

    2014-06-03

    The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.

  1. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly.

    Science.gov (United States)

    Knowles, Timothy J; Scott-Tucker, Anthony; Overduin, Michael; Henderson, Ian R

    2009-03-01

    The folding of transmembrane proteins into the outer membrane presents formidable challenges to Gram-negative bacteria. These proteins must migrate from the cytoplasm, through the inner membrane and into the periplasm, before being recognized by the beta-barrel assembly machinery, which mediates efficient insertion of folded beta-barrels into the outer membrane. Recent discoveries of component structures and accessory interactions of this complex are yielding insights into how cells fold membrane proteins. Here, we discuss how these structures illuminate the mechanisms responsible for the biogenesis of outer membrane proteins.

  2. FABRICATION AND BIOCOMPATIBILITY OF CELL OUTER MEMBRANE MIMETIC SURFACES

    Institute of Scientific and Technical Information of China (English)

    Ming-ming Zong; Yong-kuan Gong

    2011-01-01

    The surface design used for improving biocompatibility is one of the most important issues for the fabrication of medical devices. For mimicking the ideal surface structure of cell outer membrane, a large number of polymers bearing phosphorylcholine (PC) groups have been employed to modify the surfaces of biomaterials and medical devices. It has been demonstrated that the biocompatibility of the modified materials whose surface is required to interact with a living organism has been obviously improved by introducing PC groups. In this review, the fabrication strategies of cell outer membrane mimetic surfaces and their resulted biocompatibilities were summarized.

  3. Outer Membrane Vesicle Production Facilitates LPS Remodeling and Outer Membrane Maintenance in Salmonella during Environmental Transitions

    Science.gov (United States)

    Bonnington, Katherine E.

    2016-01-01

    ABSTRACT The ability of Gram-negative bacteria to carefully modulate outer membrane (OM) composition is essential to their survival. However, the asymmetric and heterogeneous structure of the Gram-negative OM poses unique challenges to the cell’s successful adaption to rapid environmental transitions. Although mechanisms to recycle and degrade OM phospholipid material exist, there is no known mechanism by which to remove unfavorable lipopolysaccharide (LPS) glycoforms, except slow dilution through cell growth. As all Gram-negative bacteria constitutively shed OM vesicles (OMVs), we propose that cells may utilize OMV formation as a way to selectively remove environmentally disadvantageous LPS species. We examined the native kinetics of OM composition during physiologically relevant environmental changes in Salmonella enterica, a well-characterized model system for activation of PhoP/Q and PmrA/B two-component systems (TCSs). In response to acidic pH, toxic metals, antimicrobial peptides, and lack of divalent cations, these TCSs modify the LPS lipid A and core, lengthen the O antigen, and upregulate specific OM proteins. An environmental change to PhoP/Q- and PmrA/B-activating conditions simultaneously induced the addition of modified species of LPS to the OM, downregulation of previously dominant species of LPS, greater OMV production, and increased OMV diameter. Comparison of the relative abundance of lipid A species present in the OM and the newly budded OMVs following two sets of rapid environmental shifts revealed the retention of lipid A species with modified phosphate moieties in the OM concomitant with the selective loss of palmitoylated species via vesiculation following exposure to moderately acidic environmental conditions. PMID:27795394

  4. Vaccination of koalas with a recombinant Chlamydia pecorum major outer membrane protein induces antibodies of different specificity compared to those following a natural live infection.

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    Kollipara, Avinash; Polkinghorne, Adam; Beagley, Kenneth W; Timms, Peter

    2013-01-01

    Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity, infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas (particularly blindness and infertility in females) would provide an important management tool to prevent further population decline of this species. An important step towards developing a vaccine in koalas is to understand the host immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cell epitopes across the Major Outer Membrane Protein (MOMP) of four C. pecorum strains/genotypes that are recognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasma antibodies from the koalas naturally infected with a C. pecorum G genotype strain recognised the epitopes located in the variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from an animal infected with a C. pecorum F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not from other genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein they produced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infected koalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserved domains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer wide cross-protection against the variety of chlamydial infections circulating in wild koala populations.

  5. Identification of outer membrane proteins of Yersinia pestis through biotinylation

    NARCIS (Netherlands)

    Smither, S.J.; Hill, J.; Baar, B.L.M. van; Hulst, A.G.; Jong, A.L. de; Titball, R.W.

    2007-01-01

    The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with

  6. LPS Remodeling Triggers Formation of Outer Membrane Vesicles in Salmonella

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    Elhenawy, Wael; Bording-Jorgensen, Michael; Valguarnera, Ezequiel; Haurat, M. Florencia; Wine, Eytan

    2016-01-01

    ABSTRACT Outer membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employing Salmonella enterica serovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in a pagL mutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane. PMID:27406567

  7. Structural basis for alginate secretion across the bacterial outer membrane

    Energy Technology Data Exchange (ETDEWEB)

    Whitney, J.C.; Robinson, H.; Hay, I. D.; Li, C.; Eckford, P. D. W.; Amaya, M. F.; Wood, L. F.; Ohman, D. E.; Bear, C. E.; Rehm, B. H.; Howell, P. L.

    2011-08-09

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  8. Structural Basis for Alginate Secretion Across the Bacterial Outer Membrane

    Energy Technology Data Exchange (ETDEWEB)

    J Whitney; I Hay; C Li; P Eckford; H Robinson; M Amaya; L Wood; D Ohman; C Bear; et al.

    2011-12-31

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  9. Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles.

    Science.gov (United States)

    Roier, Sandro; Fenninger, Judith C; Leitner, Deborah R; Rechberger, Gerald N; Reidl, Joachim; Schild, Stefan

    2013-07-01

    Pasteurella multocida is able to cause disease in humans and in a wide range of animal hosts, including fowl cholera in birds, atrophic rhinitis in pigs, and snuffles in rabbits. Together with Mannheimia haemolytica, P. multocida also represents a major bacterial causative agent of bovine respiratory disease (BRD), which is one of the most important causes for economic losses for the cattle backgrounding and feedlot industry. Commercially available vaccines only partially prevent infections caused by P. multocida and M. haemolytica. Thus, this study characterized the immunogenicity of P. multocida and M. haemolytica outer membrane vesicles (OMVs) upon intranasal immunization of BALB/c mice. Enzyme-linked immunosorbent assays (ELISA) revealed that OMVs derived from P. multocida or M. haemolytica are able to induce robust humoral and mucosal immune responses against the respective donor strain. In addition, also significant cross-immunogenic potential was observed for both OMV types. Colonization studies showed that a potential protective immune response against P. multocida is not only achieved by immunization with P. multocida OMVs, but also by immunization with OMVs derived from M. haemolytica. Immunoblot and immunoprecipitation analyses demonstrated that M. haemolytica OMVs induce a more complex immune response compared to P. multocida OMVs. The outer membrane proteins OmpA, OmpH, and P6 were identified as the three major immunogenic proteins of P. multocida OMVs. Amongst others, the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA were found to be the most important antigens of M. haemolytica OMVs. These findings are useful for the future development of broad-spectrum OMV based vaccines against BRD and other infections caused by P. multocida or M. haemolytica.

  10. Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence.

    Science.gov (United States)

    Friedrich, V; Gruber, C; Nimeth, I; Pabinger, S; Sekot, G; Posch, G; Altmann, F; Messner, P; Andrukhov, O; Schäffer, C

    2015-12-01

    Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA-2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum-free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S-layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C-terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O-glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro-inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration-dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential.

  11. Protective role of E. coli outer membrane vesicles against antibiotics.

    Science.gov (United States)

    Kulkarni, Heramb M; Nagaraj, R; Jagannadham, Medicharla V

    2015-12-01

    The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics.

  12. Assembly of β-barrel proteins into bacterial outer membranes.

    Science.gov (United States)

    Selkrig, Joel; Leyton, Denisse L; Webb, Chaille T; Lithgow, Trevor

    2014-08-01

    Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of β-barrel protein assembly into membranes, and the components of the β-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

  13. Temperate bacteriophages collected by outer membrane vesicles in Komagataeibacter intermedius.

    Science.gov (United States)

    Kharina, Alla; Podolich, Olga; Faidiuk, Iuliia; Zaika, Sergiy; Haidak, Andriy; Kukharenko, Olga; Zaets, Iryna; Tovkach, Fedor; Reva, Oleg; Kremenskoy, Maxim; Kozyrovska, Natalia

    2015-04-01

    The acetic acid bacteria have mainly relevance for bacterial cellulose production and fermented bio-products manufacture. The purpose of this study was to identify temperate bacteriophages in a cellulose-producing bacterial strain Komagataeibacter intermedius IMBG180. Prophages from K. intermedius IMBG180 were induced with mitomycin C and nalidixic acid. Transmission electron microscopy analysis exhibited tailed bacteriophages belonging to Myoviridae. A PCR assay targeting the capsid gene of the myoviruses proved phylogenetic position of induced phages. Nalidixic acid was poor inducer of prophages, however, it induced the OMV-like particles release. Size of OMVs depended on an antibiotic applied for phage induction and varied in the range of 30-80 and 120-200 nm. Inside some of them, tails of phages have been visible. Under conditions, inducing prophages, OMVs acted as the collectors of formed phage particles, using outer membrane receptors for phage detection (in this case, outer membrane siderophore receptor), and fulfilled therefore "a cleaning," as well as defensive functions, preventing bacteriophage spread outside population. This is the first description of myoviruses affiliated to K. intermedius, as well as outer membrane vesicles interaction with phages within this host.

  14. TonB-dependent outer membrane transport: going for Baroque?

    Science.gov (United States)

    Wiener, Michael C

    2005-08-01

    The import of essential organometallic micronutrients (such as iron-siderophores and vitamin B(12)) across the outer membrane of Gram-negative bacteria proceeds via TonB-dependent outer membrane transporters (TBDTs). The TBDT couples to the TonB protein, which is part of a multiprotein complex in the plasma (inner) membrane. Five crystal structures of TBDTs illustrate clearly the architecture of the protein in energy-independent substrate-free and substrate-bound states. In each of the TBDT structures, an N-terminal hatch (or plug or cork) domain occludes the lumen of a 22-stranded beta barrel. The manner by which substrate passes through the transporter (the "hatch-barrel problem") is currently unknown. Solution NMR and X-ray crystallographic structures of various TonB domains indicate a striking structural plasticity of this protein. Thermodynamic, biochemical and bacteriological studies of TonB and TBDTs indicate further that existing structures do not yet capture critical energy-dependent and in vivo conformations of the transport cycle. The reconciliation of structural and non-structural experimental data, and the unambiguous experimental elucidation of a detailed molecular mechanism of transport are current challenges for this field.

  15. Characterization and Immunogenicity of Outer Membrane Vesicles from Brucella abortus.

    Science.gov (United States)

    Kaur, Gagandeep; Singh, Satparkash; Sunil Kumar, B V; Mahajan, Kanika; Verma, Ramneek

    2016-01-01

    Bovine brucellosis is a worldwide spread zoonotic disease. The objectives of this study were characterization of outer membrane vesicles from B. abortus and to evaluate their immunogenicity in mice. For this purpose, OMVs were derived from B. abortus strain 99 using ultracentrifugation method. Isolated OMVs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transmission electron microscopy which revealed spherical 20-300 nm structures rich in proteins. OMVs also showed immuno-reactivity with mice antisera in Western blot. Further, indirect ELISA showed specific and high-titer immune responses against the antigens present in OMVs suggesting their potential for a safe acellular vaccine candidate.

  16. Genetic diversity of Chlamydia pecorum strains in wild koala locations across Australia and the implications for a recombinant C. pecorum major outer membrane protein based vaccine.

    Science.gov (United States)

    Kollipara, Avinash; Polkinghorne, Adam; Wan, Charles; Kanyoka, Pride; Hanger, Jon; Loader, Joanne; Callaghan, John; Bell, Alicia; Ellis, William; Fitzgibbon, Sean; Melzer, Alistar; Beagley, Kenneth; Timms, Peter

    2013-12-27

    The long term survival of the koala (Phascolarctos cinereus) is at risk due to a range of threatening processes. A major contributing factor is disease caused by infection with Chlamydia pecorum, which has been detected in most mainland koala populations and is associated with ocular and genital tract infections. A critical aspect for the development of vaccines against koala chlamydial infections is a thorough understanding of the prevalence and strain diversity of C. pecorum infections across wild populations. In this study, we describe the largest survey (403 koalas from eight wild populations and three wildlife hospitals) examining the diversity of C. pecorum infections. 181 of the 403 koalas tested (45%) positive for C. pecorum by species-specific quantitative PCR with infection rates ranging from 20% to 61% in the eight wild populations sampled. The ompA gene, which encodes the chlamydial major outer membrane protein (MOMP), has been the major target of several chlamydial vaccines. Based on our analysis of the diversity of MOMP amino types in the infected koalas, we conclude that, (a) there exists significant diversity of C. pecorum strains in koalas, with 10 distinct, full length C. pecorum MOMP amino types identified in the 11 koala locations sampled, (b) despite this diversity, there are predicted T and B cell epitopes in both conserved as well as variable domains of MOMP which suggest cross-amino type immune responses, and (c) a recombinant MOMP-based vaccine consisting of MOMP "F" could potentially induce heterotypic protection against a range of C. pecorum strains.

  17. HHomp—prediction and classification of outer membrane proteins

    Science.gov (United States)

    Remmert, Michael; Linke, Dirk; Lupas, Andrei N.; Söding, Johannes

    2009-01-01

    Outer membrane proteins (OMPs) are the transmembrane proteins found in the outer membranes of Gram-negative bacteria, mitochondria and plastids. Most prediction methods have focused on analogous features, such as alternating hydrophobicity patterns. Here, we start from the observation that almost all β-barrel OMPs are related by common ancestry. We identify proteins as OMPs by detecting their homologous relationships to known OMPs using sequence similarity. Given an input sequence, HHomp builds a profile hidden Markov model (HMM) and compares it with an OMP database by pairwise HMM comparison, integrating OMP predictions by PROFtmb. A crucial ingredient is the OMP database, which contains profile HMMs for over 20 000 putative OMP sequences. These were collected with the exhaustive, transitive homology detection method HHsenser, starting from 23 representative OMPs in the PDB database. In a benchmark on TransportDB, HHomp detects 63.5% of the true positives before including the first false positive. This is 70% more than PROFtmb, four times more than BOMP and 10 times more than TMB-Hunt. In Escherichia coli, HHomp identifies 57 out of 59 known OMPs and correctly assigns them to their functional subgroups. HHomp can be accessed at http://toolkit.tuebingen.mpg.de/hhomp. PMID:19429691

  18. HHomp--prediction and classification of outer membrane proteins.

    Science.gov (United States)

    Remmert, Michael; Linke, Dirk; Lupas, Andrei N; Söding, Johannes

    2009-07-01

    Outer membrane proteins (OMPs) are the transmembrane proteins found in the outer membranes of Gram-negative bacteria, mitochondria and plastids. Most prediction methods have focused on analogous features, such as alternating hydrophobicity patterns. Here, we start from the observation that almost all beta-barrel OMPs are related by common ancestry. We identify proteins as OMPs by detecting their homologous relationships to known OMPs using sequence similarity. Given an input sequence, HHomp builds a profile hidden Markov model (HMM) and compares it with an OMP database by pairwise HMM comparison, integrating OMP predictions by PROFtmb. A crucial ingredient is the OMP database, which contains profile HMMs for over 20,000 putative OMP sequences. These were collected with the exhaustive, transitive homology detection method HHsenser, starting from 23 representative OMPs in the PDB database. In a benchmark on TransportDB, HHomp detects 63.5% of the true positives before including the first false positive. This is 70% more than PROFtmb, four times more than BOMP and 10 times more than TMB-Hunt. In Escherichia coli, HHomp identifies 57 out of 59 known OMPs and correctly assigns them to their functional subgroups. HHomp can be accessed at http://toolkit.tuebingen.mpg.de/hhomp.

  19. Outer membrane proteome and antigens of Tannerella forsythia.

    Science.gov (United States)

    Veith, Paul D; O'Brien-Simpson, Neil M; Tan, Yan; Djatmiko, Deasy C; Dashper, Stuart G; Reynolds, Eric C

    2009-09-01

    Tannerella forsythia is a Gram-negative, anaerobic, fusiform bacterium implicated as a periodontal pathogen. With use of 2D PAGE, SDS PAGE, and LC-MALDI-TOF/TOF MS, 221 proteins of T. forsythia outer membrane preparations were identified, of which 197 were predicted to be localized to the cell envelope. Fifty-six proteins were reproducibly mapped by 2D PAGE and included several highly abundant proteins in the MW range 140-250 kDa that exhibited C-terminal sequence similarity to the CTD family of Porphyromonas gingivalis. Two-dimensional Western blot analyses revealed that these CTD family proteins together with several other outer membrane proteins were antigenic. The CTD family proteins exhibited a higher than expected MW, and were strongly reactive with the fluorescent glycoprotein stain, ProQ Emerald. This group included BspA and surface layer proteins A and B. TonB-dependent receptors (TDRs) (46) were identified together with 28 putative lipoproteins whose genes are immediately downstream of a TDR gene. The major OmpA-like protein was found to be TF1331. Uniquely, it was found to exist as a homodimer held together by up to three disulfide bridges as demonstrated by MS/MS of a tryptic peptide derived from unreduced TF1331.

  20. Sorting of bacterial lipoproteins to the outer membrane by the Lol system.

    Science.gov (United States)

    Narita, Shin-ichiro; Tokuda, Hajime

    2010-01-01

    Bacterial lipoproteins comprise a subset of membrane proteins with a lipid-modified cysteine residue at their amino termini through which they are anchored to the membrane. In Gram-negative bacteria, lipoproteins are localized on either the inner or the outer membrane. The Lol system is responsible for the transport of lipoproteins to the outer membrane.The Lol system comprises an inner-membrane ABC transporter LolCDE complex, a periplasmic carrier protein, LolA, and an outer membrane receptor protein, LolB. Lipoproteins are synthesized as precursors in the cytosol and then translocated across the inner membrane by the Sec translocon to the outer leaflet of the inner membrane, where lipoprotein precursors are processed to mature lipoproteins. The LolCDE complex then mediates the release of outer membrane-specific lipoproteins from the inner membrane while the inner membrane-specific lipoproteins possessing Asp at position 2 are not released by LolCDE because it functions as a LolCDE avoidance signal, causing the retention of these lipoproteins in the inner membrane. A water-soluble lipoprotein-LolA complex is formed as a result of the release reaction mediated by LolCDE. This complex traverses the hydrophilic periplasm to reach the outer membrane, where LolB accepts a lipoprotein from LolA and then catalyzes its incorporation into the inner leaflet of the outer membrane.

  1. Role of outer-membrane proteins and lipopolysaccharide in conjugation between Neisseria gonorrhoeae and Neisseria cinerea.

    Science.gov (United States)

    Genco, C A; Clark, V L

    1988-12-01

    Little is known concerning the mechanism involved in cell contact between the donor and recipient during conjugation in Neisseria gonorrhoeae. The formation of stable mating pairs during conjugation in Escherichia coli appears to require a specific protein as well as LPS in the outer membrane of the recipient cell. To attempt to identify the cell surface components necessary for conjugation in the neisseriae, we began a comparison of the outer membrane of Neisseria cinerea strains that can (Con+) and cannot (Con-) serve as recipients in conjugation with N. gonorrhoeae. There were no differences in outer-membrane protein profiles on SDS-polyacrylamide gel electrophoresis between Con+ and Con- strains that could be correlated with the ability to conjugate. However, whole outer membrane isolated from Con+ strains specifically inhibited conjugation while those from Con- strains did not. Proteolytic cleavage of outer-membrane proteins by trypsin, pronase or alpha-chymotrypsin abolished the inhibitory effect of Con+ outer membranes, suggesting that these outer membranes contained a protease-sensitive protein(s) involved in conjugation. Although periodate oxidation of Con+ outer-membrane carbohydrates did not abolish the inhibitory action of these membranes, purified LPS from both Con+ and Con- strains inhibited conjugation when added at low concentrations. These results suggest that conjugation requires the presence of a specific conjugal receptor that consists of both LPS and one or more outer-membrane proteins. Both Con+ and Con- strains contain the necessary LPS, but only Con+ strains contain the required protein(s).

  2. Characterization and immunogenicity of Kingella kingae outer-membrane proteins.

    Science.gov (United States)

    Yagupsky, Pablo; Slonim, Ariela

    2005-01-01

    In recent years, Kingella kingae has emerged as an important pediatric pathogen but the antigenicity of the organism and the host immune response have not been studied. Outer membrane proteins (OMPs) of 57 K. kingae isolates were characterized and the immune response of 19 children with invasive infections was studied by immunoblotting. Kingella kingae OMPs were remarkably similar disregarding place and time of isolation and associated clinical condition (asymptomatic carriage, bacteremia, endocarditis, septic arthritis or osteomyelitis). Most OMPs were immunogenic but the specific bands that reacted in each strain and the intensity of the reactions varied substantially. When convalescent sera were reacted with heterologous strains, bands that either were not recognized by the homologous serum or were not present in the homologous strain were visualized. These results demonstrate that OMPs of K. kingae are highly conserved but suggest that some epitopes are polymorphic, resulting in a variable pattern of immune response.

  3. Interaction of variable bacterial outer membrane lipoproteins with brain endothelium.

    Directory of Open Access Journals (Sweden)

    Gaurav Gandhi

    Full Text Available BACKGROUND: Previously we reported that the variable outer membrane lipoprotein Vsp1 from the relapsing fever spirochete Borrelia turicatae disseminates from blood to brain better than the closely related Vsp2 [1]. Here we studied the interaction between Vsp1 and Vsp2 with brain endothelium in more detail. METHODOLOGY/PRINCIPAL FINDINGS: We compared Vsp1 to Vsp2 using human brain microvascular endothelial cell (HBMEC association assays with aminoacid radiolabeled Vsp-expressing clones of recombinant Borrelia burgdorferi and lanthanide-labeled purified lipidated Vsp1 (LVsp1 and Vsp2 (LVsp2 and inoculations of the lanthanide-labeled proteins into mice. The results showed that heterologous expression of LVsp1 or LVsp2 in B. burgdorferi increased its association with HBMEC to a similar degree. Purified lanthanide-labeled lipidated Vsp1 (LVsp1 and LVsp2 by themselves were capable of associating with HBMEC. The association of LVsp1 with brain endothelium was time-dependent, saturable, and required the lipidation. The association of Vsp1 with HBMEC was inhibited by incubation at lower temperature or with excess unlabeled LVsp1 or LVsp2 but not with excess rVsp1 or mouse albumin or an anti Vsp1 monoclonal antibody. The association of LVsp2 with HBMEC and its movement from blood to brain parenchyma significantly increased in the presence of LVsp1. CONCLUSIONS/SIGNIFICANCE: Variable bacterial outer membrane lipoproteins interact with brain endothelium differently; the lipidation and variable features at the protein dome region are key modulators of this interaction.

  4. Outer membrane lipoprotein biogenesis: Lol is not the end.

    Science.gov (United States)

    Konovalova, Anna; Silhavy, Thomas J

    2015-10-05

    Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.

  5. Living on the edge: Simulations of bacterial outer-membrane proteins.

    Science.gov (United States)

    Pavlova, Anna; Hwang, Hyea; Lundquist, Karl; Balusek, Curtis; Gumbart, James C

    2016-07-01

    Gram-negative bacteria are distinguished in part by a second, outer membrane surrounding them. This membrane is distinct from others, possessing an outer leaflet composed not of typical phospholipids but rather large, highly charged molecules known as lipopolysaccharides. Therefore, modeling the structure and dynamics of proteins embedded in the outer membrane requires careful consideration of their native environment. In this review, we examine how simulations of such outer-membrane proteins have evolved over the last two decades, culminating most recently in detailed, highly accurate atomistic models of the outer membrane. We also draw attention to how the simulations have coupled with experiments to produce novel insights unattainable through a single approach. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

  6. The novel chlamydial adhesin CPn0473 mediates the lipid raft-dependent uptake of Chlamydia pneumoniae.

    Science.gov (United States)

    Fechtner, Tim; Galle, Jan N; Hegemann, Johannes H

    2016-08-01

    Chlamydiae are Gram-negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down-stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473-coupled fluorescent latex beads adhere to human epithelial HEp-2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp-2 cells with rCPn0473 does not attenuate adhesion but promotes dose-dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473-dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae.

  7. The novel chlamydial adhesin CPn0473 mediates the lipid raft‐dependent uptake of Chlamydia pneumoniae

    Science.gov (United States)

    Fechtner, Tim; Galle, Jan N.

    2016-01-01

    Summary Chlamydiae are Gram‐negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down‐stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473‐coupled fluorescent latex beads adhere to human epithelial HEp‐2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp‐2 cells with rCPn0473 does not attenuate adhesion but promotes dose‐dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473‐dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae. PMID:26780295

  8. Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles.

    Science.gov (United States)

    Cahill, Bethaney K; Seeley, Kent W; Gutel, Dedra; Ellis, Terri N

    2015-11-01

    Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane.

  9. Topological analysis of Chlamydia trachomatis L2 outer membrane protein 2

    DEFF Research Database (Denmark)

    Mygind, P; Christiansen, Gunna; Birkelund, Svend

    1998-01-01

    Using monospecific polyclonal antisera to different parts of Chlamydia trachomatis L2 outer membrane protein 2 (Omp2), we show that the protein is localized at the inner surface of the outer membrane. Omp2 becomes immunoaccessible when Chlamydia elementary bodies are treated with dithiothreitol...

  10. Molecular identification of chlamydial cause of abortion in small ruminants in Jordan.

    Science.gov (United States)

    Ababneh, Huthaifa Salah; Ababneh, Mustafa Mohammed Kheir; Hananeh, Wael Mahmoud; Alsheyab, Fawzi Mohammad; Jawasreh, Khaleel Ibraheem; Al-Gharaibeh, Moath Ahmad; Ababneh, Mohammed Mahmoud

    2014-12-01

    Chlamydophila abortus (Ch. abortus) is the etiological agent of ovine enzootic abortion (OEA) and one of the most common infectious agents of abortion in small ruminants worldwide. RFLP-PCR analysis of the outer membrane protein gene (OMP2 gene) was used for diagnosis and characterization of chlamydial causes of abortion in small ruminants in Jordan. Sixty-six placental tissues and 15 vaginal swabs were collected from aborted ewes and does to identify cause of abortion in Jordan. Thirty-eight placental samples (58 %) and 13 vaginal swabs (87 %) were positive for chlamydial DNA. Shedding of bacteria in vaginal swabs was detected within 7 days after abortion. The results of this study showed that chlamydiosis is one of the important causes of abortion in small ruminants in Jordan. In addition, vaginal swab is an excellent sample for molecular diagnosis of chlamydiosis. DNA sequencing and RFLP analysis of the OMP2 reveal that all chlamydial cause of abortion in small ruminants in Jordan are due to Ch. abortus. While, Ch. pecorum was not detected in any sample. OMP2 gene of the isolated Jordanian strain was identical (100 %) to Ch. abortus FAS strain. In conclusion, Ch. abortus is an important cause of abortion in Jordan; vaginal swab within 7 days of abortion can be used for molecular diagnosis of chlamydiosis in small ruminants.

  11. Outer membrane vesicles – offensive weapons or good Samaritans?

    Directory of Open Access Journals (Sweden)

    Ingar Olsen

    2015-04-01

    Full Text Available Outer membrane vesicles (OMVs from Gram-negative bacteria were first considered as artifacts and were followed with disbelief and bad reputation. Later, their existence was accepted and they became characterized as bacterial bombs, virulence bullets, and even decoys. Today, we know that OMVs also can be involved in cell–cell signaling/communication and be mediators of immune regulation and cause disease protection. Furthermore, OMVs represent a distinct bacterial secretion pathway selecting and protecting their cargo, and they can even be good Samaritans providing nutrients to the gut microbiota maintaining commensal homeostasis beneficial to the host. The versatility in functions of these nanostructures is remarkable and includes both defense and offense. The broad spectrum of usability does not stop with that, as it now seems that OMVs can be used as vaccines and adjuvants or vehicles engineered for drug treatment of emerging and new diseases not only caused by bacteria but also by virus. They may even represent new ways of selective drug treatment.

  12. Outer membrane vesicles - offensive weapons or good Samaritans?

    Science.gov (United States)

    Olsen, Ingar; Amano, Atsuo

    2015-01-01

    Outer membrane vesicles (OMVs) from Gram-negative bacteria were first considered as artifacts and were followed with disbelief and bad reputation. Later, their existence was accepted and they became characterized as bacterial bombs, virulence bullets, and even decoys. Today, we know that OMVs also can be involved in cell-cell signaling/communication and be mediators of immune regulation and cause disease protection. Furthermore, OMVs represent a distinct bacterial secretion pathway selecting and protecting their cargo, and they can even be good Samaritans providing nutrients to the gut microbiota maintaining commensal homeostasis beneficial to the host. The versatility in functions of these nanostructures is remarkable and includes both defense and offense. The broad spectrum of usability does not stop with that, as it now seems that OMVs can be used as vaccines and adjuvants or vehicles engineered for drug treatment of emerging and new diseases not only caused by bacteria but also by virus. They may even represent new ways of selective drug treatment.

  13. Proteomic Analysis of Outer Membrane Proteins from Salmonella Enteritidis Strains with Different Sensitivity to Human Serum

    Science.gov (United States)

    Dudek, Bartłomiej; Krzyżewska, Eva; Kapczyńska, Katarzyna; Rybka, Jacek; Pawlak, Aleksandra; Korzekwa, Kamila; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

    2016-01-01

    Differential analysis of outer membrane composition of S. Enteritidis strains, resistant to 50% normal human serum (NHS) was performed in order to find factors influencing the resistance to higher concentrations of NHS. Ten S. Enteritidis clinical strains, resistant to 50% NHS, all producing very long lipopolysaccharide, were subjected to the challenge of 75% NHS. Five extreme strains: two resistant and three sensitive to 75% NHS, were chosen for the further analysis of outer membrane proteins composition. Substantial differences were found in the levels of particular outer membrane proteins between resistant and sensitive strains, i.e. outer membrane protease E (PgtE) was present mainly in resistant strains, while sensitive strains possessed a high level of flagellar hook-associated protein 2 (FliD) and significantly higher levels of outer membrane protein A (OmpA). PMID:27695090

  14. The Power of Asymmetry: Architecture and Assembly of the Gram-Negative Outer Membrane Lipid Bilayer.

    Science.gov (United States)

    Henderson, Jeremy C; Zimmerman, Shawn M; Crofts, Alexander A; Boll, Joseph M; Kuhns, Lisa G; Herrera, Carmen M; Trent, M Stephen

    2016-09-08

    Determining the chemical composition of biological materials is paramount to the study of natural phenomena. Here, we describe the composition of model gram-negative outer membranes, focusing on the predominant assembly, an asymmetrical bilayer of lipid molecules. We also give an overview of lipid biosynthetic pathways and molecular mechanisms that organize this material into the outer membrane bilayer. An emphasis is placed on the potential of these pathways as targets for antibiotic development. We discuss deviations in composition, through bacterial cell surface remodeling, and alternative modalities to the asymmetric lipid bilayer. Outer membrane lipid alterations of current microbiological interest, such as lipid structures found in commensal bacteria, are emphasized. Additionally, outer membrane components could potentially be engineered to develop vaccine platforms. Observations related to composition and assembly of gram-negative outer membranes will continue to generate novel discoveries, broaden biotechnologies, and reveal profound mysteries to compel future research.

  15. Need for TolC, an Escherichia coli outer membrane protein, in the secretion of heat-stable enterotoxin I across the outer membrane.

    Science.gov (United States)

    Yamanaka, H; Nomura, T; Fujii, Y; Okamoto, K

    1998-09-01

    Escherichia coli heat-stable enterotoxin Ip (STIp) is a typical extracellular toxin consisting of 18 amino acid residues synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. STIp synthesized in the cytoplasm must cross the inner and outer membranes to migrate into the extracellular environment. Previous studies showed that the precursor translocates across the inner membrane utilizing the general export pathway consisting of Sec proteins. However, it remains unclear how it crosses the outer membrane. In this study, we examined the effects of mutation of the tolC gene which encodes an E. coli outer membrane protein, TolC, on the release of STIp into the extracellular environment. The mutation reduced the amount of STIp released into culture supernatant and increased the amount of STIp accumulated in the periplasm. This indicates that TolC mediates the translocation of STIp across the outer membrane. The inability to transfer STIp in the periplasm into the culture supernatant was restored by introduction of the tolC gene into the mutant cells. In the mouse intestinal loop assay, living cells of the mutants did not show a positive response, but wild-type cells did. These results showed that TolC is involved in the translocation of STIp across the outer membrane.

  16. CTA1-DD is an effective adjuvant for targeting anti-chlamydial immunity to the murine genital mucosa.

    Science.gov (United States)

    Cunningham, Kelly A; Carey, Alison J; Lycke, Nils; Timms, Peter; Beagley, Kenneth W

    2009-07-01

    Chlamydia trachomatis is a significant human pathogen with potentially severe disease sequelae in the genital tract, including infertility. A successful vaccine will need to effectively target immunity to the genital mucosa. Intranasal immunisation with cholera toxin (CT) can target immunity to the genital tract, but has the potential to cause neurological side effects. CTA1-DD is a non-toxic potent mucosal adjuvant which combines the enzymatic properties of CT, with a B cell targeting moiety. Here, we demonstrate that intranasal immunisation with CTA1-DD and chlamydial Major Outer Membrane Protein (MOMP) results in the induction of neutralising systemic and mucosal antibodies, and reduces the level of chlamydial shedding following intravaginal challenge with Chlamydia muridarum. Thus, CTA1-DD is an effective adjuvant for vaccine development against Chlamydia trachomatis, and possibly also a range of other genital pathogens.

  17. Assessing the Outer Membrane Insertion and Folding of Multimeric Transmembrane β-Barrel Proteins.

    Science.gov (United States)

    Leo, Jack C; Oberhettinger, Philipp; Linke, Dirk

    2015-01-01

    In addition to the cytoplasmic membrane, Gram-negative bacteria have a second lipid bilayer, the outer membrane, which is the de facto barrier between the cell and the extracellular milieu. Virtually all integral proteins of the outer membrane form β-barrels, which are inserted into the outer membrane by the BAM complex. Some outer membrane proteins, like the porins and trimeric autotransporter adhesins, are multimeric. In the former case, the porin trimer consists of three individual β-barrels, whereas in the latter, the single autotransporter β-barrel domain is formed by three separate polypeptides. This chapter reviews methods to investigate the folding and membrane insertion of multimeric OMPs and further explains the use of a BamA depletion strain to study the effects of the BAM complex on multimeric OMPs in E. coli.

  18. Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB

    OpenAIRE

    Okuda, Suguru; Tokuda, Hajime

    2009-01-01

    Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detai...

  19. Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex

    DEFF Research Database (Denmark)

    Mygind, Per H; Christiansen, Gunna; Roepstorff, P;

    2000-01-01

    The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis....... By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH....

  20. Salmonellae PhoPQ regulation of the outer membrane to resist innate immunity.

    Science.gov (United States)

    Dalebroux, Zachary D; Miller, Samuel I

    2014-02-01

    Salmonellae sense host cues to regulate properties important for bacterial survival and replication within host tissues. The PhoPQ two-component regulatory system senses phagosome acidification and cationic antimicrobial peptides (CAMP) to regulate the protein and lipid contents of the bacterial envelope that comprises an inner and outer membrane. PhoPQ-regulated lipid components of the outer membrane include lipopolysaccharides and glycerophospholipids. Envelope proteins regulated by PhoPQ, include: components of virulence associated secretion systems, the flagellar apparatus, membrane transport systems, and proteins that are likely structural components of the outer membrane. PhoPQ alteration of the bacterial surface results in increased bacterial resistance to CAMP and decreased detection by the innate immune system. This review details the molecular complexity of the bacterial cell envelope and highlights the outer membrane lipid bilayer as an environmentally regulated bacterial organelle.

  1. ROLE OF OUTER MEMBRANE PROTEINS IN IMIPENEM DIFFUSION IN PSEUDOMONAS AERUGINOSA

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    The present study identified the properties of porins in the outer membrane in Pseudomonas aeruginosa,and showed the role of outer membrane in determining imipenem diffusion in Pseudomonas aeruginosa. The molecular weight of the major outer membrane protein was analyzed by SDS-PAGE. The purification of the porins in Pseudomonas aeruginosa was achieved by DEAE ion - exchange HPLC. The purified outer membrane proteins were reconstituted with phosphatidylcholine and dicetylphosphate into membrane vesicles, and were tested by the liposomes swelling method for the diffusion of imipenem. The permeability assay showed that OprC (70 kD), OprD2 (46kD), and OprE(43 kD) were the channel- forming proteins. But only OprD2 was thought to be the likely route of imipenem diffusion.

  2. OmcF, a Putative c-Type Monoheme Outer Membrane Cytochrome Required for the Expression of Other Outer Membrane Cytochromes in Geobacter sulfurreducens

    OpenAIRE

    2005-01-01

    Outer membrane cytochromes are often proposed as likely agents for electron transfer to extracellular electron acceptors, such as Fe(III). The omcF gene in the dissimilatory Fe(III)-reducing microorganism Geobacter sulfurreducens is predicted to code for a small outer membrane monoheme c-type cytochrome. An OmcF-deficient strain was constructed, and its ability to reduce and grow on Fe(III) citrate was found to be impaired. Following a prolonged lag phase (150 h), the OmcF-deficient strain de...

  3. Outer membrane proteins induced by iron deficiency in Anabaena sp.PCC 7120

    Institute of Scientific and Technical Information of China (English)

    Yanling Dong; Xudong Xu

    2009-01-01

    Iron deficiency can induce cyanobacteria to synthesize siderophore receptor proteins on the outer membrane to enhance the uptake of iron. In this study, an outer membrane of high purity was prepared from Anabaena sp. PCC 7120 based on aqueous polymer two-phase partitioning and discontinuous sucrose density ultra-centrifugation, and the induction of outer membrane proteins by iron deficiency was investigated using 2-D gel electrophoresis. At least five outer membrane proteins were newly synthesized or significantly up-regulated in cells transferred to iron-deficient conditions, which were all identified to be siderophore receptor proteins according to MALDI-TOF-MS analyses. Bacterial luciferase reporter genes luxAB were employed to monitor the transcription of the encoding genes. The genes were induced by iron deficiency at the transcriptional level in different responsive modes. Luciferase activity expressed from an iron-regulated promoter may be used as a bioreporter for utilizable iron in natural water samples.

  4. Presence of methyl sterol and bacteriohopanepolyol in an outer-membrane preparation from Methylococcus capsulatus (Bath)

    Science.gov (United States)

    Jahnke, Linda L.; Stan-Lotter, Helga; Kato, Katharine; Hochstein, Lawrence I.

    1992-01-01

    Cytoplasmic/intracytoplasmic and outer membrane preparations of Methylococcus capsulatus (Bath) were isolated by sucrose density gradient centrifugation of a total membrane fraction prepared by disruption using a French pressure cell. The cytoplasmic and/or intracytoplasmic membrane fraction consisted of two distinct bands, Ia and Ib (buoyant densities 1.16 and 1.18 g ml (exp -1), respectively) that together contained 57% of the protein, 68% of the phospholipid, 73% of the ubiquinone and 89% of the CN-sensitive NADH oxidase activity. The only apparent difference between these two cytoplasmic bands was a much higher phospholipid content for Ia. The outer membrane fraction (buoyant density 1.23-1.24 g ml (exp -1)) contained 60% of the lipopolysaccharide-associated, beta-hydroxypalmitic acid, 74% of the methylsterol, and 66% of the bacteriohopanepolyol (BHP); phospholipid to methyl sterol or BHP ratios were 6:1. Methanol dehydrogenase activity and a c-type cytochrome were also present in this outer membrane fraction. Phospholipase A activity was present in borh the cytoplasmic membrane and outer membrane fractions. The unique distribution of cyclic triterpenes may reflect a specific role in conferring outer membrane stability in this methanotrophic bacterium.

  5. A Mathematical Model for Diffusion-Controlled Monolithic Matrix Coated with outer Membrane System

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A release model for diffusion-controlled monolithic matrix coated with outer membrane system is proposed and solved by using the refined double integral method. The calculated results are in satisfactory agreement with the experimental release data. The present model can be well used to describe the release process for all cd/cs values. In addition, the release effects of the monolithic matrix coated with outer membrane system are discussed theoretically.

  6. The Chloroplast Outer Envelope Membrane: The Edge of Light and Excitement

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The chloroplast is surrounded by a double-membrane envelope at which proteins, ions, and numerous metabolites Including nucleotides, amino acids, fatty acids, and carbohydrates are exchanged between the two aqueous phases, the cytoplasm and the chloroplast stroma. The chloroplast envelope is also the location where the biosynthesis and accumulation of various lipids take place. By contrast to the inner membrane, which contains a number of specific transporters and acts as the permeability barrier, the chloroplast outer membrane has often been considered a passive compartment derived from the phagosomal membrane. However, the presence of galactoglycerolipids and β-barrel membrane proteins support the common origin of the outer membranes of the chloroplast envelope and extant cyanobacteria. Furthermore, recent progress in the field underlines that the chloroplast outer envelope plays important roles not only for translocation of various molecules, but also for regulation of metabolic activities and signaling processes. The chloroplast outer envelope membrane offers various interesting and challenging questions that are relevant to the understanding of organelle biogenesis, plant growth and development, and also membrane biology in general.

  7. Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division

    NARCIS (Netherlands)

    Gray, A.N.; Egan, A.J.F.; van 't Veer, I.L.; Verheul, J.; Colavin, A.; Koumoutsi, A.; Biboy, J.; Altelaar, A.F.M.; Damen, M.J.; Huang, K.C.; Simorre, J.P.; Breukink, E.; den Blaauwen, T.; Typas, A.; Gross, C.A.; Vollmer, W.

    2015-01-01

    To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope ma

  8. A Hidden Markov Model method, capable of predicting and discriminating β-barrel outer membrane proteins

    Directory of Open Access Journals (Sweden)

    Hamodrakas Stavros J

    2004-03-01

    Full Text Available Abstract Background Integral membrane proteins constitute about 20–30% of all proteins in the fully sequenced genomes. They come in two structural classes, the α-helical and the β-barrel membrane proteins, demonstrating different physicochemical characteristics, structure and localization. While transmembrane segment prediction for the α-helical integral membrane proteins appears to be an easy task nowadays, the same is much more difficult for the β-barrel membrane proteins. We developed a method, based on a Hidden Markov Model, capable of predicting the transmembrane β-strands of the outer membrane proteins of gram-negative bacteria, and discriminating those from water-soluble proteins in large datasets. The model is trained in a discriminative manner, aiming at maximizing the probability of correct predictions rather than the likelihood of the sequences. Results The training has been performed on a non-redundant database of 14 outer membrane proteins with structures known at atomic resolution; it has been tested with a jacknife procedure, yielding a per residue accuracy of 84.2% and a correlation coefficient of 0.72, whereas for the self-consistency test the per residue accuracy was 88.1% and the correlation coefficient 0.824. The total number of correctly predicted topologies is 10 out of 14 in the self-consistency test, and 9 out of 14 in the jacknife. Furthermore, the model is capable of discriminating outer membrane from water-soluble proteins in large-scale applications, with a success rate of 88.8% and 89.2% for the correct classification of outer membrane and water-soluble proteins respectively, the highest rates obtained in the literature. That test has been performed independently on a set of known outer membrane proteins with low sequence identity with each other and also with the proteins of the training set. Conclusion Based on the above, we developed a strategy, that enabled us to screen the entire proteome of E. coli for

  9. Characterization of the Outer Membrane Protein OprF of Pseudomonas aeruginosa in a Lipopolysaccharide Membrane by Computer Simulation

    Energy Technology Data Exchange (ETDEWEB)

    Straatsma, TP; Soares, Thereza A.

    2009-02-01

    The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded anti-parallel β-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared to simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared to the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel.

  10. Chlamydial hemagglutinin identified as lipopolysaccharide.

    OpenAIRE

    Watkins, N G; Caldwell, H D; Hackstadt, T

    1987-01-01

    Chlamydial lipopolysaccharide (LPS) agglutinated mouse and rabbit erythrocytes but not human, guinea pig, or pronghorn antelope erythrocytes. Hemagglutination was not specific for Chlamydia spp., as rough LPSs from Coxiella burnetii and Escherichia coli also agglutinated erythrocytes from the same animal species. Nonagglutinated and agglutinated erythrocytes bound equivalent amounts of LPS, indicating that hemagglutination was not due to a specific interaction of chlamydial LPS with erythrocy...

  11. Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

    Directory of Open Access Journals (Sweden)

    Tatini Rakshit

    Full Text Available Rhodopsin forms nanoscale domains (i.e., nanodomains in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.

  12. Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

    Science.gov (United States)

    Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J

    2010-12-01

    Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

  13. Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival of Vibrio cholerae and Suppresses Viability of Acanthamoeba castellanii

    Directory of Open Access Journals (Sweden)

    Soni Priya Valeru

    2014-01-01

    Full Text Available Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive inside Acanthamoeba castellanii. It has been shown that V. cholerae expresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA and outer membrane vesicles (OMVs in survival of V. cholerae alone and during its interaction with A. castellanii. The results showed that an OmpA mutant of V. cholerae survived longer than wild-type V. cholerae when cultivated alone. Cocultivation with A. castellanii enhanced the survival of both bacterial strains and OmpA protein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of the OmpA mutant of V. cholerae decreased the viability of A. castellanii and this bacterial strain released more OMVs than wild-type V. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from the OmpA mutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule for OmpA in survival of V. cholerae and OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment.

  14. Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

    Science.gov (United States)

    Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

    2016-04-15

    Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae.

  15. Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Liting Lim

    Full Text Available Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane.

  16. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    Science.gov (United States)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

    2011-06-01

    Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

  17. Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum

    Science.gov (United States)

    Goodman, Christopher D.; McFadden, Geoffrey I.

    2016-01-01

    Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane. PMID:27442138

  18. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    Institute of Scientific and Technical Information of China (English)

    YUAN Ye; WANG Xiuli; GUO Sheping; QIU xuemei

    2011-01-01

    Gram-negative vibrio parahaemolyticus is a common pathogen in humans and marine animals.The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host.Thus,the outer membrane proteins are an ideal target for vaccines.We amplified a complete outer membrane protein gene (ompW) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells.The gene coded for a protein that was 42.78 kDa.We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V.parahaemolyticus.In addition,the purified OmpW protein can be used for further functional and structural studies.

  19. Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology.

    Science.gov (United States)

    Niemann, Moritz; Wiese, Sebastian; Mani, Jan; Chanfon, Astrid; Jackson, Christopher; Meisinger, Chris; Warscheid, Bettina; Schneider, André

    2013-02-01

    Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

  20. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

    Directory of Open Access Journals (Sweden)

    Deirdre R Ducken

    Full Text Available Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to

  1. Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

    DEFF Research Database (Denmark)

    Juul, Nicolai Stefan; Timmerman, E; Gevaert, K;

    2007-01-01

    that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we...... compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full...

  2. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

    Science.gov (United States)

    Ducken, Deirdre R; Brown, Wendy C; Alperin, Debra C; Brayton, Kelly A; Reif, Kathryn E; Turse, Joshua E; Palmer, Guy H; Noh, Susan M

    2015-01-01

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines

  3. A conserved small RNA promotes silencing of the outer membrane protein YbfM

    DEFF Research Database (Denmark)

    Rasmussen, Anders Aamann; Johansen, Jesper; Nielsen, Jesper S;

    2009-01-01

    In the past few years an increasing number of small non-coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the post-transcriptional level. These RNAs act under various growth and stress conditions, suggesting that one...... important physiological role of regulatory RNA molecules in Gram-negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP-sRNA network by showing that the expression of the outer membrane protein YbfM is silenced by a conserved s...

  4. Proteomic and genomic analysis reveals novel Campylobacter jejuni outer membrane proteins and potential heterogeneity.

    Science.gov (United States)

    Watson, Eleanor; Sherry, Aileen; Inglis, Neil F; Lainson, Alex; Jyothi, Dushyanth; Yaga, Raja; Manson, Erin; Imrie, Lisa; Everest, Paul; Smith, David G E

    2014-09-01

    Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC-ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith-Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.

  5. Green Modification of Outer Selective P84 Nanofiltration (NF) Hollow Fiber Membranes for Cadmium Removal

    KAUST Repository

    Gao, Jie

    2015-10-26

    Outer-selective thin-film composite (TFC) hollow fiber membranes are normally made from interfacial polymerization of m-phenylenediamine (MPD) and trimesoyl chloride (TMC). However, the removal of excess MPD solution and the large consumption of alkane solvents are their technical bottlenecks. In this study, green methods to prepare the outer selective TFC hollow fiber membranes were explored by firstly modifying the membrane substrate with polyethyleneimine (PEI) and then by water soluble small molecules such as glutaraldehyde (GA) and epichlorohydrin (ECH). Using P84 polyimide as the substrate, not only do these modifications decrease substrate\\'s pore size, but also vary surface charge by making the membranes less positively charged. As a result, the resultant membranes have higher rejections against salts such as Na2SO4, NaCl and MgSO4. The PEI and then GA modified membrane has the best separation performance with a NaCl rejection over 90% and a pure water permeability (PWP) of 1.74±0.01 Lm−2bar−1h−1. It also shows an impressive rejection to CdCl2 (94%) during long-term stability tests. The CdCl2 rejection remains higher than 90% at operating temperatures from 5 to 60 °C. This study may provide useful insights for green manufacturing of outer-selective nanofiltration (NF) hollow fiber membranes.

  6. Outer membrane biogenesis in Helicobacter pylori: A deviation from the paradigm

    Directory of Open Access Journals (Sweden)

    George W. Liechti

    2012-04-01

    Full Text Available The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM. Lipopolysaccharide (LPS and numerous outer membrane proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its outer membrane profile limits the effectiveness of vaccines that use any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ε- proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ε-proteobacteria, while the inner and outer membrane associated apparatus of LPS, lipoprotein, and OM protein transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to

  7. Formation of Mitochondrial Outer Membrane Derived Protrusions and Vesicles in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Akihiro Yamashita

    Full Text Available Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs, while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles. Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN. The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence.

  8. Chlamydial plasmids and bacteriophages.

    Science.gov (United States)

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  9. Analysis of proteins in Chlamydia trachomatis L2 outer membrane complex, COMC

    DEFF Research Database (Denmark)

    Birkelund, Svend; Morgan-Fisher, Marie; Timmerman, Evy;

    2009-01-01

    amino groups of in vivo generated proteolytic cleavage sites facilitated identification of such sites in known outer membrane proteins (MOMPs). Our results further support a proposed prediction of the topology of the MOMPs. Furthermore, a previously unknown MOMP, CTL0626 (Ct372), was assigned as an MOMP...

  10. The Motion of a Single Molecule, the Lambda-Receptor, in the Bacterial Outer Membrane

    DEFF Research Database (Denmark)

    Oddershede, Lene; Dreyer, Jakob Kisbye; Grego, Sonia

    2002-01-01

    Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo...

  11. Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

    DEFF Research Database (Denmark)

    Koehler, JF; Birkelund, Svend; Stephens, RS

    1992-01-01

    The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis and cap...... the contribution of the MOMP variable segments to the topographical interactions which determine the antigenic structure responsible for human immune response....

  12. The beta-barrel outer membrane protein assembly complex of Neisseria meningitidis.

    NARCIS (Netherlands)

    Volokhina, E.B.; Beckers, F.; Tommassen, J.; Bos, M.P.

    2009-01-01

    The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the beta-barrel asse

  13. Comparison of outer membrane protein and biochemical profiles of Haemophilus aegyptius and Haemophilus influenzae biotype III.

    Science.gov (United States)

    Carlone, G M; Sottnek, F O; Plikaytis, B D

    1985-11-01

    Haemophilus aegyptius and Haemophilus influenzae biotype III are morphologically and biochemically similar; however, their outer membrane protein (Sarkosyl insoluble) profiles are distinct. Of 18 strains of H. aegyptius examined, 15 had a type 1 protein profile, and 3 had a type 2 profile, whereas the 5 strains of H. influenzae biotype III examined had three other protein profile types. All Haemophilus strains examined had 31- and 76-kilodalton (kDa) proteins and minor proteins with molecular masses between 20 and 100 kDa. H. aegyptius, with a type 1 protein profile, had major outer membrane proteins with apparent molecular masses of 27, 35.5, and 41.5 kDa, and H. aegyptius, with a type 2 protein profile, had 26-, 29-, 39.5-, and 41-kDa proteins. The type strain of H. influenzae biotype III had three major outer membrane proteins with apparent molecular masses of 29, 38.5 and 40 kDa. Four other strains designated as H. influenzae biotype III had major outer membrane proteins between 27 and 41.5 kDa representing two additional protein profiles.

  14. Analysis of the humoral immune response to Chlamydia outer membrane protein 2

    DEFF Research Database (Denmark)

    Mygind, P; Christiansen, Gunna; Persson, K;

    1998-01-01

    The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated...

  15. OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations.

    Science.gov (United States)

    Benz, Roland; Jones, Michael D; Younas, Farhan; Maier, Elke; Modi, Niraj; Mentele, Reinhard; Lottspeich, Friedrich; Kleinekathöfer, Ulrich; Smit, John

    2015-01-01

    Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures

  16. The properties of the outer membrane localized Lipid A transporter LptD

    Energy Technology Data Exchange (ETDEWEB)

    Haarmann, Raimund; Ibrahim, Mohamed; Stevanovic, Mara; Bredemeier, Rolf; Schleiff, Enrico, E-mail: schleiff@bio.uni-frankfurt.d [JWGU Frankfurt/Main, Cluster of Excellence Macromolecular Complexes, Center of Membrane Proteomics, Department of Biosciences, Molecular Cell Biology, Max-von-Laue Strasse 9, D-60439 Frankfurt (Germany)

    2010-11-17

    Gram-negative bacteria are surrounded by a cell wall including the outer membrane. The outer membrane is composed of two distinct monolayers where the outer layer contains lipopolysaccharides (LPS) with the non-phospholipid Lipid A as the core. The synthesis of Lipid A is initiated in the cytosol and thereby the molecule has to be transported across the inner and outer membranes. The {beta}-barrel lipopolysaccharide-assembly protein D (LptD) was discovered to be involved in the transfer of Lipid A into the outer membrane of Gram-negative bacteria. At present the molecular procedure of lipid transfer across the outer membrane remains unknown. Here we approached the functionality of the transfer system by an electrophysiological analysis of the outer membrane protein from Escherichia coli named ecLptD. In vitro the protein shows cation selectivity and has an estimated pore diameter of about 1.8 nm. Addition of Lipid A induces a transition of the open state to a sub-conductance state with two independent off-rates, which might suggest that LptD is able to bind and transport the molecule in vitro. To generalize our findings with respect to the Lipid A transport system of other Gram-negative bacteria we have explored the existence of the proteins involved in this pathway by bioinformatic means. We were able to identify the membrane-inserted components of the Lipid A transport system in all Gram-negative bacteria, whereas the periplasmic components appear to be species-specific. The LptD proteins of different bacteria are characterized by their periplasmic N-terminal domain and a C-terminal barrel region. The latter shows distinct sequence properties, particularly in LptD proteins of cyanobacteria, and this specific domain can be found in plant proteins as well. By electrophysiological experiments on LptD from Anabaena sp. PCC 7120 we are able to confirm the functional relation of anaLptD to Lipid A transport.

  17. OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations.

    Directory of Open Access Journals (Sweden)

    Roland Benz

    Full Text Available Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two

  18. Outer Membrane Protein Folding and Topology from a Computational Transfer Free Energy Scale.

    Science.gov (United States)

    Lin, Meishan; Gessmann, Dennis; Naveed, Hammad; Liang, Jie

    2016-03-02

    Knowledge of the transfer free energy of amino acids from aqueous solution to a lipid bilayer is essential for understanding membrane protein folding and for predicting membrane protein structure. Here we report a computational approach that can calculate the folding free energy of the transmembrane region of outer membrane β-barrel proteins (OMPs) by combining an empirical energy function with a reduced discrete state space model. We quantitatively analyzed the transfer free energies of 20 amino acid residues at the center of the lipid bilayer of OmpLA. Our results are in excellent agreement with the experimentally derived hydrophobicity scales. We further exhaustively calculated the transfer free energies of 20 amino acids at all positions in the TM region of OmpLA. We found that the asymmetry of the Gram-negative bacterial outer membrane as well as the TM residues of an OMP determine its functional fold in vivo. Our results suggest that the folding process of an OMP is driven by the lipid-facing residues in its hydrophobic core, and its NC-IN topology is determined by the differential stabilities of OMPs in the asymmetrical outer membrane. The folding free energy is further reduced by lipid A and assisted by general depth-dependent cooperativities that exist between polar and ionizable residues. Moreover, context-dependency of transfer free energies at specific positions in OmpLA predict regions important for protein function as well as structural anomalies. Our computational approach is fast, efficient and applicable to any OMP.

  19. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    Science.gov (United States)

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

  20. Both phototropin 1 and 2 localize on the chloroplast outer membrane with distinct localization activity.

    Science.gov (United States)

    Kong, Sam-Geun; Suetsugu, Noriyuki; Kikuchi, Shingo; Nakai, Masato; Nagatani, Akira; Wada, Masamitsu

    2013-01-01

    Chloroplasts change their position to adapt cellular activities to fluctuating environmental light conditions. Phototropins (phot1 and phot2 in Arabidopsis) are plant-specific blue light photoreceptors that perceive changes in light intensity and direction, and mediate actin-based chloroplast photorelocation movements. Both phot1 and phot2 regulate the chloroplast accumulation response, while phot2 is mostly responsible for the regulation of the avoidance response. Although it has been widely accepted that distinct intracellular localizations of phototropins are implicated in the specificity, the mechanism underlying the phot2-specific avoidance response has remained elusive. In this study, we examined the relationship of the phot2 localization pattern to the chloroplast photorelocation movement. First, the fusion of a nuclear localization signal with phot2, which effectively reduced the amount of phot2 in the cytoplasm, retained the activity for both the accumulation and avoidance responses, indicating that membrane-localized phot2 but not cytoplasmic phot2 is functional to mediate the responses. Importantly, some fractions of phot2, and of phot1 to a lesser extent, were localized on the chloroplast outer membrane. Moreover, the deletion of the C-terminal region of phot2, which was previously shown to be defective in blue light-induced Golgi localization and avoidance response, affected the localization pattern on the chloroplast outer membrane. Taken together, these results suggest that dynamic phot2 trafficking from the plasma membrane to the Golgi apparatus and the chloroplast outer membrane might be involved in the avoidance response.

  1. A modular BAM complex in the outer membrane of the alpha-proteobacterium Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Khatira Anwari

    Full Text Available Mitochondria are organelles derived from an intracellular alpha-proteobacterium. The biogenesis of mitochondria relies on the assembly of beta-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an alpha-proteobacterium, and the BAM (beta-barrel assembly machinery complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A approximately 150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane.

  2. The role of outer membrane in Serratia marcescens intrinsic resistance to antibiotics.

    Science.gov (United States)

    Sánchez, L; Ruiz, N; Leranoz, S; Viñas, M; Puig, M

    1997-09-01

    Three different porins from Serratia marcescens were described. They were named Omp1, Omp2 and Omp3 and their molecular weights were 42, 40 and 39 kDa respectively. Omp2 and Omp3 showed osmoregulation and thermoregulation in a similar way to OmpC and OmpF of Escherichia coli. Permeability coefficients of the outer membrane of this species were calculated following the Zimmermann and Rosselet method. P values were similar to those obtained in Escherichia coli, which suggests that the chromosomal beta-lactamase would play a major role in the resistance of Serratia marcescens to beta-lactam antibiotics. Both MIC values and permeabilities were modified by salycilates and acetylsalycilate. Synergism between the outer membrane and the beta-lactamase was also evaluated. When bacteria grew in the presence of a beta-lactam in the medium, the beta-lactamase accounted for most of the resistance.

  3. Proteomic and genomic analysis reveals novel Campylobacter jejuni outer membrane proteins and potential heterogeneity

    Directory of Open Access Journals (Sweden)

    Eleanor Watson

    2014-09-01

    Full Text Available Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC–ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith–Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.

  4. Membrane recycling at the infranuclear pole of the outer hair cell

    Science.gov (United States)

    Harasztosi, Csaba; Harasztosi, Emese; Gummer, Anthony W.

    2015-12-01

    Rapid endocytic activity of outer hair cells (OHCs) in the guinea-pig cochlea has been already studied using the fluorescent membrane marker FM1-43. It was demonstrated that vesicles were endocytosed at the apical pole of OHCs and transcytosed to the basolateral membrane and through a central strand towards the nucleus. The significance of endocytic activity in the infranuclear region is still not clear. Therefore, in this study endocytic activity at the synaptic pole of OHCs was investigated. Confocal laser scanning microscopy was used to visualize dye uptake of OHCs isolated from the guinea-pig cochlea. Signal intensity changes were quantified in the apical and basal poles relative to the signal at the membrane. Data showed no significant difference in fluorescent signal intensity changes between the opposite poles of the OHC. These results suggest that endocytic activities in both the basal and the apical poles contribute equally to the membrane recycling of OHCs.

  5. Thermotropic phase transitions in model membranes of the outer skin layer based on ceramide 6

    Science.gov (United States)

    Gruzinov, A. Yu.; Kiselev, M. A.; Ermakova, E. V.; Zabelin, A. V.

    2014-01-01

    The lipid intercellular matrix stratum corneum of the outer skin layer is a multilayer membrane consisting of a complex mixture of different lipids: ceramides, fatty acids, cholesterol, and its derivatives. The basis of the multilayer membrane is the lipid bilayer, i.e., a two-dimensional liquid crystal. Currently, it is known that the main way of substance penetration through the skin is the lipid matrix. The complexity of the actual biological system does not allow reliable direct study of its properties; therefore, system modeling is often used. Phase transitions in the lipid system whose composition simulates the native lipid matrix are studied by the X-ray synchrotron radiation diffraction method.

  6. Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

    OpenAIRE

    Alakomi, Hanna-Leena

    2007-01-01

    Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intri...

  7. Epitope mapping of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi.

    OpenAIRE

    Lachumanan, R; Devi, S; Cheong, Y M; Rodda, S J; Pang, T.

    1993-01-01

    Binding studies of 160 overlapping, synthetic octapeptides from the hydrophilic regions of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi with sera from patients with scrub typhus revealed 15 immunodominant peptides which are recognized by all the sera tested. Further analysis of the specificity of peptide binding with five of these peptides indicated that the peptides showed significantly stronger binding to scrub typhus patients' sera than they did to sera from patients ...

  8. Outer Membrane Components of the Tad (Tight Adherence) Secreton of Aggregatibacter actinomycetemcomitans▿ †

    OpenAIRE

    Clock, Sarah A.; Paul J Planet; Perez, Brenda A.; Figurski, David H.

    2007-01-01

    Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were sta...

  9. Comparison of outer membrane protein and biochemical profiles of Haemophilus aegyptius and Haemophilus influenzae biotype III.

    OpenAIRE

    Carlone, G M; Sottnek, F O; Plikaytis, B. D.

    1985-01-01

    Haemophilus aegyptius and Haemophilus influenzae biotype III are morphologically and biochemically similar; however, their outer membrane protein (Sarkosyl insoluble) profiles are distinct. Of 18 strains of H. aegyptius examined, 15 had a type 1 protein profile, and 3 had a type 2 profile, whereas the 5 strains of H. influenzae biotype III examined had three other protein profile types. All Haemophilus strains examined had 31- and 76-kilodalton (kDa) proteins and minor proteins with molecular...

  10. Distinct constrictive processes, separated in time and space,divide Caulobacter inner and outer membranes

    Energy Technology Data Exchange (ETDEWEB)

    Judd, Ellen M.; Comolli, Luis R.; Chen, Joseph C.; Downing,Kenneth H.; Moerner, W.E.; McAdams, Harley H.

    2005-05-01

    Cryo-electron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner, and then the outer, membrane in a manner distinctly different from septum-forming bacteria. The smallest observed pre-fission constrictions were 60 nm for both the inner and outer membrane. FLIP experiments had previously shown cytoplasmic compartmentalization, when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments, occurring 18 min before daughter cell separation in a 135 min cell cycle. Here, we used FLIP experiments with membrane-bound and periplasmic fluorescent proteins to show that (1) periplasmic compartmentalization occurs after cytoplasmic compartmentalization, consistent with the cryoEM observations, and (2) inner membrane and periplasmic proteins can diffuse past the FtsZ constriction site, indicating that the cell division machinery does not block membrane diffusion.

  11. Differential proteomic analysis of outer membrane enriched extracts of Bacteroides fragilis grown under bile salts stress.

    Science.gov (United States)

    Boente, Renata F; Pauer, Heidi; Silva, Deborah N S; Filho, Joaquim Santos; Sandim, Vanessa; Antunes, Luis Caetano M; Ferreira, Rosana Barreto Rocha; Zingali, Russolina B; Domingues, Regina M C P; Lobo, Leandro A

    2016-06-01

    Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses.

  12. The participation of outer membrane proteins in the bacterial sensitivity to nanosilver.

    Science.gov (United States)

    Kędziora, Anna; Krzyżewska, Eva; Dudek, Bartłomiej; Bugla-Płoskońska, Gabriela

    2016-01-01

    The presented study is to analyze the participation of outer membrane proteins of Gram- negative bacteria in sensitivity to silver nanomaterials. The mechanism of interaction of silver with the bacterial cell is best described in this group of microorganisms. There are several theories regarding the effectiveness of antimicrobial ions and nanosilver, and at the indicated differences in the way they work. Outer membrane proteins of Gram-negative bacteria are involved in the procurement of silver from the environment and contribute to the development mechanisms of resistance to nanometals. They are measurable parameter in the field of cell phenotypic response to the presence of Gram-negative bacteria in the environment silver nanoforms: its properties, chemical composition, content or times of action. Proteomic methods (including two dimensional electrophoresis and MALDI‑TOF MS) are therefore relevant techniques for determining the susceptibility of bacteria to silver and the changes taking place in the outer membrane under the influence: uptime/exposure and physical and chemical parameters of silver nanomaterials. Many products containing nanosilver is still in the research phase in terms of physico‑chemical characteristics and biological activity, others have been already implemented in many industries. During the very fast nanotechnology developing and introduction to the market products based on the nanosilver the bacterial answer to nanosilver is needed.

  13. Detection of apoptosis through the lipid order of the outer plasma membrane leaflet.

    Science.gov (United States)

    Darwich, Zeinab; Klymchenko, Andrey S; Kucherak, Oleksandr A; Richert, Ludovic; Mély, Yves

    2012-12-01

    Cell plasma membranes of living cells maintain their asymmetry, so that the outer leaflet presents a large quantity of sphingomyelin, which is critical for formation of ordered lipid domains. Here, a recently developed probe based on Nile Red (NR12S) was applied to monitor changes in the lipid order specifically at the outer leaflet of cell membranes. Important key features of NR12S are its ratiometric response exclusively to lipid order (liquid ordered vs. liquid disordered phase) and not to surface charge, the possibility of using it at very low concentrations (10-20nM) and the very simple staining protocol. Cholesterol extraction, oxidation and sphingomyelin hydrolysis were found to red shift the emission spectrum of NR12S, indicating a decrease in the lipid order at the outer plasma membrane leaflet. Remarkably, apoptosis induced by three different agents (actinomycin D, camptothecin, staurosporine) produced very similar spectroscopic effects, suggesting that apoptosis also significantly decreases the lipid order at this leaflet. The applicability of NR12S to detect apoptosis was further validated by fluorescence microscopy and flow cytometry, using the ratio between the blue and red parts of its emission band. Thus, for the first time, an environment-sensitive probe, sensitive to lipid order, is shown to detect apoptosis, suggesting a new concept in apoptosis sensing.

  14. Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

    Institute of Scientific and Technical Information of China (English)

    GUO Peng; WANG Na; LIU Yong-jie; LU Cheng-ping

    2014-01-01

    Aeromonas hydrophila isolates from clinical cases (n=43) were tested against 8 antimicrobial agents and typed by outer membrane protein (OMP) pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin (MICs, 16 mg mL-1) and sulfamonomethoxine (MICs, 64 mg mL-1), but susceptible to norlfoxacin (MICs, 0.5 mg mL-1). There was a high incidence of resistance to erythromycin (90.70%) and tylosin (93.02%), while a low incidences of resistance to ciprofloxacin (2.33%), enrofloxacin (2.33%) and florfenicol (4.65%). Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective proifles. The strains with the similar OMP proifles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to lforfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to lforfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to lfuoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

  15. Structure Prediction of Outer Membrane Protease Protein of Salmonella typhimurium Using Computational Techniques

    Directory of Open Access Journals (Sweden)

    Rozina Tabassum

    2016-03-01

    Full Text Available Salmonella typhimurium, a facultative gram-negative intracellular pathogen belonging to family Enterobacteriaceae, is the most frequent cause of human gastroenteritis worldwide. PgtE gene product, outer membrane protease emerges important in the intracellular phases of salmonellosis. The pgtE gene product of S. typhimurium was predicted to be capable of proteolyzing T7 RNA polymerase and localize in the outer membrane of these gram negative bacteria. PgtE product of S. enterica and OmpT of E. coli, having high sequence similarity have been revealed to degrade macrophages, causing salmonellosis and other diseases. The three-dimensional structure of the protein was not available through Protein Data Bank (PDB creating lack of structural information about E protein. In our study, by performing Comparative model building, the three dimensional structure of outer membrane protease protein was generated using the backbone of the crystal structure of Pla of Yersinia pestis, retrieved from PDB, with MODELLER (9v8. Quality of the model was assessed by validation tool PROCHECK, web servers like ERRAT and ProSA are used to certify the reliability of the predicted model. This information might offer clues for better understanding of E protein and consequently for developmet of better therapeutic treatment against pathogenic role of this protein in salmonellosis and other diseases.

  16. Monoclonal antibodies against the iron regulated outer membrane Proteins of Acinetobacter baumannii are bactericidal

    Directory of Open Access Journals (Sweden)

    Goel Vikas

    2001-08-01

    Full Text Available Abstract Background Iron is an important nutrient required by all forms of life.In the case of human hosts,the free iron availability is 10-18M,which is far less than what is needed for the survival of the invading bacterial pathogen.To survive in such conditions, bacteria express new proteins in their outer membrane and also secrete iron chelators called siderophores. Results/ Discussion Acinetobacter baumannii ATCC 19606, a nosocomial pathogen which grows under iron restricted conditions, expresses four new outer membrane proteins,with molecular weight ranging from 77 kDa to 88 kDa, that are called Iron Regulated Outer Membrane Proteins (IROMPs. We studied the functional and immunological properties of IROMPs expressed by A.baumanii ATCC 19606.The bands corresponding to IROMPs were eluted from SDS-PAGE and were used to immunize BALB/c mice for the production of monoclonal antibodies. Hybridomas secreting specific antibodies against these IROMPs were selected after screening by ELISA and their reactivity was confirmed by Western Blot. The antibodies then generated belonged to IgM isotype and showed bactericidical and opsonising activities against A.baumanii in vitro.These antibodies also blocked siderophore mediated iron uptake via IROMPs in bacteria. Conclusion This proves that iron uptake via IROMPs,which is mediated through siderophores,may have an important role in the survival of A.baumanii inside the host,and helps establishing the infection.

  17. Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production

    DEFF Research Database (Denmark)

    Pedersen, K.; Gram, Lone; Austin, D.A.;

    1997-01-01

    The virulence of 18 strains of Vibrio anguillarum serogroup 01 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only...... strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it...

  18. Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

    Directory of Open Access Journals (Sweden)

    Miranda Lo

    Full Text Available BACKGROUND: Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. METHODOLOGY/PRINCIPAL FINDINGS: To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS. We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. CONCLUSIONS/SIGNIFICANCE: This is the first study to compare transcriptional and translational responses to temperature

  19. Transport rectification in nanopores with outer membranes modified with surface charges and polyelectrolytes.

    Science.gov (United States)

    Tagliazucchi, Mario; Rabin, Yitzhak; Szleifer, Igal

    2013-10-22

    This work reports a comprehensive theoretical study of the transport-rectification properties of cylindrical nanopores with neutral inner walls and chemically modified outer membrane. The chemical species on the two outer sides of the membrane have charges of opposite sign and can be either surface-confined species (i.e., surface charges) or polyelectrolyte brushes. The advantage of this design over other types of rectifying nanopores is that it requires controlling the composition of the outer walls of the pore (which are easy to access) rather than the inner walls, thus simplifying the fabrication process. Ion-current rectification in nanopores with charged outer walls is ascribed to applied-potential-induced changes in the ionic concentration within the pore. The rectification efficiency is studied as a function of pore length, radius, surface charge and bulk electrolyte concentration. An analytical model is derived for the case of surface-confined charges that predicts the current-potential curves in very good agreement with the numerical calculations. Neutral nanopores with polyelectrolyte-modified outer walls have two distinct advantages compared to surface-charged systems: (i) they exhibit higher rectification factors due to the large charge density immobilized by the polyelectrolyte brushes, and (ii) the applied potential deforms the polyelectrolyte chains toward the oppositely charged electrode. This deformation brings the polyelectrolyte brushes into the pore in the low conductivity state and expels them from the pore in the high conductivity regime. Calculations of the potentials of mean-force suggest that the applied-field-induced conformational changes can be used to control the translocation of cargoes larger than ions, such as proteins and nanoparticles.

  20. Outer Mitochondrial Membrane Localization of Apoptosis-Inducing Factor: Mechanistic Implications for Release

    Directory of Open Access Journals (Sweden)

    Seong-Woon Yu

    2009-10-01

    Full Text Available Poly(ADP-ribose polymerase-1-dependent cell death (known as parthanatos plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor, but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.

  1. Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

    Directory of Open Access Journals (Sweden)

    Seong‑Woon Yu

    2009-11-01

    Full Text Available Poly(ADP-ribose polymerase-1-dependent cell death (known as parthanatos plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor, but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.

  2. Appoptosin interacts with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

    Science.gov (United States)

    Zhang, Cuilin; Shi, Zhun; Zhang, Lingzhi; Zhou, Zehua; Zheng, Xiaoyuan; Liu, Guiying; Bu, Guojun; Fraser, Paul E; Xu, Huaxi; Zhang, Yun-Wu

    2016-03-01

    Mitochondrial morphology is regulated by fusion and fission machinery. Impaired mitochondria dynamics cause various diseases, including Alzheimer's disease. Appoptosin (encoded by SLC25A38) is a mitochondrial carrier protein that is located in the mitochondrial inner membrane. Appoptosin overexpression causes overproduction of reactive oxygen species (ROS) and caspase-dependent apoptosis, whereas appoptosin downregulation abolishes β-amyloid-induced mitochondrial fragmentation and neuronal death during Alzheimer's disease. Herein, we found that overexpression of appoptosin resulted in mitochondrial fragmentation in a manner independent of its carrier function, ROS production or caspase activation. Although appoptosin did not affect levels of mitochondrial outer-membrane fusion (MFN1 and MFN2), inner-membrane fusion (OPA1) and fission [DRP1 (also known as DNM1L) and FIS1] proteins, appoptosin interacted with MFN1 and MFN2, as well as with the mitochondrial ubiquitin ligase MITOL (also known as MARCH5) but not OPA1, FIS1 or DRP1. Appoptosin overexpression impaired the interaction between MFN1 and MFN2, and mitochondrial fusion. By contrast, co-expression of MFN1, MITOL and a dominant-negative form of DRP1, DRP1(K38A), partially rescued appoptosin-induced mitochondrial fragmentation and apoptosis, whereas co-expression of FIS1 aggravated appoptosin-induced apoptosis. Together, our results demonstrate that appoptosin can interact with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

  3. Identification of immunogenic outer membrane proteins of Haemophilus influenzae type b in the infant rat model system

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, E.J.; Frisch, C.F.; McDade, R.L. Jr.; Johnston, K.H.

    1981-06-01

    Outer membrane proteins of Haemophilus influenzae type b which are immunogenic in infant rats were identified by a radioimmunoprecipitation method. Intact cells of H. influenzae type b were radioiodinated by a lactoperoxidase-catalyzed procedure, and an outer membrane-containing fraction was prepared from these cells. These radioiodinated outer membranes were mixed with sera obtained from rats convalescing from systemic H. influenzae type b disease induced at 6 days of age, and the resultant (antibody-outer membrane protein antigen) complexes were extracted from these membranes by treatment with nonionic detergent and ethylenediaminetetraacetic acid. These soluble antibody-antigen complexes were isolated by means of adsorption to protein A-bearing staphylococci, and the radioiodinated protein antigens were identified by gel electrophoresis followed by autoradiography. Infant rats were shown to mount a readily detectable antibody response to several different proteins present in the outer membrane of H. influenzae type b. Individual infant rats were found to vary both qualitatively and quantitatively in their immune response to these immunogenic outer membrane proteins.

  4. Components of SurA required for outer membrane biogenesis in uropathogenic Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Kristin M Watts

    Full Text Available BACKGROUND: SurA is a periplasmic peptidyl-prolyl isomerase (PPIase and chaperone of Escherichia coli and other Gram-negative bacteria. In contrast to other PPIases, SurA appears to have a distinct role in chaperoning newly synthesized porins destined for insertion into the outer membrane. Previous studies have indicated that the chaperone activity of SurA rests in its "core module" (the N- plus C-terminal domains, based on in vivo envelope phenotypes and in vitro binding and protection of non-native substrates. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we determined the components of SurA required for chaperone activity using in vivo phenotypes relevant to disease causation by uropathogenic E. coli (UPEC, namely membrane resistance to permeation by antimicrobials and maturation of the type 1 pilus usher FimD. FimD is a SurA-dependent, integral outer membrane protein through which heteropolymeric type 1 pili, which confer bladder epithelial binding and invasion capacity upon uropathogenic E. coli, are assembled and extruded. Consistent with prior results, the in vivo chaperone activity of SurA in UPEC rested primarily in the core module. However, the PPIase domains I and II were not expendable for wild-type resistance to novobiocin in broth culture. Steady-state levels of FimD were substantially restored in the UPEC surA mutant complemented with the SurA N- plus C-terminal domains. The addition of PPIase domain I augmented FimD maturation into the outer membrane, consistent with a model in which domain I enhances stability of and/or substrate binding by the core module. CONCLUSIONS/SIGNIFICANCE: Our results confirm the core module of E. coli SurA as a potential target for novel anti-infective development.

  5. Outer Membrane Proteins Derived from Non-cyanobacterial Lineage Cover the Peptidoglycan of Cyanophora paradoxa Cyanelles and Serve as a Cyanelle Diffusion Channel.

    Science.gov (United States)

    Kojima, Seiji; Muramoto, Koji; Kusano, Tomonobu

    2016-09-16

    The cyanelle is a primitive chloroplast that contains a peptidoglycan layer between its inner and outer membranes. Despite the fact that the envelope structure of the cyanelle is reminiscent of Gram-negative bacteria, the Cyanophora paradoxa genome appears to lack genes encoding homologs of putative peptidoglycan-associated outer membrane proteins and outer membrane channels. These are key components of Gram-negative bacterial membranes, maintaining structural stability and regulating permeability of outer membrane, respectively. Here, we discovered and characterized two dominant peptidoglycan-associated outer membrane proteins of the cyanelle (∼2 × 10(6) molecules per cyanelle). We named these proteins CppF and CppS (cyanelle peptidoglycan-associated proteins). They are homologous to each other and function as a diffusion channel that allows the permeation of compounds with Mr outer membrane components, providing a physical link with the underlying peptidoglycan layer and functioning as a diffusion route for various small substances across the outer membrane.

  6. Resistance of Escherichia coli to nourseothricin (streptothricin): sensitization of resistant strains by abolition of its outer membrane resistance.

    Science.gov (United States)

    Seltmann, G

    1992-01-01

    The polycationic antibiotic, nourseothricin, represents a mixture of several streptothricins, mainly D and F. The molecular weight of the latter compound amounts to 486. Obviously, although very slowly, it can pass the outer membrane via the porin pores. It has been shown earlier that nourseothricin is able to generate some kind of channels into the outer membrane through which it can pass the cell wall. On the other hand, there were indications that resistant strains containing a streptothricin-inactivating acetyl transferase possess an additional protecting system, namely a reduced penetrability of the outer membrane. In this study, it could be shown that such strains indeed could be rendered sensitive by damaging the barrier function of the outer membrane.

  7. Use of a purified outer membrane protein F (porin) preparation of Pseudomonas aeruginosa as a protective vaccine in mice.

    OpenAIRE

    Gilleland, H. E.; Parker, M G; Matthews, J.M.; Berg, R D

    1984-01-01

    The outer membrane protein F (porin) from the PAO1 strain of Pseudomonas aeruginosa was purified by two different methods. One procedure involved separation by column chromatography of proteins extracted from isolated outer membranes, whereas the other involved extraction from gels after slab polyacrylamide gel electrophoresis of proteins extracted from cell envelopes. Both procedures yielded protein F preparations which successfully immunized mice from subsequent challenge with the PAO1 stra...

  8. Cytotoxic effects of Kingella kingae outer membrane vesicles on human cells.

    Science.gov (United States)

    Maldonado, R; Wei, R; Kachlany, S C; Kazi, M; Balashova, N V

    2011-01-01

    Kingella kingae is an emerging pathogen causing osteoarticular infections in pediatric patients. Electron microscopy of K. kingae clinical isolates revealed the heterogeneously-sized membranous structures blebbing from the outer membrane that were classified as outer membrane vesicles (OMVs). OMVs purified from the secreted fraction of a septic arthritis K. kingae isolate were characterized. Among several major proteins, K. kingae OMVs contained virulence factors RtxA toxin and PilC2 pilus adhesin. RtxA was also found secreted as a soluble protein in the extracellular environment indicating that the bacterium may utilize different mechanisms for the toxin delivery. OMVs were shown to be hemolytic and possess some leukotoxic activity while high leukotoxicity was detected in the non-hemolytic OMV-free component of the secreted fraction. OMVs were internalized by human osteoblasts and synovial cells. Upon interaction with OMVs, the cells produced increased levels of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL-6) suggesting that these cytokines might be involved in the signaling response of infected joint and bone tissues during natural K. kingae infection. This study is the first report of OMV production by K. kingae and demonstrates that OMVs are a complex virulence factor of the organism causing cytolytic and inflammatory effects on host cells.

  9. Evolution of outer membrane beta-barrels from an ancestral beta beta hairpin.

    Science.gov (United States)

    Remmert, M; Biegert, A; Linke, D; Lupas, A N; Söding, J

    2010-06-01

    Outer membrane beta-barrels (OMBBs) are the major class of outer membrane proteins from Gram-negative bacteria, mitochondria, and plastids. Their transmembrane domains consist of 8-24 beta-strands forming a closed, barrel-shaped beta-sheet around a central pore. Despite their obvious structural regularity, evidence for an origin by duplication or for a common ancestry has not been found. We use three complementary approaches to show that all OMBBs from Gram-negative bacteria evolved from a single, ancestral beta beta hairpin. First, we link almost all families of known single-chain bacterial OMBBs with each other through transitive profile searches. Second, we identify a clear repeat signature in the sequences of many OMBBs in which the repeating sequence unit coincides with the structural beta beta hairpin repeat. Third, we show that the observed sequence similarity between OMBB hairpins cannot be explained by structural or membrane constraints on their sequences. The third approach addresses a longstanding problem in protein evolution: how to distinguish between a very remotely homologous relationship and the opposing scenario of "sequence convergence." The origin of a diverse group of proteins from a single hairpin module supports the hypothesis that, around the time of transition from the RNA to the protein world, proteins arose by amplification and recombination of short peptide modules that had previously evolved as cofactors of RNAs.

  10. SNAREs Interact with Retinal Degeneration Slow and Rod Outer Segment Membrane Protein-1 during Conventional and Unconventional Outer Segment Targeting.

    Science.gov (United States)

    Zulliger, Rahel; Conley, Shannon M; Mwoyosvi, Maggie L; Stuck, Michael W; Azadi, Seifollah; Naash, Muna I

    2015-01-01

    Mutations in the photoreceptor protein peripherin-2 (also known as RDS) cause severe retinal degeneration. RDS and its homolog ROM-1 (rod outer segment protein 1) are synthesized in the inner segment and then trafficked into the outer segment where they function in tetramers and covalently linked larger complexes. Our goal is to identify binding partners of RDS and ROM-1 that may be involved in their biosynthetic pathway or in their function in the photoreceptor outer segment (OS). Here we utilize several methods including mass spectrometry after affinity purification, in vitro co-expression followed by pull-down, in vivo pull-down from mouse retinas, and proximity ligation assay to identify and confirm the SNARE proteins Syntaxin 3B and SNAP-25 as novel binding partners of RDS and ROM-1. We show that both covalently linked and non-covalently linked RDS complexes interact with Syntaxin 3B. RDS in the mouse is trafficked from the inner segment to the outer segment by both conventional (i.e., Golgi dependent) and unconventional secretory pathways, and RDS from both pathways interacts with Syntaxin3B. Syntaxin 3B and SNAP-25 are enriched in the inner segment (compared to the outer segment) suggesting that the interaction with RDS/ROM-1 occurs in the inner segment. Syntaxin 3B and SNAP-25 are involved in mediating fusion of vesicles carrying other outer segment proteins during outer segment targeting, so could be involved in the trafficking of RDS/ROM-1.

  11. Modulation of cytokines and transcription factors (T-Bet and GATA3 in CD4 enriched cervical cells of Chlamydia trachomatis infected fertile and infertile women upon stimulation with chlamydial inclusion membrane proteins B and C

    Directory of Open Access Journals (Sweden)

    Salhan Sudha

    2009-08-01

    Full Text Available Abstract Background Chlamydial Inclusion membrane proteins (Incs, are involved in biochemical interactions with host cells and infecting Chlamydiae. We have previously reported the role of two Chlamydia trachomatis (CT Incs, namely IncB and IncC in generating host immunity in CT infected women. Emerging data shows involvement of Inc stimulated CD4 positive T cells in aiding host immunity in infected fertile and infertile women through the secretion of interferon gamma. However the lack of data on the intra-cytokine interplay to these Incs in infected cell milieu prompted us to investigate further. Methods A total of 14 CT-positive fertile, 18 CT-positive infertile women and 25 uninfected controls were enrolled in this study. CD8 depleted, CD4 enriched cervical cells were isolated and upon stimulation with IncB and IncC, modulation of cytokines (Interleukin (IL-1 Beta, IL-4, IL-5, IL-6, IL-10, Interferon-gamma, IL-12, IL-23, Tumor Necrosis Factor-alpha and Granulocyte macrophage colony-stimulating factor (GM-CSF and T cell lineage regulating transcription factors T-Bet and GATA3 was determined by real-time reverse-transcriptase (RT-PCR and ELISA. Results Significant higher expression (P Conclusion Overall our data shows that CT IncB and IncC are able to upregulate expression of cytokines, namely interferon-gamma, IL-12, IL-23 and GM-CSF in CT-positive fertile women while expression of IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 were upregulated in CT-positive infertile women. Our study also suggests that Incs are able to modulate expression of T cell lineage determinants indicating their involvement in regulation of immune cells.

  12. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane.

    Science.gov (United States)

    Alakomi, H L; Skyttä, E; Saarela, M; Mattila-Sandholm, T; Latva-Kala, K; Helander, I M

    2000-05-01

    The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.

  13. Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form

    Directory of Open Access Journals (Sweden)

    Oscarsson Jan

    2008-01-01

    Full Text Available Abstract Background Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. Results The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S and in its spontaneous laboratory variant (D7SS resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 μm, AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05–0.2 μm were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. Conclusion Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.

  14. Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane.

    Science.gov (United States)

    Wang, Yan; Andole Pannuri, Archana; Ni, Dongchun; Zhou, Haizhen; Cao, Xiou; Lu, Xiaomei; Romeo, Tony; Huang, Yihua

    2016-05-06

    The partially de-N-acetylated poly-β-1,6-N-acetyl-d-glucosamine (dPNAG) polymer serves as an intercellular biofilm adhesin that plays an essential role for the development and maintenance of integrity of biofilms of diverse bacterial species. Translocation of dPNAG across the bacterial outer membrane is mediated by a tetratricopeptide repeat-containing outer membrane protein, PgaA. To understand the molecular basis of dPNAG translocation, we determined the crystal structure of the C-terminal transmembrane domain of PgaA (residues 513-807). The structure reveals that PgaA forms a 16-strand transmembrane β-barrel, closed by four loops on the extracellular surface. Half of the interior surface of the barrel that lies parallel to the translocation pathway is electronegative, suggesting that the corresponding negatively charged residues may assist the secretion of the positively charged dPNAG polymer. In vivo complementation assays in a pgaA deletion bacterial strain showed that a cluster of negatively charged residues proximal to the periplasm is necessary for biofilm formation. Biochemical analyses further revealed that the tetratricopeptide repeat domain of PgaA binds directly to the N-deacetylase PgaB and is critical for biofilm formation. Our studies support a model in which the positively charged PgaB-bound dPNAG polymer is delivered to PgaA through the PgaA-PgaB interaction and is further targeted to the β-barrel lumen of PgaA potentially via a charge complementarity mechanism, thus priming the translocation of dPNAG across the bacterial outer membrane.

  15. Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

    OpenAIRE

    Alakomi, H.-L.; Skyttä, E.; Saarela, M; Mattila-Sandholm, T.; Latva-Kala, K.; Helander, I M

    2000-01-01

    The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH...

  16. Dissociation of outer membrane for Escherichia coli cell caused by cerium nitrate

    Institute of Scientific and Technical Information of China (English)

    陈爱美; 施庆珊; 冯劲; 欧阳友生; 陈仪本; 谭绍早

    2010-01-01

    The biological effect of cerium nitrate on the outer membrane(OM) of Escherichia coli(E.coli) cell was studied,and the antim-icrobial mechanism of rare earth elements was explored.The antimicrobial effect of cerium nitrate on E.coli cell was valued by plate count method,and the morphology change of E.coli cell was observed with scanning electron microscopy(SEM) and transmission electron microscopy(TEM).The results showed that the E.coli cell suspension was flocculated when the concentration of Ce(NO3)3?6H2O...

  17. Discovery of an archetypal protein transport system in bacterial outer membranes.

    Science.gov (United States)

    Selkrig, Joel; Mosbahi, Khedidja; Webb, Chaille T; Belousoff, Matthew J; Perry, Andrew J; Wells, Timothy J; Morris, Faye; Leyton, Denisse L; Totsika, Makrina; Phan, Minh-Duy; Celik, Nermin; Kelly, Michelle; Oates, Clare; Hartland, Elizabeth L; Robins-Browne, Roy M; Ramarathinam, Sri Harsha; Purcell, Anthony W; Schembri, Mark A; Strugnell, Richard A; Henderson, Ian R; Walker, Daniel; Lithgow, Trevor

    2012-04-01

    Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.

  18. Calcium pump in the disk membranes isolated from bovine retinal rod outer segments.

    Science.gov (United States)

    Panfoli, I; Morelli, A; Pepe, I M

    1994-08-01

    The existence of a Ca2+ pump in rod outer segment disks of bovine retina is strongly suggested by the isolation on sodium dodecyl sulfate polyacrylamide gel electrophoresis of a hydroxylamine-sensitive phosphorylated intermediate (E-P) of molecular mass of about 100 kDa as well as by measurements of active calcium transport and adenosine 5'-triphosphate (ATP) hydrolysis. Active Ca2+ uptake by disks was dependent on the presence of Mg(2+)-ATP, was inhibited by vanadate or lanthanum and appeared poorly sensitive to calmodulin. ATP hydrolysis by disk membranes was a function of free Ca2+ concentration in the absence of exogenous Mg2+. The presence of a Ca2+ pump on disk membranes is discussed in terms of its possible role in Ca2+ ion buffering during photoreceptor cell functioning.

  19. Rhizobium strains differ considerably in outer membrane permeability and polymyxin B resistance.

    Science.gov (United States)

    Komaniecka, Iwona; Zamłyńska, Katarzyna; Zan, Radosław; Staszczak, Magdalena; Pawelec, Jarosław; Seta, Irena; Choma, Adam

    2016-01-01

    Six rhizobium (Rhizobium leguminosarum bv. Trifolii TA1, Sinorhizobium meliloti 1021, Mesorhizobium huakuii IFO 15243(T), Ochrobactrum lupini LUP 21(T), Bradyrhizobium japonicum USDA110 and B. elkanii USDA 76) and two Escherichia coli strains (E. coli ATCC 25922 and E. coli HB 101) were compared in respect to polymyxin B and EDTA resistance, as well as bacterial outer membrane (OM) permeability to a fluorescent hydrophobic agent (N-phenyl-1-naphthylamine - NPN). TEM (Transmission Electron Microscopy) and a microbial test demonstrated that all the rhizobia were much more resistant to polymyxin B in comparison with E. coli strains. EDTA and polymyxin B enhance permeability of B. japonicum and O. lupini OM. Other rhizobia incorporated NPN independently of the presence of membrane-deteriorating agents; however, the level of fluorescence (measured as NPN absorption) was strain dependent.

  20. Knowledge of Chlamydial Infection among University Students.

    Science.gov (United States)

    White, David M.; Felts, W. Michael

    1989-01-01

    Results of a study designed to assess knowledge about chlamydial infection among university students (N=258) revealed serious gaps in knowledge. Data suggests that students held several misconceptions, or had no knowledge, regarding symptoms, transmission, treatment, and testing for chlamydial infection. (IAH)

  1. Deuterium Labeling Strategies for Creating Contrast in Structure-Function Studies of Model Bacterial Outer Membranes Using Neutron Reflectometry.

    Science.gov (United States)

    Le Brun, Anton P; Clifton, Luke A; Holt, Stephen A; Holden, Peter J; Lakey, Jeremy H

    2016-01-01

    Studying the outer membrane of Gram-negative bacteria is challenging due to the complex nature of its structure. Therefore, simplified models are required to undertake structure-function studies of processes that occur at the outer membrane/fluid interface. Model membranes can be created by immobilizing bilayers to solid supports such as gold or silicon surfaces, or as monolayers on a liquid support where the surface pressure and fluidity of the lipids can be controlled. Both model systems are amenable to having their structure probed by neutron reflectometry, a technique that provides a one-dimensional depth profile through a membrane detailing its thickness and composition. One of the strengths of neutron scattering is the ability to use contrast matching, allowing molecules containing hydrogen and those enriched with deuterium to be highlighted or matched out against the bulk isotopic composition of the solvent. Lipopolysaccharides, a major component of the outer membrane, can be isolated for incorporation into model membranes. Here, we describe the deuteration of lipopolysaccharides from rough strains of Escherichia coli for incorporation into model outer membranes, and how the use of deuterated materials enhances structural analysis of model membranes by neutron reflectometry.

  2. Molecular and virulence characteristics of an outer membrane-associated RTX exoprotein in Pasteurella pneumotropica

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    Kawamoto Eiichi

    2011-03-01

    Full Text Available Abstract Background Pasteurella pneumotropica is a ubiquitous bacterium that is frequently isolated from laboratory rodents and causes various clinical symptoms in immunodeficient animals. Currently two RTX toxins, PnxIA and PnxIIA, which are similar to hemolysin-like high-molecular-weight exoproteins are known in this species. In this study, we identified and analyzed a further RTX toxin named PnxIIIA and the corresponding type I secretion system. Results The RTX exoprotein, PnxIIIA, contains only a few copies of the RTX repeat-like sequence and 3 large repeat sequences that are partially similar to the outer membrane protein found in several prokaryotes. Recombinant PnxIIIA protein (rPnxIIIA was cytotoxic toward J774A.1 mouse macrophage cells, whereas cytotoxicity was attenuated by the addition of anti-CD11a monoclonal antibody. rPnxIIIA could bind to extracellular matrices (ECMs and cause hemagglutination of sheep erythrocytes. Binding was dependent on the 3 large repeat sequences in PnxIIIA. Protein interaction analyses indicated that PnxIIIA is mainly localized in the outer membrane of P. pneumotropica ATCC 35149 in a self-assembled oligomeric form. PnxIIIA is less cytotoxic to J774A.1 cells than PnxIA and PnxIIA. Conclusions The results implicate that PnxIIIA is located on the cell surface and participates in adhesion to ECMs and enhanced hemagglutination in the rodent pathogen P. pneumotropica.

  3. An outer membrane receptor of Neisseria meningitidis involved in zinc acquisition with vaccine potential.

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    Michiel Stork

    Full Text Available Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.

  4. Expression,Characterization and Immunogenicity of a Major Outer Membrane Protein from Vibrio alginolyticus

    Institute of Scientific and Technical Information of China (English)

    Ronghua QIAN; Wuying CHU; Zhijuan MAO; Chongwen ZHANG; Yongwei WEI; Lian YU

    2007-01-01

    Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine animals.During infection and induction of the host immune response,outer membrane proteins of bacteria play an important role.In this study,an outer membrane protein gene (ompW) was cloned from V. alginolyticus and expressed in Escherichia coli.The 645 bp open reading frame (ORF) encodes a protein of 214 amino acid residues with a predicted molecular weight of 23.3 kDa.The amino acid sequence showed a high identity with that of Photobacterium damselae(96.2%) and Vibrio parahaemolyticus(94.4%).The alignment analysis indicated that OmpW was highly conserved.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the gene was over-expressed in E.coli BL21(DE3).Western blot analysis revealed that the expressed protein had immunoreactivity.The recombinant protein was purified by affinity chromatography on Ni-NTA Superflow resin.Large yellow croaker vaccinated with the purified OmpW showed significantly increased antibody to OmpW,which could resist the infection by V.alginolyticus.A specific antibody was detected by enzyme-linked immunosorbent assay.This study suggested that the conserved OmpW could be an effective vaccine candidate against infection by V.alginolyticus.

  5. Wzi is an outer membrane lectin that underpins group 1 capsule assembly in Escherichia coli.

    Science.gov (United States)

    Bushell, Simon R; Mainprize, Iain L; Wear, Martin A; Lou, Hubing; Whitfield, Chris; Naismith, James H

    2013-05-07

    Many pathogenic bacteria encase themselves in a polysaccharide capsule that provides a barrier to the physical and immunological challenges of the host. The mechanism by which the capsule assembles around the bacterial cell is unknown. Wzi, an integral outer-membrane protein from Escherichia coli, has been implicated in the formation of group 1 capsules. The 2.6 Å resolution structure of Wzi reveals an 18-stranded β-barrel fold with a novel arrangement of long extracellular loops that blocks the extracellular entrance and a helical bundle that plugs the periplasmic end. Mutagenesis shows that specific extracellular loops are required for in vivo capsule assembly. The data show that Wzi binds the K30 carbohydrate polymer and, crucially, that mutants functionally deficient in vivo show no binding to K30 polymer in vitro. We conclude that Wzi is a novel outer-membrane lectin that assists in the formation of the bacterial capsule via direct interaction with capsular polysaccharides.

  6. Wzi Is an Outer Membrane Lectin that Underpins Group 1 Capsule Assembly in Escherichia coli

    Science.gov (United States)

    Bushell, Simon R.; Mainprize, Iain L.; Wear, Martin A.; Lou, Hubing; Whitfield, Chris; Naismith, James H.

    2013-01-01

    Summary Many pathogenic bacteria encase themselves in a polysaccharide capsule that provides a barrier to the physical and immunological challenges of the host. The mechanism by which the capsule assembles around the bacterial cell is unknown. Wzi, an integral outer-membrane protein from Escherichia coli, has been implicated in the formation of group 1 capsules. The 2.6 Å resolution structure of Wzi reveals an 18-stranded β-barrel fold with a novel arrangement of long extracellular loops that blocks the extracellular entrance and a helical bundle that plugs the periplasmic end. Mutagenesis shows that specific extracellular loops are required for in vivo capsule assembly. The data show that Wzi binds the K30 carbohydrate polymer and, crucially, that mutants functionally deficient in vivo show no binding to K30 polymer in vitro. We conclude that Wzi is a novel outer-membrane lectin that assists in the formation of the bacterial capsule via direct interaction with capsular polysaccharides. PMID:23623732

  7. Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT from Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    Uhlin Bernt

    2009-10-01

    Full Text Available Abstract Background Background: Cytolethal distending toxin (CDT is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment. Results Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50% of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form. Conclusion Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC to the surrounding environment, including infected host tissue.

  8. Bacterial origin of a mitochondrial outer membrane protein translocase: new perspectives from comparative single channel electrophysiology.

    Science.gov (United States)

    Harsman, Anke; Niemann, Moritz; Pusnik, Mascha; Schmidt, Oliver; Burmann, Björn M; Hiller, Sebastian; Meisinger, Chris; Schneider, André; Wagner, Richard

    2012-09-07

    Mitochondria are of bacterial ancestry and have to import most of their proteins from the cytosol. This process is mediated by Tom40, an essential protein that forms the protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic translocase of the outer mitochondrial membrane (ATOM), a protein that shows similarities to both eukaryotic Tom40 and bacterial protein translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other protein translocases reveals that ATOM closely resembles bacterial-type protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes.

  9. Structure of BamA, an essential factor in outer membrane protein biogenesis.

    Science.gov (United States)

    Albrecht, Reinhard; Schütz, Monika; Oberhettinger, Philipp; Faulstich, Michaela; Bermejo, Ivan; Rudel, Thomas; Diederichs, Kay; Zeth, Kornelius

    2014-06-01

    Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous β-barrel with impaired β1-β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.

  10. Structural and functional importance of outer membrane proteins in Vibrio cholerae flagellum.

    Science.gov (United States)

    Bari, Wasimul; Lee, Kang-Mu; Yoon, Sang Sun

    2012-08-01

    Vibrio cholerae has a sheath-covered monotrichous flagellum that is known to contribute to virulence. Although the structural organization of the V. cholerae flagellum has been extensively studied, the involvement of outer membrane proteins as integral components in the flagellum still remains elusive. Here we show that flagella produced by V. cholerae O1 El Tor strain C6706 were two times thicker than those from two other Gram-negative bacteria. A C6706 mutant strain (SSY11) devoid of two outer membrane proteins (OMPs), OmpU and OmpT, produced thinner flagella. SSY11 showed significant defects in the flagella-mediated motility as compared to its parental strain. Moreover, increased shedding of the flagella-associated proteins was observed in the culture supernatant of SSY11. This finding was also supported by the observation that culture supernatants of the SSY11 strain induced the production of a significantly higher level of IL-8 in human colon carcinoma HT29 and alveolar epithelial A549 cells than those of the wild-type C6706 strain. These results further suggest a definite role of these two OMPs in providing the structural integrity of the V. cholerae flagellum as part of the surrounding sheath.

  11. Immunoproteomic Analysis ofBordetella bronchisepticaOuter Membrane Proteins and Identiifcation of New Immunogenic Proteins

    Institute of Scientific and Technical Information of China (English)

    JI Quan-an

    2014-01-01

    Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine ofB. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted fromB. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identiifed by matrix-assisted laser desorption/ionization time of lfight-mass spectrometry (MALDI-TOF-MS), a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins forB. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB (B. bronchiseptica strain isolated from a infected rabbit). The mortality of mice was 80% compared to 100% of positive controls. The identiifcation of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.

  12. Intranasal immunization with nontypeable Haemophilus influenzae outer membrane vesicles induces cross-protective immunity in mice.

    Directory of Open Access Journals (Sweden)

    Sandro Roier

    Full Text Available Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.

  13. Proteomic analysis of Lawsonia intracellularis reveals expression of outer membrane proteins during infection.

    Science.gov (United States)

    Watson, Eleanor; Alberdi, M Pilar; Inglis, Neil F; Lainson, Alex; Porter, Megan E; Manson, Erin; Imrie, Lisa; Mclean, Kevin; Smith, David G E

    2014-12-05

    Lawsonia intracellularis is the aetiological agent of the commercially significant porcine disease, proliferative enteropathy. Current understanding of host-pathogen interaction is limited due to the fastidious microaerophilic obligate intracellular nature of the bacterium. In the present study, expression of bacterial proteins during infection was investigated using a mass spectrometry approach. LC-ESI-MS/MS analysis of two isolates of L. intracellularis from heavily-infected epithelial cell cultures and database mining using fully annotated L. intracellularis genome sequences identified 19 proteins. According to the Clusters of Orthologous Groups (COG) functional classification, proteins were identified with roles in cell metabolism, protein synthesis and oxidative stress protection; seven proteins with putative or unknown function were also identified. Detailed bioinformatic analyses of five uncharacterised proteins, which were expressed by both isolates, identified domains and motifs common to other outer membrane-associated proteins with important roles in pathogenesis including adherence and invasion. Analysis of recombinant proteins on Western blots using immune sera from L. intracellularis-infected pigs identified two proteins, LI0841 and LI0902 as antigenic. The detection of five outer membrane proteins expressed during infection, including two antigenic proteins, demonstrates the potential of this approach to interrogate L. intracellularis host-pathogen interactions and identify novel targets which may be exploited in disease control.

  14. Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane

    Directory of Open Access Journals (Sweden)

    Philip Michael Day

    2014-10-01

    Full Text Available Translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75 is the core component of the chloroplast protein import machinery. It belongs to the Omp85 family whose members exist in various Gram-negative bacteria, mitochondria and chloroplasts of eukaryotes. Chloroplasts of Viridiplantae contain another Omp85 homolog called outer envelope protein 80 (OEP80, whose exact function is unknown. In addition, the Arabidopsis thaliana genome encodes truncated forms of Toc75 and OEP80. Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85, Toc75 and OEP80 are inconsistent. The bipartite targeting sequence-dependent sorting of Toc75 has been demonstrated but the targeting mechanisms of other chloroplast Omp85 homologs remain largely unexplored. This study was aimed to address these unresolved issues in order to further our understanding of chloroplast evolution. Sequence alignments and recently determined structures of bacterial Omp85 homologs were used to predict structures of chloroplast Omp85 homologs. The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80. Phylogenetic analyses using Omp85 homologs from various cyanobacteria and chloroplasts provided strong support for the grouping of Toc75 and OEP80 sister to cyanoOmp85. However, this support was diminished when the analysis included Omp85 homologs from other bacteria and mitochondria. Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

  15. Surface display of a borrelial lipoprotein on meningococcal outer membrane vesicles.

    Science.gov (United States)

    Salverda, Merijn L M; Meinderts, Sanne M; Hamstra, Hendrik-Jan; Wagemakers, Alex; Hovius, Joppe W R; van der Ark, Arno; Stork, Michiel; van der Ley, Peter

    2016-02-17

    Outer Membrane Vesicles (OMVs) are gaining attention as vaccine candidates. The successful expression of heterologous antigens in OMVs, with the OMV functioning both as adjuvant and delivery vehicle, has greatly enhanced their vaccine potential. Since there are indications that surface exposed antigens might induce a superior immune response, targeting of heterologous antigens to the OMV surface is of special interest. Several systems for surface display of heterologous antigens on OMVs have been developed. However, these systems have not been used to display lipidated membrane-associated proteins known as lipoproteins, which are emerging as key targets for protective immunity. We were therefore interested to see whether we could express a foreign lipoprotein on the outer surface of OMVs. When outer surface protein A (OspA), a borrelial surface-exposed lipoprotein, was expressed in meningococci, it was found that although OspA was present in OMVs, it was no longer surface-exposed. Therefore, a set of fusions of OspA to different regions of factor H binding protein (fHbp), a meningococcal surface-exposed lipoprotein, were designed and tested for their surface-exposure. An N-terminal part of fHbp was found to be necessary for the successful surface display of OspA on meningococcal OMVs. When mice were immunized with this set of OMVs, an OspA-specific antibody response was only elicited by OMVs with clearly surface-exposed OspA, strengthening the idea that the exact positioning of an antigen in the OMV affects the immune response. This method for the surface display of heterologous lipoproteins on OMVs is a step forward in the development of OMVs as a vaccine platform.

  16. Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

    Directory of Open Access Journals (Sweden)

    Annamari Paino

    Full Text Available Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI, was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control

  17. Outer-selective thin film composite (TFC) hollow fiber membranes for osmotic power generation

    KAUST Repository

    Le, Ngoc Lieu

    2016-01-14

    The pressure-retarded osmosis (PRO) process is a green technique for power generation to respond the world\\'s need of energy sustainability. In this study, we have developed the vital component of the process, i.e. membrane, in the configuration of the outer-selective thin-film composite (TFC) hollow fiber, which is more practical than other configurations in the real applications. The support layer morphology and the formation of the selective polyamide layer have been optimized for a good PRO performance. The results show that the bore fluid with higher amount of the solvent N-methyl-2-pyrrolidone leads to full finger-like hollow fibers, which provide higher flux but lower pressure tolerance. The addition of higher amount of diethylene glycol into the dope solution, improves the pore formation and suppresses the macrovoid formation, while properly lowering the take-up speed increases their wall thickness and pressure tolerance. A simple alcohol-pre-wetting approach on the fiber support leads to a smooth and thin polyamide layer, which is favorable for a high water flux and power density. Its efficiency follows this order: n-propanol>ethanol>methanol>water. The n-propanol pre-wetted TFC membrane can tolerate 17 bar with a peak power density of 9.59 W/m2 at room temperature, using 1 M NaCl solution as the draw solution and DI water as feed. This work demonstrates the potential of outer-selective TFC hollow fiber membranes for energy conversion via PRO process, provides useful database to fabricate suitable support morphology and raise a simple technique to practically form a thin and smooth polyamide layer.

  18. Discovery of Salmonella virulence factors translocated via outer membrane vesicles to murine macrophages.

    Science.gov (United States)

    Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N; Heffron, Fred

    2011-06-01

    Salmonella enterica serovar Typhimurium, an intracellular pathogen and leading cause of food-borne illness, encodes a plethora of virulence effectors. Salmonella virulence factors are translocated into host cells and manipulate host cellular activities, providing a more hospitable environment for bacterial proliferation. In this study, we report a new set of virulence factors that is translocated into the host cytoplasm via bacterial outer membrane vesicles (OMV). PagK (or PagK1), PagJ, and STM2585A (or PagK2) are small proteins composed of ∼70 amino acids and have high sequence homology to each other (>85% identity). Salmonella lacking all three homologues was attenuated for virulence in a mouse infection model, suggesting at least partial functional redundancy among the homologues. While each homologue was translocated into the macrophage cytoplasm, their translocation was independent of all three Salmonella gene-encoded type III secretion systems (T3SSs)-Salmonella pathogenicity island 1 (SPI-1) T3SS, SPI-2 T3SS, and the flagellar system. Selected methods, including direct microscopy, demonstrated that the PagK-homologous proteins were secreted through OMV, which were enriched with lipopolysaccharide (LPS) and outer membrane proteins. Vesicles produced by intracellular bacteria also contained lysosome-associated membrane protein 1 (LAMP1), suggesting the possibility of OMV convergence with host cellular components during intracellular trafficking. This study identified novel Salmonella virulence factors secreted via OMV and demonstrated that OMV can function as a vehicle to transfer virulence determinants to the cytoplasm of the infected host cell.

  19. Prestin-driven cochlear amplification is not limited by the outer hair cell membrane time constant.

    Science.gov (United States)

    Johnson, Stuart L; Beurg, Maryline; Marcotti, Walter; Fettiplace, Robert

    2011-06-23

    Outer hair cells (OHCs) provide amplification in the mammalian cochlea using somatic force generation underpinned by voltage-dependent conformational changes of the motor protein prestin. However, prestin must be gated by changes in membrane potential on a cycle-by-cycle basis and the periodic component of the receptor potential may be greatly attenuated by low-pass filtering due to the OHC time constant (τ(m)), questioning the functional relevance of this mechanism. Here, we measured τ(m) from OHCs with a range of characteristic frequencies (CF) and found that, at physiological endolymphatic calcium concentrations, approximately half of the mechanotransducer (MT) channels are opened at rest, depolarizing the membrane potential to near -40 mV. The depolarized resting potential activates a voltage-dependent K+ conductance, thus minimizing τ(m) and expanding the membrane filter so there is little receptor potential attenuation at the cell's CF. These data suggest that minimal τ(m) filtering in vivo ensures optimal activation of prestin.

  20. Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin.

    Science.gov (United States)

    Collin, Séverine; Guilvout, Ingrid; Nickerson, Nicholas N; Pugsley, Anthony P

    2011-05-01

    The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane.

  1. Outer membrane vesicles of Gallibacterium anatis induce protective immunity in egg-laying hens

    DEFF Research Database (Denmark)

    Pors, Susanne Elisabeth; Pedersen, Ida Just; Skjerning, Ragnhild Bager

    2016-01-01

    Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have...... previously shown promising results in protection against infections and we hypothesized that OMVs could serve as an immunogen to protect egg-laying hens against G. anatis. To investigate the immunogenic potential of G. anatis OMVs, two in vivo studies in egg-laying hens were made. The trials assessedthe...... degree of protection provided by immunization with G. anatis OMV against challenge and the IgY responses in serum after immunization and challenge, respectively. A total of 64 egg-laying hens were included in the trials. OMVs for immunization were produced and purified from a high-producing G. anatis...

  2. Iron repressible outer membrane proteins of Moraxella bovis and demonstration of siderophore-like activity.

    Science.gov (United States)

    Fenwick, B; Rider, M; Liang, J; Brightman, A

    1996-02-01

    Moraxella bovis (strain Epp 63), grown in RPMI 1640 medium supplemented with desferrioxamine mesylate (0.05 mg/ml) resulted in cell free culture supernatants with an increased chromeazurol-S response indicating the presence of high affinity iron binding ligand(s). Supernatants of cultures where growth occurred in tryptic soy broth, RPMI 1640, or RPMI 1640-desferrioxamine supplemented with ferrous sulfate (10 micrograms/ml) were negative on the chromeazurol-S test. Growth of M. bovis in RPMI 1640 or RPMI 1640-desferrioxamine medium induced the expression of previously unrecognized outer membrane proteins whose expression was repressed when the medium was supplemented with iron and which were not produced when growth occurred in tryptic soy broth.

  3. An immunoproteomic approach for characterization of the outer membrane proteins of Salmonella Gallinarum.

    Science.gov (United States)

    Cho, Youngjae; Sun, Jisun; Han, Jang Hyuck; Jang, Joo Hyun; Kang, Zheng Wu; Hahn, Tae-Wook

    2014-03-01

    Salmonella enterica serovar Gallinarum (SG) is an important pathogen that causes fowl typhoid in chickens. In order to investigate SG outer membrane proteins (OMPs) as potential vaccine candidate proteins, we established a proteomic map and database of antigenic SG-OMPs. A total of 174 spots were detected by 2DE. Twenty-two antigen-reactive spots were identified as nine specific proteins using PMF. OmpA was the most abundant protein among all of the identified OMPs, and it exhibited seven protein species. We conducted Western blot analysis for the SG-OMPs in order to determine which proteins were cross-reactive to the serovars Salmonella Enteritidis, Salmonella Typhimurium, and SG. Our results indicated that OmpA was considered to be an antigenic cross-reactive protein among the three serovars. This study sheds new light on our understanding of cross-protection among Salmonella serovars.

  4. Prokaryotic Expression and Identification of Outer Membrane Protein 2 of Chlamydia trachomatis

    Institute of Scientific and Technical Information of China (English)

    陈超群; 吴移谋; 李忠玉; 朱翠明; 尹卫国

    2004-01-01

    Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 × His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coil M15, providing an efficient and simple system for assaying the immunological properties of Omp2.

  5. In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli.

    Science.gov (United States)

    Larsen, Ray A; Letain, Tracy E; Postle, Kathleen

    2003-07-01

    Gram-negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in each membrane, a process called shuttling. A key feature of shuttling is that the amino-terminal signal anchor must quit its association with the cytoplasmic membrane, and TonB becomes associated solely with the outer membrane. However, the initial studies did not exclude the possibility that TonB was artifactually pulled from the cytoplasmic membrane by the fractionation process. To resolve this ambiguity, we devised a method to test whether the extreme TonB amino-terminus, located in the cytoplasm, ever became accessible to the cys-specific, cytoplasmic membrane-impermeant molecule, Oregon Green(R) 488 maleimide (OGM) in vivo. A full-length TonB and a truncated TonB were modified to carry a sole cysteine at position 3. Both full-length TonB and truncated TonB (consisting of the amino-terminal two-thirds) achieved identical conformations in the cytoplasmic membrane, as determined by their abilities to cross-link to the cytoplasmic membrane protein ExbB and their abilities to respond conformationally to the presence or absence of proton motive force. Full-length TonB could be amino-terminally labelled in vivo, suggesting that it was periplasmically exposed. In contrast, truncated TonB, which did not associate with the outer membrane, was not specifically labelled in vivo. The truncated TonB also acted as a control for leakage of OGM across the cytoplasmic membrane. Further, the extent of labelling for full-length TonB correlated roughly with the proportion of TonB found at the outer membrane. These findings suggest that TonB does indeed disengage from the cytoplasmic membrane during energy transduction and shuttle to the outer membrane.

  6. Detection of β-Lactamases and Outer Membrane Porins among Klebsiella pneumoniae Strains Isolated in Iran

    Directory of Open Access Journals (Sweden)

    Ali Hashemi

    2014-01-01

    Full Text Available This descriptive study was accomplished on 83 K. pneumoniae strains isolated from two hospitals in Tehran, Iran. Antibiotic susceptibility tests were performed by disc diffusion and broth microdilution methods. ESBLs, MBL, Amp-C, and KPC producing strains were detected by phenotypic confirmatory test, combination disk diffusion test (CDDT, Amp-C detection kit, and modified Hodge test, respectively. OXA-48, NDM-1, and CTX-M-15 genes were detected by PCR and sequencing methods. The outer membrane porins such as OmpK35 and OmpK36 were analysed by SDS-PAGE, PCR, and sequencing methods. From 83 K. pneumoniae isolates, 48 (57.5%, 3 (3.5%, 23 (28%, and 5 (6% were ESBL, MBL, Amp-C, and KPC positive, respectively. The CTX-M-15 gene was detected in 30 (62.5% and OXA-48 gene was found in 2 (4.1% of the 48 ESBL-producing isolates. Two isolates harboured both OXA-48 and CTX-M-15; NDM-1 gene was not detected in this study. Outer membrane porin, OmpK35, was detected in 30 (62.5% of 48 ESBL-producing isolates while OmpK36 was found in 35 (72.91% of 48 ESBL-producing isolates. In this study, fosfomycin and tigecycline were more effective than other antibiotics. The high prevalence of β-lactamase-producing K. pneumoniae detected in this study is of great concern, which requires infection control measures including antibacterial management and identification of β-lactamases-producing isolates.

  7. Outer membrane protein DsrA is the major fibronectin-binding determinant of Haemophilus ducreyi.

    Science.gov (United States)

    Leduc, Isabelle; White, C Dinitra; Nepluev, Igor; Throm, Robert E; Spinola, Stanley M; Elkins, Christopher

    2008-04-01

    The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains.

  8. Epoxide-mediated differential packaging of Cif and other virulence factors into outer membrane vesicles.

    Science.gov (United States)

    Ballok, Alicia E; Filkins, Laura M; Bomberger, Jennifer M; Stanton, Bruce A; O'Toole, George A

    2014-10-01

    Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions.

  9. Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    Flora Tomasello; Angela Messina; Lydia Lartigue; Laura Schembri; Chantal Medina; Simona Reina; Didier Thorava; Marc Crouzet; Francois Ichas; Vito De Pinto; Francesca De Giorgi

    2009-01-01

    Voltage-dependent anion channel (VDAC)l is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that over-expression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 ex-pression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-M1M crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-XL, indicative of PTP opera-tion. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.

  10. Folding outer membrane proteins independently of the β-barrel assembly machinery: an assembly pathway for multimeric complexes?

    Science.gov (United States)

    Huysmans, Gerard H M

    2016-06-15

    Since the discovery of the essential role of the β-barrel assembly machinery (BAM) for the membrane insertion of outer membrane proteins (OMPs) that are unrelated in sequence, members of this universally conserved family dominate discussions on OMP assembly in bacteria, mitochondria and chloroplasts. However, several multimeric bacterial OMPs assemble independently of the catalyzing BAM-component BamA. Recent progress on this alternative pathway is reviewed here, and a model for BAM-independent assembly for multimeric OMPs is proposed in which monomer delivery to the membrane and stable prepore formation are key steps towards productive membrane insertion.

  11. Localization of cytochromes in the outer membrane of Desulfovibrio vulgaris (Hildenborough) and their role in anaerobic biocorrosion.

    Science.gov (United States)

    Van Ommen Kloeke, F; Bryant, R D; Laishley, E J

    1995-12-01

    A protocol was developed whereby the outer and cytoplasmic membranes of the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) were isolated and partially characterized. The isolated outer membrane fractions from cultures grown under high (100 ppm) and low (5 ppm) Fe2+ conditions were compared by SDS-PAGE electrophoresis, and showed that several protein bands were derepressed under the low iron conditions, most notably at 50 kDa, and 77.5 kDa. Outer membrane isolated from low iron cultured cells was found to contain two proteins, 77.5 kDa and 62.5 kDa in size, that reacted with a heme-specific stain and were referred to as high molecular weight cytochromes. Studies conducted on the low iron isolated outer membrane by a phosphate/mild steel hydrogen evolution system showed that addition of the membrane fraction caused an immediate acceleration in H2 production. A new model for the anaerobic biocorrosion of mild steel is proposed.

  12. Direct observation of the uptake of outer membrane proteins by the periplasmic chaperone Skp.

    Directory of Open Access Journals (Sweden)

    Zhi-Xin Lyu

    Full Text Available The transportation of membrane proteins through the aqueous subcellular space is an important and challenging process. Its molecular mechanism and the associated structural change are poorly understood. Periplasmic chaperones, such as Skp in Escherichia coli, play key roles in the transportation and protection of outer membrane proteins (OMPs in Gram-negative bacteria. The molecular mechanism through which Skp interacts with and protects OMPs remains mysterious. Here, a combined experimental and molecular dynamics simulation study was performed to gain the structural and dynamical information in the process of OMPs and Skp binding. Stopped-flow experiments on site specific mutated and labeled Skp and several OMPs, namely OmpC, the transmembrane domain of OmpA, and OmpF, allowed us to obtain the mechanism of OMP entering the Skp cavity, and molecular dynamics simulations yielded detailed molecular interactions responsible for this process. Both experiment and simulation show that the entrance of OMP into Skp is a highly directional process, which is initiated by the interaction between the N-terminus of OMP and the bottom "tentacle" domain of Skp. The opening of the more flexible tentacle of Skp, the non-specific electrostatic interactions between OMP and Skp, and the constant formation and breaking of salt bridges between Skp and its substrate together allow OMP to enter Skp and gradually "climb" into the Skp cavity in the absence of an external energy supply.

  13. Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    Jennifer M Bomberger

    2009-04-01

    Full Text Available Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane-derived vesicles (OMV secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including beta-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP-mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.

  14. Dual effect of local anesthetics on the function of excitable rod outer segment disk membrane

    Energy Technology Data Exchange (ETDEWEB)

    Mashimo, T.; Abe, K.; Yoshiya, I.

    1986-04-01

    The effects of local anesthetics and a divalent cation, Ca2+, on the function of rhodopsin were estimated from the measurements of light-induced proton uptake. The light-induced proton uptake by rhodopsin in the rod outer segment disk membrane was enhanced at lower pH (4) but depressed at higher pHs (6 to 8) by the tertiary amine local anesthetics lidocaine, bupivacaine, tetracaine, and dibucaine. The order of local anesthetic-induced depression of the proton uptake followed that of their clinical anesthetic potencies. The depression of the proton uptake versus the concentration of the uncharged form of local anesthetic nearly describes the same curve for small and large dose of added anesthetic. Furthermore, a neutral local anesthetic, benzocaine, depressed the proton uptake at all pHs between 4 and 7. These results indicate that the depression of the proton uptake is due to the effect of only the uncharged form. It is hypothesized that the uncharged form of local anesthetics interacts hydrophobically with the rhodopsin in the disk membrane. The dual effect of local anesthetics on the proton uptake, on the other hand, suggests that the activation of the function of rhodopsin may be caused by the charged form. There was no significant change in the light-induced proton uptake by rhodopsin when 1 mM of Ca2+ was introduced into the disk membrane at varying pHs in the absence or presence of local anesthetics. This fact indicates that Ca2+ ion does not influence the diprotonating process of metarhodopsin; neither does it interfere with the local anesthetic-induced changes in the rhodopsin molecule.

  15. Multicomponent Moraxella catarrhalis outer membrane vesicles induce an inflammatory response and are internalized by human epithelial cells

    NARCIS (Netherlands)

    Schaar, V.; Vries, S.P. de; Perez Vidakovics, M.L.; Bootsma, H.J.; Larsson, L.; Hermans, P.W.M.; Bjartell, A.; Morgelin, M.; Riesbeck, K.

    2011-01-01

    Moraxella catarrhalis is an emerging human respiratory pathogen in patients with chronic obstructive pulmonary disease (COPD) and in children with acute otitis media. The specific secretion machinery known as outer membrane vesicles (OMVs) is a mechanism by which Gram-negative pathogens interact wit

  16. Structural studies of two outer membrane proteins: OmpT from Escherichia coli and NspA from Neisseria meningitidis

    NARCIS (Netherlands)

    Rutten, L.

    2003-01-01

    This Thesis describes the three-dimensional structures of two outer membrane proteins (OMPs), OmpT and NspA, from two pathogenic Gram-negative bacteria. These structures reveal information about the functioning of these proteins and can potentially be used for the design of antimicrobial drugs or va

  17. Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis Implications for Vaccine Development

    NARCIS (Netherlands)

    Waterbeemd, van de B.; Zomer, G.; IJssel, van den J.; Keulen, van L.; Eppink, M.H.M.; Ley, de P.; Pol, van der L.A.

    2013-01-01

    Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use a

  18. The major outer membrane proteins of enterobacteriaceae. Their immunological relatedness and their possible role in bacterial opsonization

    NARCIS (Netherlands)

    Hofstra, Harmen

    1981-01-01

    This thesis deals with immunological investigations of the major outer membrane proteins of the Enterobacteriaceae as a new group of enterobacterial common envelope antigens, and with some aspects of the possible role of antibodies, prepared against these proteins, in host defense mechanisms. ... Zi

  19. A conserved aromatic residue in the autochaperone domain of the autotransporter Hbp is critical for initiation of outer membrane translocation

    NARCIS (Netherlands)

    Soprova, Z.; Sauri, A.; van Ulsen, P.; Tame, J.R.H.; den Blaauwen, T.; Jong, W.S.P.; Luirink, J.

    2010-01-01

    Autotransporters are bacterial virulence factors that share a common mechanism by which they are transported to the cell surface. They consist of an N-terminal passenger domain and a C-terminal β-barrel, which has been implicated in translocation of the passenger across the outer membrane (OM). The

  20. 1H, 13C and 15N assignment of the GNA1946 outer membrane lipoprotein from Neisseria meningitidis

    NARCIS (Netherlands)

    Neumoin, A.; Leonchiks, A.; Petit, P.; Vuillard, L.; Pizza, M.; Soriani, M.; Boelens, R.; Bonvin, A.M.J.J.

    2011-01-01

    GNA1946 (Genome-derived Neisseria Antigen 1946) is a highly conserved exposed outer membrane lipoprotein from Neisseria meningitidis bacteria of 287 amino acid length (31 kDa). Although the structure of NMB1946 has been solved recently by X-Ray crystallography, understanding the behaviour of GNA1946

  1. Uncoupler resistance in E. coli Tuv and Cuv is due to the exclusion of uncoupler by the outer membrane

    DEFF Research Database (Denmark)

    Haworth, Robert S.; Jensen, Peter Ruhdal; Michelsen, Ole;

    1990-01-01

    The uncoupler resistant bacterial strains E. coli Tuv and Cuv share the high deoxycholate sensitivity of the parent strain, Doc S. However, both Tuv and Cuv show greater resistance than Doc S to other detergents. Measurement of the periplasmic volume indicates that the outer membrane of Doc S...

  2. Photoreceptor Outer Segment on Internal Limiting Membrane after Macular Hole Surgery: Implications for Pathogenesis

    Directory of Open Access Journals (Sweden)

    Michael E. Grinton

    2015-09-01

    Full Text Available Purpose: This report presents a case, which highlights key principles in the pathophysiology of macular holes. It has been hypothesized that anteroposterior (AP and tangential vitreous traction on the fovea are the primary underlying factors causing macular holes [Nischal and Pearson; in Kanski and Bowling: Clinical Ophthalmology: A Systemic Approach, 2011, pp 629-631]. Spectral domain optical coherence tomography (OCT has subsequently corroborated this theory in part but shown that AP vitreofoveal traction is the more common scenario [Steel and Lotery: Eye 2013;27:1-21]. Methods: This study was conducted as a single case report. Results: A 63-year old female presented to her optician with blurred and distorted vision in her left eye. OCT showed a macular hole with a minimum linear diameter of 370 µm, with persistent broad vitreofoveal attachment on both sides of the hole edges. The patient underwent combined left phacoemulsification and pars plana vitrectomy, internal limiting membrane (ILM peel and gas injection. The ILM was examined by electron microscopy and showed the presence of a cone outer segment on the retinal side. Post-operative OCT at 11 weeks showed a closed hole with recovery of the foveal contour and good vision. Conclusion: Our case shows the presence of a photoreceptor outer segment on the retinal side of the ILM and reinforces the importance of tangential traction in the development of some macula holes. The case highlights the theory of transmission of inner retinal forces to the photoreceptors via Müller cells and how a full thickness macular hole defect can occur in the absence of AP vitreomacular traction.

  3. Photoreceptor Outer Segment on Internal Limiting Membrane after Macular Hole Surgery: Implications for Pathogenesis

    Science.gov (United States)

    Grinton, Michael E.; Sandinha, Maria T.; Steel, David H.W.

    2015-01-01

    Purpose This report presents a case, which highlights key principles in the pathophysiology of macular holes. It has been hypothesized that anteroposterior (AP) and tangential vitreous traction on the fovea are the primary underlying factors causing macular holes [Nischal and Pearson; in Kanski and Bowling: Clinical Ophthalmology: A Systemic Approach, 2011, pp 629–631]. Spectral domain optical coherence tomography (OCT) has subsequently corroborated this theory in part but shown that AP vitreofoveal traction is the more common scenario [Steel and Lotery: Eye 2013;27:1–21]. Methods This study was conducted as a single case report. Results A 63-year old female presented to her optician with blurred and distorted vision in her left eye. OCT showed a macular hole with a minimum linear diameter of 370 µm, with persistent broad vitreofoveal attachment on both sides of the hole edges. The patient underwent combined left phacoemulsification and pars plana vitrectomy, internal limiting membrane (ILM) peel and gas injection. The ILM was examined by electron microscopy and showed the presence of a cone outer segment on the retinal side. Post-operative OCT at 11 weeks showed a closed hole with recovery of the foveal contour and good vision. Conclusion Our case shows the presence of a photoreceptor outer segment on the retinal side of the ILM and reinforces the importance of tangential traction in the development of some macula holes. The case highlights the theory of transmission of inner retinal forces to the photoreceptors via Müller cells and how a full thickness macular hole defect can occur in the absence of AP vitreomacular traction. PMID:26557083

  4. Expression, processing, and localization of PmpD of Chlamydia trachomatis Serovar L2 during the chlamydial developmental cycle.

    Directory of Open Access Journals (Sweden)

    Andrey O Kiselev

    Full Text Available BACKGROUND: While families of polymorphic membrane protein (pmp genes have been identified in several Chlamydia species, their function remains mostly unknown. These proteins are of great interest, however, because of their location in the outer membrane and possible role in chlamydial virulence. METHODOLOGY/PRINCIPAL FINDING: We analyzed the relative transcription of the pmpD gene, a member of the pmp gene family in C. trachomatis serovar L2, and its protein product translation and processing during the chlamydial developmental cycle. By real-time reverse transcription polymerase chain reaction, the pmpD gene was found to be upregulated at 16 to 24 four hours after infection. Using polyclonal antibodies generated against the predicted passenger domain of PmpD, we demonstrated that it is initially localized on the surface of reticulate bodies, followed by its secretion outside Chlamydia starting at 24 hours after infection. In elementary bodies, we found a approximately 157 kDa PmpD only inside the cell. Both events, the upregulation of pmpD gene transcription and PmpD protein processing and secretion, are coincidental with the period of replication and differentiation of RBs into EBs. We also demonstrated that, in the presence of penicillin, the cleavage and secretion of the putative passenger domain was suppressed. CONCLUSION/SIGNIFICANCE: Our results are in agreement with the general concept that PmpD is an autotransporter protein which is post-translationally processed and secreted in the form of the putative passenger domain outside Chlamydia at mid- to- late point after infection, coinciding with the development of RBs into EBs.

  5. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA.

    Science.gov (United States)

    Liu, Yimo; Wang, Zheming; Liu, Juan; Levar, Caleb; Edwards, Marcus J; Babauta, Jerome T; Kennedy, David W; Shi, Zhi; Beyenal, Haluk; Bond, Daniel R; Clarke, Thomas A; Butt, Julea N; Richardson, David J; Rosso, Kevin M; Zachara, John M; Fredrickson, James K; Shi, Liang

    2014-12-01

    The multi-heme, outer membrane c-type cytochrome (c-Cyt) OmcB of Geobacter sulfurreducens was previously proposed to mediate electron transfer across the outer membrane. However, the underlying mechanism has remained uncharacterized. In G. sulfurreducens, the omcB gene is part of two tandem four-gene clusters, each is predicted to encode a transcriptional factor (OrfR/OrfS), a porin-like outer membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (OmaB/OmaC) and an outer membrane c-Cyt (OmcB/OmcC) respectively. Here, we showed that OmbB/OmbC, OmaB/OmaC and OmcB/OmcC of G. sulfurreducens PCA formed the porin-cytochrome (Pcc) protein complexes, which were involved in transferring electrons across the outer membrane. The isolated Pcc protein complexes reconstituted in proteoliposomes transferred electrons from reduced methyl viologen across the lipid bilayer of liposomes to Fe(III)-citrate and ferrihydrite. The pcc clusters were found in all eight sequenced Geobacter and 11 other bacterial genomes from six different phyla, demonstrating a widespread distribution of Pcc protein complexes in phylogenetically diverse bacteria. Deletion of ombB-omaB-omcB-orfS-ombC-omaC-omcC gene clusters had no impact on the growth of G. sulfurreducens PCA with fumarate but diminished the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite. Complementation with the ombB-omaB-omcB gene cluster restored the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite.

  6. A conformational landscape for alginate secretion across the outer membrane of Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Jingquan [Trinity College, Dublin (Ireland); Rouse, Sarah L. [University of Oxford, South Parks Road, Oxford (United Kingdom); Li, Dianfan; Pye, Valerie E.; Vogeley, Lutz; Brinth, Alette R.; El Arnaout, Toufic [Trinity College, Dublin (Ireland); Whitney, John C.; Howell, P. Lynne [The Hospital for Sick Children, Toronto, Ontario (Canada); University of Toronto, Toronto, Ontario (Canada); Sansom, Mark S. P. [University of Oxford, South Parks Road, Oxford (United Kingdom); Caffrey, Martin, E-mail: martin.caffrey@tcd.ie [Trinity College, Dublin (Ireland)

    2014-08-01

    Crystal structures of the β-barrel porin AlgE reveal a mechanism whereby alginate is exported from P. aeruginosa for biofilm formation. The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common β-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.

  7. A conformational landscape for alginate secretion across the outer membrane of Pseudomonas aeruginosa.

    Science.gov (United States)

    Tan, Jingquan; Rouse, Sarah L; Li, Dianfan; Pye, Valerie E; Vogeley, Lutz; Brinth, Alette R; El Arnaout, Toufic; Whitney, John C; Howell, P Lynne; Sansom, Mark S P; Caffrey, Martin

    2014-08-01

    The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common β-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.

  8. Reconceptualizing the chlamydial inclusion as a pathogen-specified parasitic organelle: an expanded role for Inc proteins.

    Science.gov (United States)

    Moore, Elizabeth R; Ouellette, Scot P

    2014-01-01

    Chlamydia is an obligate intracellular pathogen that develops in the host cell in a vacuole termed the chlamydial inclusion. The prevailing concept of the chlamydial inclusion is of a parasitophorous vacuole. Here, the inclusion is the recipient of one-way host-pathogen interactions thus draining nutrients from the cell and negatively impacting it. While Chlamydia orchestrates some aspects of cell function, recent data indicate host cells remain healthy up until, and even after, chlamydial egress. Thus, while Chlamydia relies on the host cell for necessary metabolites, the overall function of the host cell, during chlamydial growth and development, is not grossly disturbed. This is consistent with the obligate intracellular organism's interest to maintain viability of its host. To this end, Chlamydia expresses inclusion membrane proteins, Incs, which serve as molecular markers for the inclusion membrane. Incs also contribute to the physical structure of the inclusion membrane and facilitate host-pathogen interactions across it. Given the function of Incs and the dynamic interactions that occur at the inclusion membrane, we propose that the inclusion behaves similarly to an organelle-albeit one that benefits the pathogen. We present the hypothesis that the chlamydial inclusion acts as a pathogen-specified parasitic organelle. This representation integrates the inclusion within existing subcellular trafficking pathways to divert a subset of host-derived metabolites thus maintaining host cell homeostasis. We review the known interactions of the chlamydial inclusion with the host cell and discuss the role of Inc proteins in the context of this model and how this perspective can impact the study of these proteins. Lessons learnt from the chlamydial pathogen-specified parasitic organelle can be applied to other intracellular pathogens. This will increase our understanding of how intracellular pathogens engage the host cell to establish their unique developmental niches.

  9. Outer membrane vesicles derived from Escherichia coli induce systemic inflammatory response syndrome.

    Directory of Open Access Journals (Sweden)

    Kyong-Su Park

    Full Text Available Sepsis, characterized by a systemic inflammatory state that is usually related to Gram-negative bacterial infection, is a leading cause of death worldwide. Although the annual incidence of sepsis is still rising, the exact cause of Gram-negative bacteria-associated sepsis is not clear. Outer membrane vesicles (OMVs, constitutively secreted from Gram-negative bacteria, are nano-sized spherical bilayered proteolipids. Using a mouse model, we showed that intraperitoneal injection of OMVs derived from intestinal Escherichia coli induced lethality. Furthermore, OMVs induced host responses which resemble a clinically relevant condition like sepsis that was characterized by piloerection, eye exudates, hypothermia, tachypnea, leukopenia, disseminated intravascular coagulation, dysfunction of the lungs, hypotension, and systemic induction of tumor necrosis factor-alpha and interleukin-6. Our study revealed a previously unidentified causative microbial signal in the pathogenesis of sepsis, suggesting OMVs as a new therapeutic target to prevent and/or treat severe sepsis caused by Gram-negative bacterial infection.

  10. Structural characterization of outer membrane components of the type IV pili system in pathogenic Neisseria.

    Directory of Open Access Journals (Sweden)

    Samta Jain

    Full Text Available Structures of the type IV pili secretin complexes from Neisseria gonorrhoeae and Neisseria meningitidis, embedded in outer membranes were investigated by transmission electron microscopy. Single particle averaging revealed additional domains not observed previously. Secretin complexes of N. gonorrhoeae showed a double ring structure with a 14-15-fold symmetry in the central ring, and a 14-fold symmetry of the peripheral ring with 7 spikes protruding. In secretin complexes of N. meningitidis, the spikes were absent and the peripheral ring was partly or completely lacking. When present, it had a 19-fold symmetry. The structures of the complexes in several pil mutants were determined. Structures obtained from the pilC1/C2 adhesin and the pilW minor pilin deletion strains were similar to wild-type, whereas deletion of the homologue of N. meningitidis PilW resulted in the absence of secretin structures. Remarkably, the pilE pilin subunit and pilP lipoprotein deletion mutants showed a change in the symmetry of the peripheral ring from 14 to 19 and loss of spikes. The pilF ATPase mutant also lost the spikes, but maintained 14-fold symmetry. These results show that secretin complexes contain previously unidentified large and flexible extra domains with a probable role in stabilization or assembly of type IV pili.

  11. Immunogenicity of Coxiella burnetii whole cells and their outer membrane components.

    Science.gov (United States)

    Gajdosová, E; Kovácová, E; Toman, R; Skultéty, L; Lukácová, M; Kazár, J

    1994-12-01

    The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole cells (Cb I and Cb II) and their outer membrane components (OMC), i.e. phase I trichloroacetic acid extract (TCAE), phase I 29 K protein (PRO), phase I and II lipopolysaccharides (LPS I, LPS II), polysaccharides (PS I, PS II), and lipid A (LA I, LA II), were compared. The highest immune response was observed in BALB/c mice by Cb I in both humoral immunity and lymphocyte transformation assays, and in the protective effect as well. The immune response was also significant by Cb II, but their protective capacity was low. The OMC reacted variously. Only TCAE and PRO gave a high value of humoral immunity evaluated by the serological methods. All OMC reacted in the haemolytic plaque assay giving different responses. Lymphoproliferation of splenocytes was positive with all OMC using both Cb I and Cb II antigens with the exception of PS I and PS II in the case of Cb II antigen. The induction of protection against infectious Cb I was demonstrated after immunization with TCAE, PRO, and LPS I. Other OMC did not induce protection against this agent.

  12. Immunoproteomic Profiling of Bordetella pertussis Outer Membrane Vesicle Vaccine Reveals Broad and Balanced Humoral Immunogenicity.

    Science.gov (United States)

    Raeven, René H M; van der Maas, Larissa; Tilstra, Wichard; Uittenbogaard, Joost P; Bindels, Tim H E; Kuipers, Betsy; van der Ark, Arno; Pennings, Jeroen L A; van Riet, Elly; Jiskoot, Wim; Kersten, Gideon F A; Metz, Bernard

    2015-07-01

    The current resurgence of whooping cough is alarming, and improved pertussis vaccines are thought to offer a solution. Outer membrane vesicle vaccines (omvPV) are potential vaccine candidates, but omvPV-induced humoral responses have not yet been characterized in detail. The purpose of this study was to determine the antigen composition of omvPV and to elucidate the immunogenicity of the individual antigens. Quantitative proteome analysis revealed the complex composition of omvPV. The omvPV immunogenicity profile in mice was compared to those of classic whole cell vaccine (wPV), acellular vaccine (aPV), and pertussis infection. Pertussis-specific antibody levels, antibody isotypes, IgG subclasses, and antigen specificity were determined after vaccination or infection by using a combination of multiplex immunoassays, two-dimensional immunoblotting, and mass spectrometry. The vaccines and infection raised strong antibody responses, but large quantitative and qualitative differences were measured. The highest antibody levels were obtained by omvPV. All IgG subclasses (IgG1/IgG2a/IgG2b/IgG3) were elicited by omvPV and in a lower magnitude by wPV, but not by aPV (IgG1) or infection (IgG2a/b). The majority of omvPV-induced antibodies were directed against Vag8, BrkA, and LPS. The broad and balanced humoral response makes omvPV a promising pertussis vaccine candidate.

  13. Construction and Immunogenicity of Recombinant Swinepox Virus Expressing Outer Membrane Protein L of Salmonella.

    Science.gov (United States)

    Fang, Yizhen; Lin, Huixing; Ma, Zhe; Fan, Hongjie

    2016-07-28

    Salmonella spp. are gram-negative flagellated bacteria that cause a variety of diseases in humans and animals, ranging from mild gastroenteritis to severe systemic infection. To explore development of a potent vaccine against Salmonella infections, the gene encoding outer membrane protein L (ompL) was inserted into the swinepox virus (SPV) genome by homologous recombination. PCR, western blot, and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL. The immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model. Forty mice were assigned to four groups, which were immunized with rSPV-OmpL, inactive Salmonella (positive control), wildtype SPV (wtSPV; negative control), or PBS (challenge control), respectively. The OmpLspecific antibody in the rSPV-OmpL-immunized group increased dramatically and continuously over time post-vaccination, and was present at a significantly higher level than in the positive control group (p Salmonella typhimurium CVCC542, eight out of ten mice in the rSPV-OmpLvaccinated group were protected, whereas all the mice in the negative control and challenge control groups died within 3 days. Passive immune protection assays showed that hyperimmune sera against OmpL could provide mice with effective protection against challenge from S. typhimurium. The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection.

  14. Characterization and Expression of Outer Membrane Protein AI Gene of Aeromonas veronii

    Institute of Scientific and Technical Information of China (English)

    Wang Hai-juan; Wang Li

    2015-01-01

    The outer membrane protein, ompA, ofAeromonas veronii has a role in the virulence of the organism and is a potential candidate for vaccine development. In this study, ompAⅠofAeromonas veronii strain WA106 was cloned and sequenced, then, it was expressed inEscherichia coli BL21. The nucleotide sequence of ompAⅠgene was 1 023 base pairs (GenBank Accession NO.KC748024), which showed 100% homology with that ofA. veronii (NO.AB290200.1). This predicted protein was composed of 340 amino acid residues. Its molecular weight was 35.78 ku and isoelectric point was 5.18. The protein was a hydrophilic protein containing alpha helix and random coil with percentage of 35.0% and 49.7%, respectively. The tertiary structure, quaternary structure prediction showed that ompAⅠprotein contained two peptide chains. SDS-PAGE showed that the actual value of the fusion protein was consistent with the expected result. It will facilitate further study of the role of ompAⅠprotein.

  15. Lymphocytic proliferative response to outer-membrane proteins isolated from Salmonella.

    Science.gov (United States)

    González, C R; Isibasi, A; Ortiz-Navarrete, V; Paniagua, J; García, J A; Blanco, F; Kumate, J

    1993-01-01

    Porins isolated from Salmonella typhi have been demonstrated to protect against the challenge with this bacteria in mice. The mechanism has not been clarified, but could be associated with activation of both humoral and cellular immunity. In order to evaluate the induction of specific T cell responses, the lymphocytic proliferation to porins isolated from Salmonella typhimurium, Salmonella typhi and Escherichia coli was examined by 3H-thymidine incorporation assay in mice immunized with three different antigens: acetone-killed S. typhimurium, its porins, or outer-membrane proteins (OMPs) isolated from S. typhi. Higher proliferative responses were observed in mice immunized with porins and OMPs compared with those which received the acetone-killed bacteria. Although cross-reactivity was observed between porins, they were not mitogenic. Moreover, porins were able to activate T lymphocytes isolated from mice immunized with S. typhi OMPs. These results suggest that T cell activation, through the release of lymphokines, may play a role in the induction of protective immunity with porins.

  16. Trapped translocation intermediates establish the route for export of capsular polysaccharides across Escherichia coli outer membranes

    Science.gov (United States)

    Nickerson, Nicholas N.; Mainprize, Iain L.; Hampton, Lauren; Jones, Michelle L.; Naismith, James H.; Whitfield, Chris

    2014-01-01

    The outer membrane (OM) of Gram-negative bacteria is designed to exclude potentially harmful molecules. This property presents a challenge for bacteria that must secrete proteins and large glycoconjugates to grow, divide, and persist. Proteins involved in trafficking such molecules have been identified, but their precise roles are often unresolved due to the difficulty in capturing “snapshots” during the export pathway. Wza is the prototype for the large family of OM polysaccharide export proteins. In Escherichia coli, Wza is essential for the assembly of a capsule, a protective surface coat composed of long-chain polysaccharides. Wza creates an octameric α-helical channel spanning the OM, but the bulk of the protein exists as a large periplasmic structure enclosing an extensive lumen. Residues within the lumen of Wza were targeted for site-specific incorporation of the UV photo–cross-linkable unnatural amino acid p-benzoyl-l-phenylalanine. Using this in vivo photo–cross-linking strategy, we were able to trap polysaccharide translocation intermediates within the lumen of Wza, providing the first unequivocal evidence to our knowledge that nascent capsular polysaccharide chains exit the cell through the Wza portal. PMID:24843147

  17. Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles.

    Science.gov (United States)

    Ojima, Yoshihiro; Nguyen, Minh Hong; Yajima, Reiki; Taya, Masahito

    2015-09-01

    Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.

  18. Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response.

    Science.gov (United States)

    Lee, Je Chul; Lee, Eun Jeoung; Lee, Jung Hwa; Jun, So Hyun; Choi, Chi Won; Kim, Seung Il; Kang, Sang Sun; Hyun, Sunghee

    2012-06-01

    Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community-acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)-1β and IL-8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram-negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response.

  19. Immunodetection of outer membrane proteins by flow cytometry of isolated mitochondria.

    Science.gov (United States)

    Pickles, Sarah; Arbour, Nathalie; Vande Velde, Christine

    2014-09-18

    Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

  20. Dataset of the proteome of purified outer membrane vesicles from the human pathogen Aggregatibacter actinomycetemcomintans

    Directory of Open Access Journals (Sweden)

    Thomas Kieselbach

    2017-02-01

    Full Text Available The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen, which is linked to aggressive forms of periodontitis and can be associated with endocarditis. The outer membrane vesicles (OMVs of this species contain effector proteins such as cytolethal distending toxin (CDT and leukotoxin (LtxA, which they can deliver into human host cells. The OMVs can also activate innate immunity through NOD1- and NOD2-active pathogen-associated molecular patterns. This dataset provides a proteome of highly purified OMVs from A. actinomycetemcomitans serotype e strain 173. The experimental data do not only include the raw data of the LC-MS/MS analysis of four independent preparations of purified OMVs but also the mass lists of the processed data and the Mascot.dat files from the database searches. In total 501 proteins are identified, of which 151 are detected in at least three of four independent preparations. In addition, this dataset contains the COG definitions and the predicted subcellular locations (PSORTb 3.0 for the entire genome of A. actinomycetemcomitans serotype e strain SC1083, which is used for the evaluation of the LC-MS/MS data. These data are deposited in ProteomeXchange in the public dataset PXD002509. In addition, a scientific interpretation of this dataset by Kieselbach et al. (2015 [2] is available at http://dx.doi.org/10.1371/journal.pone.0138591.

  1. Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

    Science.gov (United States)

    Nguyen, Minh Hong; Yajima, Reiki; Taya, Masahito

    2015-01-01

    Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells. PMID:26092467

  2. Immunopotentiation of outer membrane protein through anti-idiotype Pasteurella multocida vaccine in rabbits.

    Science.gov (United States)

    Arif, Javid; Rahman, Sajjad-Ur; Arshad, Muhammad; Akhtar, Pervez

    2013-11-01

    Pasteurella multocida was isolated from cattle affected with haemorrhagic septicaemia and characterized on the basis of morphological, cultural and biochemical tests. Bacterial outer membrane proteins (OMPs) were extracted with 1% Sarkosyl method. P. multocida anti-idiotype vaccine prepared from OMPs (21.3 mg per 100 ml), was evaluated and compared with bacterin supplemented with 10% OMPs and plain alum-adsorbed bacterin in rabbit models. It was observed that OMPs-anti-idiotype vaccine induced high levels of antibody titres (geomean titres -GMT) detected using indirect haemagglutination (IHA) test. The OMPs anti-idiotype antibody titres of 168.9 GMT were obtained to 42.2 GMT in OMPs supplemented bacterin on 21 days post vaccination, while the plain bacterin had the least titre of 27.9 GMT. The OMPs-anti-idiotype vaccine provoked better immunogenic response in terms of highest GMT titres and long lasting effect in rabbits and 100% protection against the challenge with homologous strain of P. multocida,while 88% protection was obtained in rabbits, given OMPs supplemented bacterin.

  3. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens.

    Science.gov (United States)

    Cecil, Jessica D; O'Brien-Simpson, Neil M; Lenzo, Jason C; Holden, James A; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T; Yan, Yan; Caruso, Frank; Reynolds, Eric C

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis.

  4. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

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    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-10-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for (/sup 3/H)diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with (/sup 3/H)flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines.

  5. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens.

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    Jessica D Cecil

    Full Text Available Highly purified outer membrane vesicles (OMVs of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis.

  6. Role of MINOS in Mitochondrial Membrane Architecture : Cristae Morphology and Outer Membrane Interactions Differentially Depend on Mitofilin Domains

    NARCIS (Netherlands)

    Zerbes, Ralf M.; Bohnert, Maria; Stroud, David A.; von der Malsburg, Karina; Kram, Anita; Oeljeklaus, Silke; Warscheid, Bettina; Becker, Thomas; Wiedemann, Nils; Veenhuis, Marten; van der Klei, Ida J.; Pfanner, Nikolaus; van der Laan, Martin

    2012-01-01

    The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein com

  7. Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

    KAUST Repository

    Ates, Louis S.

    2015-05-04

    Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow

  8. Specific association of lectin LecB with the surface of Pseudomonas aeruginosa: role of outer membrane protein OprF.

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    Horst Funken

    Full Text Available The fucose binding lectin LecB affects biofilm formation and is involved in pathogenicity of Pseudomonas aeruginosa. LecB resides in the outer membrane and can be released specifically by treatment of an outer membrane fraction with fucose suggesting that it binds to specific ligands. Here, we report that LecB binds to the outer membrane protein OprF. In an OprF-deficient P. aeruginosa mutant, LecB is no longer detectable in the membrane but instead in the culture supernatant indicating a specific interaction between LecB and OprF.

  9. Plasmolysis and cell shape depend on solute outer-membrane permeability during hyperosmotic shock in E. coli.

    Science.gov (United States)

    Pilizota, Teuta; Shaevitz, Joshua W

    2013-06-18

    The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.

  10. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

    Science.gov (United States)

    Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  11. Targeting and Assembly of Components of the TOC Protein Import Complex at the Chloroplast Outer Envelope Membrane

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    Lynn G.L. Richardson

    2014-06-01

    Full Text Available The translocon at the outer envelope membrane of chloroplasts (TOC initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  12. Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique

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    Appleton Hazel

    2010-02-01

    Full Text Available Abstract Background Salmonella enterica serovar Typhimurium (S. Typhimurium is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI™ FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS and identified from mass spectral database searches. Results In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX. Conclusion Using a multi-step digest approach the LPI™ technique enables the incorporation of a

  13. Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody.

    Science.gov (United States)

    Zheng, W Y; Wang, Y; Zhang, Z C; Yan, F

    2015-10-05

    We examined the immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. Genomic DNA from the M5 strain of goat Brucella was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T-1. The expression and immunological characteristics of the fusion protein GST-omp31 were subjected to preliminary western blot detection with goat Brucella rabbit immune serum. The Brucella immunized BALB/c mouse serum was detected using purified protein. The high-potency mouse splenocytes and myeloma Sp2/0 cells were fused. Positive clones were screened by enzyme-linked immunosorbent assay to establish a hybridoma cell line. Mice were inoculated intraperitoneally with hybridoma cells to prepare ascites. The mAb was purified using the n-caprylic acid-ammonium sulfate method. The characteristics of mAb were examined using western blotting and enzyme-linked immunosorbent assay. A 680-base pair band was observed after polymerase chain reaction. Enzyme digestion identification and sequencing showed that the pGEX-4T-1-omp31 prokaryotic expression vector was successfully established; a target band of approximately 57 kDa with an apparent molecular weight consistent with the size of the target fusion protein. At 25°C, the expression of soluble expression increased significantly; the fusion protein GST-omp31 was detected by western blotting. Anti-omp31 protein mAb was obtained from 2 strains of Brucella. The antibody showed strong specificity and sensitivity and did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus pyocyaneus. The pGEX-4T-1-omp31 prokaryotic expression vector was successfully established and showed good immunogenicity. The antibody also showed strong specificity and good sensitivity.

  14. A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles.

    Science.gov (United States)

    Leitner, Deborah R; Lichtenegger, Sabine; Temel, Philipp; Zingl, Franz G; Ratzberger, Desiree; Roier, Sandro; Schild-Prüfert, Kristina; Feichter, Sandra; Reidl, Joachim; Schild, Stefan

    2015-01-01

    Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs) derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS)-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB) independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I, and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo.

  15. Heterogeneous interactome between Litopenaeus vannamei plasma proteins and Vibrio parahaemolyticus outer membrane proteins.

    Science.gov (United States)

    Liu, Xiang; She, Xin-Tao; Zhu, Qing-Feng; Li, Hui; Peng, Xuan-Xian

    2013-01-01

    A great loss has been suffered by microbial infectious diseases under intensive shrimp farming in recent years. In this background, the understanding of shrimp innate immunity becomes an importantly scientific issue, but little is known about the heterogeneous protein-protein interaction between pathogenic cells and hosts, which is a key step for the invading microbes to infect internet organs through bloodstream. In the present study, bacterial outer membrane (OM) protein array and pull-down approaches are used to isolate both Vibrio parahaemolyticus OM proteins that bind to shrimp serum proteins and the shrimp serum proteins that interact with bacterial cells, respectively. Three interacting shrimp serum proteins, hemocyanin, β-1,3-glucan binding protein and LV_HP_RA36F08r and thirty interacting OM proteins were determined. They form 63 heterogeneous protein-protein interactions. Nine out of the 30 OM proteins were randomly demonstrated to be up-regulated or down-regulated when bacterial cells were cultured with shrimp sera, indicating the biological significance of the network. The interesting findings uncover the complexity of struggle between host immunity and bacterial infection. Compared with our previous report on heterogeneous interactome between fish grill and bacterial OM proteins, the present study further extends the investigation from lower vertebrates to invertebrates and develops a bacterial OM protein array to identify the OM proteins bound with shrimp serum proteins, which elevates the frequencies of the bound OM proteins. Our results highlight the way to determine and understand the heterogeneous interaction between hosts and microbes.

  16. Identification of Outer Membrane and Exoproteins of Carbapenem-Resistant Multilocus Sequence Type 258 Klebsiella pneumoniae.

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    Amanda J Brinkworth

    Full Text Available Carbapenem-resistant Klebsiella pneumoniae strains have emerged as a cause of life-threatening infections in susceptible individuals (e.g., transplant recipients and critically ill patients. Strains classified as multilocus sequence type (ST 258 are among the most prominent causes of carbapenem-resistant K. pneumoniae infections worldwide, but the basis for the success of this lineage remains incompletely determined. To gain a more comprehensive view of the molecules potentially involved in the success of ST258, we used a proteomics approach to identify surface-associated and culture supernatant proteins produced by ST258. Protein samples were prepared from varied culture conditions in vitro, and were analyzed by a combination of two-dimensional electrophoresis and liquid chromatography followed by tandem mass spectrometry (LC-MS/MS. We identified a total of 193 proteins in outer membrane preparations from bacteria cultured in Luria-Bertani broth (LB or RPMI 1640 tissue culture media (RPMI. Compared with LB, several iron-acquisition proteins, including IutA, HmuR, HmuS, CirA, FepA, FitA, FoxA, FhuD, and YfeX, were more highly expressed in RPMI. Of the 177 proteins identified in spent media, only the fimbrial subunit, MrkA, was predicted to be extracellular, a finding that suggests few proteins (or a limited quantity are freely secreted by ST258. Notably, we discovered 203 proteins not reported in previous K. pneumoniae proteome studies. In silico modeling of proteins with unknown function revealed several proteins with beta-barrel transmembrane structures typical of porins, as well as possible host-interacting proteins. Taken together, these findings contribute several new targets for the mechanistic study of drug-resistance and pathogenesis by ST258 K. pneumoniae isolates.

  17. Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yong; Liu, Liguo; Fu, Hua; Wei, Candong, E-mail: weicando@ipbcams.ac.cn; Jin, Qi, E-mail: zdsys@vip.sina.com

    2014-10-31

    Highlights: • We utilized mTRAQ-based quantification to study protein changes in Congo red-induced OMVs. • A total of 148 proteins were identified in S. flexneri-derived OMVs. • Twenty-eight and five proteins are significantly up- and down-regulated in the CR-induced OMV, respectively. • The result implied that a special sorting mechanism of particular proteins into OMVs may exist. • Key node proteins in the protein interaction network might be important for pathogenicity. - Abstract: The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein–protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria.

  18. Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages.

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N.; Heffron, Fred

    2011-06-01

    We have previously shown that the regulators SpvR, FruR, IHF, PhoP/PhoQ, SsrA/SsrB, SlyA, Hnr, RpoE, SmpB, CsrA, RpoS, Crp, OmpR/EnvZ, and Hfq are essential for Salmonella Typhimurium virulence in mice. Here we use quantitative LC-MS-based proteomics profiling of in-frame deletion mutants of these 14 regulators to identify proteins that are coordinately regulated by these virulence regulators and are thus presumably novel factors contributing to Salmonella pathogenesis. Putative candidate proteins from proteomics analysis were determined, which exhibited similar abundance profiles to those of Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS) proteins. A subset of 5 proteins including STM0082, STM1548, PdgL, STM1633, and STM3595 was selected for further analysis. All 5 proteins were expressed inside macrophage cells and STM0082 (SrfN) was secreted into host cytoplasm. Furthermore, deletion of STM0082 attenuated virulence in mice when administered intraperitoneally as determined by competitive index. srfN transcription was positively regulated by SsrAB, however, secretion was independent of SPI-2 TTSS as well as SPI-1 TTSS and flagella. Proteins including PagK and STM2585A, which are positively regulated by PhoP/PhoQ, have sec signal peptides as predicted for SrfN and were secreted into macrophage cytoplasm regardless of SPI-2 TTSS. Isolation of outer membrane vesicles (OMVs) revealed the presence of SrfN, PagK, and STM2585A inside vesicle compartments. This result is the first case showing delivery of virulence effectors via OMVs in S. Typhimurium. Moreover, Hfq regulation of SrfN translation suggests that small non-coding RNAs may be responsible for regulating effector protein expression.

  19. The Role of Helicobacter pylori Outer Membrane Proteins in Adherence and Pathogenesis

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    Armelle Ménard

    2013-08-01

    Full Text Available Helicobacter pylori is one of the most successful human pathogens, which colonizes the mucus layer of the gastric epithelium of more than 50% of the world’s population. This curved, microaerophilic, Gram-negative bacterium induces a chronic active gastritis, often asymptomatic, in all infected individuals. In some cases, this gastritis evolves to more severe diseases such as peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. H. pylori has developed a unique set of factors, actively supporting its successful survival and persistence in its natural hostile ecological niche, the human stomach, throughout the individual’s life, unless treated. In the human stomach, the vast majority of H. pylori cells are motile in the mucus layer lining, but a small percentage adheres to the epithelial cell surfaces. Adherence to the gastric epithelium is important for the ability of H. pylori to cause disease because this intimate attachment facilitates: (1 colonization and persistence, by preventing the bacteria from being eliminated from the stomach, by mucus turnover and gastric peristalsis; (2 evasion from the human immune system and (3 efficient delivery of proteins into the gastric cell, such as the CagA oncoprotein. Therefore, bacteria with better adherence properties colonize the host at higher densities. H. pylori is one of the most genetically diverse bacterial species known and is equipped with an extraordinarily large set of outer membrane proteins, whose role in the infection and persistence process will be discussed in this review, as well as the different receptor structures that have been so far described for mucosal adherence.

  20. Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells.

    Science.gov (United States)

    March, Catalina; Moranta, David; Regueiro, Verónica; Llobet, Enrique; Tomás, Anna; Garmendia, Junkal; Bengoechea, José A

    2011-03-25

    Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-Δwca(K2)ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfα, kc, and il6 than the wild type. ompA mutants activated NF-κB, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-κB-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-κB, whereas 52145-Δwca(K2)ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung.

  1. Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC from V. cholerae strains.

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    Sridhar Elluri

    Full Text Available Outer membrane vesicles (OMVs released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC, is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and

  2. PRODUCTION AND CHARACTERIZATION OF MONOCLONAL AND POLYCLONAL ANTIBODY AGAINST RECOMBINANT OUTER MEMBRANE PROTEIN

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    Mahdi Fasihi-Ramandi

    2014-01-01

    Full Text Available There are many studies related to immunological and molecular methods for diagnosis of Vibrio cholera (V. cholerae. However, most assays dependent on enrichment of culture of bacteria, which need more time and involves the use of costly equipment and reagents. In this study Balb/c mice were immunized with recombinant Outer Membrane Protein (rOMPw of vibrio cholerae and splenocytes of hyper immunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using rOMPw as coating antigen. The monoclonal antibodies from ascitic fluids were purified and its reaction with rOMPw was assessed by ELISA. Polyclonal antibodies were also produced by immunization of rabbits with the above mentioned antigen. The rabbit sera was affinity purified using Hi-Trap protein G column. The result showed that monoclonal antibody specific to rOMPw has been successfully generated. The monoclonal antibody reacted with recombinant OMPw in ELISA and immunonoblat method. Rabbit polyclonal antibody was also bound to rOMPw by ELISA. The results of agglutination test with whole bacteria also showed that both mouse monoclonal and rabbit polyclonal antibodies reacted with whole vibrio cholera but not other related bacteria. The purpose of this study was to check out if anti OMPw antibodies could use as diagnostic assay for detection of V. cholerae. Our results demonstrated that anti recombinant OMPw monoclonal and polyclonal antibodies are able to diagnose whole bacteria in pure culture using agglutination test but not by home made immunochromatic strip test.

  3. A Meningococcal Outer Membrane Vesicle Vaccine Incorporating Genetically Attenuated Endotoxin Dissociates Inflammation From Immunogenicity

    Directory of Open Access Journals (Sweden)

    David J. Dowling

    2016-12-01

    Full Text Available Background. Group B Neisseria meningitidis, an endotoxin-producing gram-negative bacterium, causes the highest incidence of group B meningococcus (MenB disease in the first year of life. The Bexsero vaccine is indicated in Europe from 8 weeks of age. Endotoxin components of outer membrane vesicles (OMVs or soluble lipopolysaccharide (LPS represent a potential source of inflammation and residual reactogenicity. The purpose of this study was to compare novel candidate MenB vaccine formulations with licensed vaccines, including Bexsero, using age-specific in vitro culture systems.Methods. OMVs from wild type and inactivated lpxL1 gene mutant N. meningitidis strains were characterized in human neonatal and adult in vitro whole blood assays and dendritic cell arrays. OMVs were benchmarked against licensed vaccines, including Bexsero and whole cell pertussis formulations, with respect to Th-polarizing cytokine and PGE2 production, as well as cell surface activation markers (HLA-DR, CD86, CCR7. OMV immunogenicity was assessed in mice.Results. ΔlpxLI native OMVs demonstrated significantly less cytokine induction in human blood and DCs than Bexsero and most of the other pediatric vaccines (e.g., PedvaxHib, EasyFive, Bacillus Calmette–Guérin (BCG tested. Despite a much lower inflammatory profile in vitro than Bexsero, ΔlpxLI native OMVs still had moderate DC maturing ability and induced robust anti-N. meningitidis antibody responses after murine immunization.Conclusions. A meningococcal vaccine comprised of attenuated LPS-based OMVs with a limited inflammatory profile in vitro induces robust antigen-specific immunogenicity in vivo.

  4. Meningococcal Outer Membrane Vesicle Composition-Dependent Activation of the Innate Immune Response.

    Science.gov (United States)

    Zariri, Afshin; Beskers, Joep; van de Waterbeemd, Bas; Hamstra, Hendrik Jan; Bindels, Tim H E; van Riet, Elly; van Putten, Jos P M; van der Ley, Peter

    2016-10-01

    Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen-associated molecular patterns, including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines, detergent treatment is generally used to reduce the LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared the effects of standard OMV isolation methods using detergent or EDTA with those of genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1 and pagL) on the in vitro induction of innate immune responses. The use of detergent decreased both Toll-like receptor 4 (TLR4) and TLR2 activation by OMVs, while the LPS modifications reduced only TLR4 activation. Mutational removal of PorB or lipoprotein factor H binding protein (fHbp), two proteins known to trigger TLR2 signaling, had no effect, indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent-treated OMVs and lpxL1 OMVs showed similar reductions of cytokine profiles in the human monocytic cell line MM6 and human dendritic cells (DCs). OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of proinflammatory cytokines interleukin-6 (IL-6) and IL-1β but not of IP-10, a typical TRIF-dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment.

  5. Expression and Purification of the Major Outer Membrane Protein of Chlamydia Trachomatis in Prokaryotic Cell

    Institute of Scientific and Technical Information of China (English)

    李忠玉; 吴移谋; 陈超群; 万艳平; 朱翠明

    2004-01-01

    To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis ( C.trachomatis ) and to express the fusion protein in E. coli BL21, the MOMP gene was amphfied by polymerase chain reaction (PCR) from genome of C. trachomatis serovar D. The fragment was cloned into the prokaryotic expression vector pET-22b( + ) after digestion with BamH Ⅰ and Not Ⅰ and transformed into E. coli XL1-Blue. Recombinants were selected by enzyme digestion and sequencing and the recombinant plasmid with MOMP gene was then transformed into E. coli BL21 with IPTG to express the target gene. The expression recombinant proteins were purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. It was found that a 1.2 kb MOMP gene was isolated. The DNA sequence of MOMP was found to be just the same as the sequence published by GenBank. A recombinant plasmid containing MOMP gene was constructed to express the fusion proteins in E.coli. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 47 kDa that was consistent with the theoretical predicted value, and the specificity of the expressed protein was conformed by Western blot. It concluded that the MOMP gene could be expressed in the prokaryotic system, by which it provided the foundation for the future studies on the biological activities of C. trachomatis and for the development of vaccine against this pathogen.

  6. Effects of salicylate on shape, electromotility and membrane characteristics of isolated outer hair cells from guinea pig cochlea.

    Science.gov (United States)

    Shehata, W E; Brownell, W E; Dieler, R

    1991-01-01

    A reversible tinnitus and hearing loss have long been known to result from large doses of salicylate. Cochlear electrophysiology and otoacoustic emission studies suggest that the drug may interfere with outer hair cell electromotility. Exposure of isolated outer hair cells to sodium salicylate concentrations ranging from 0.05 to 10 mM reveals a dose dependent, reversible loss of turgidity and dimunition of electromotility. There was also a change in membrane conductance with salicylate superfusion that occurred later in time from the onset of shape and electromotility changes. There was no evidence of dose dependence for the change in membrane conductance, nor was the change reversible. The changes in shape and electromotility that we observe in vitro may impair cochlear partition movements in vivo and could account, at least in part, for the salicylate-induced hearing loss and effects on otoacoustic emissions.

  7. Chlamydial Pneumonitis: A Creepy Neonatal Disease

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    Kam Lun Hon

    2013-01-01

    Full Text Available We present a case of neonatal chlamydial pneumonitis to illustrate that a high index of suspicion is necessary to make the diagnosis so that treatment can be promptly instituted. The child was afebrile and the only symptom was a cough. The respiratory equations are calculated to understand the respiratory physiology. There was no overt abnormality with ventilation, oxygenation, compliance, resistance, or ventilation-perfusion mismatch despite radiographic abnormality. The literature is searched to review if treatment with a systemic macrolide antibiotic is needed in an otherwise asymptomatic neonate with chlamydial pneumonitis.

  8. Evaluation of a multisubunit recombinant polymorphic membrane protein and major outer membrane protein T cell vaccine against Chlamydia muridarum genital infection in three strains of mice.

    Science.gov (United States)

    Yu, Hong; Karunakaran, Karuna P; Jiang, Xiaozhou; Brunham, Robert C

    2014-08-06

    An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of Chlamydia muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH+MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population.

  9. Plasmolysis and Cell Shape Depend on Solute Outer-Membrane Permeability during Hyperosmotic Shock in E. coli

    OpenAIRE

    Pilizota, Teuta; Shaevitz, Joshua W.

    2013-01-01

    The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on t...

  10. Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice

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    Qiong Liu

    2016-03-01

    Full Text Available Lipopolysaccharide (LPS is a major virulence factor present in the outer membrane of Salmonella enterica serovar Typhimurium (S. Typhimurium. Outer membrane proteins (OMPs from Salmonella show high immunogenicity and provide protection against Salmonella infection, and truncated LPS alters the outer membrane composition of the cell wall. In our previous study, we demonstrated that Salmonella mutants carrying truncated LPS failed to induce strong immune responses and cross-reaction to other enteric bacteria, due to their high attenuation and low colonization in the host. Therefore, we plan to investigate whether outer membrane proteins from Salmonella mutants with truncated LPS resulting from a series of nonpolar mutations, including ∆waaC12, ∆waaF15, ∆waaG42, ∆rfaH49, ∆waaI43, ∆waaJ44, ∆waaL46, ∆wbaP45 and ∆wzy-48, affect immunogenicity and provide protection against diverse Salmonella challenge. In this study, the immunogenicity and cross-protection efficiency of purified OMPs from all mutants were investigated to explore a potential OMP vaccine to protect against homologous or heterologous serotype Salmonella challenge. The results demonstrated that OMPs from three Salmonella mutants (∆waaC12, ∆waaJ44 and ∆waaL46 induced higher immune responses and provided good protection against homologous S. Typhimurium. The OMPs from these three mutants were also selected to determine the cross-protective efficacy against homologous and heterologous serotype Salmonella. Our results indicated that the mutant ∆waaC12 can elicit higher cross-reactivity and can provide good protection against S. Choleraesuis and S. Enteritidis infection and that the cross-reactivity may be ascribed to an antigen of approximately 18.4–30 kDa.

  11. Bordetella pertussis lipid A glucosamine modification confers resistance to cationic antimicrobial peptides and increases resistance to outer membrane perturbation.

    Science.gov (United States)

    Shah, Nita R; Hancock, Robert E W; Fernandez, Rachel C

    2014-08-01

    Bordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

  12. Antigen sequence typing of outer membrane protein (fetA gene of Neisseria meningitidis serogroup A from Delhi & adjoining areas

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    S Dwivedi

    2014-01-01

    Full Text Available Background & objectives: Meningitis caused by Neisseria meningitidis is a fatal disease. Meningococcal meningitis is an endemic disease in Delhi and irregular pattern of outbreaks has been reported in India. All these outbreaks were associated with serogroup A. Detailed molecular characterization of N. meningitidis is required for the management of this fatal disease. In this study, we characterized antigenic diversity of surface exposed outer membrane protein (OMP FetA antigen of N. meningitidis serogroup A isolates obtained from cases of invasive meningococcal meningitis in Delhi, India. Methods: Eight isolates of N. meningitidis were collected from cerebrospinal fluid during October 2008 to May 2011 from occasional cases of meningococcal meningitis. Seven isolates were from outbreaks of meningococcal meningitis in 2005-2006 in Delhi and its adjoining areas. These were subjected to molecular typing of fetA gene, an outer membrane protein gene. Results: All 15 N. meningitides isolates studied were serogroup A. This surface exposed porin is putatively under immune pressure. Hence as a part of molecular characterization, genotyping was carried out to find out the diversity in outer membrane protein (FetA gene among the circulating isolates of N. meningitidis. All 15 isolates proved to be of the same existing allele type of FetA variable region (VR when matched with global database. The allele found was F3-1 for all the isolates. Interpretation & conclusions: There was no diversity reported in the outer membrane protein FetA in the present study and hence this protein appeared to be a stable molecule. More studies on molecular characterization of FetA antigen are required from different serogroups circulating in different parts of the world.

  13. Immunization of Mice With Vibrio cholerae Outer-Membrane Vesicles Protects Against Hyperinfectious Challenge and Blocks Transmission

    OpenAIRE

    Bishop, Anne L.; Tarique, Abdullah A.; Patimalla, Bharathi; Calderwood, Stephen B.; Qadri, Firdausi; Camilli, Andrew

    2011-01-01

    Background. Vibrio cholerae excreted by cholera patients is “hyperinfectious” (HI), which can be modeled by passage through infant mice. Immunization of adult female mice with V. cholerae outer-membrane vesicles (OMVs) passively protects suckling mice from challenge. Although V. cholerae is unable to colonize protected pups, the bacteria survive passage and have the potential to be transmitted to susceptible individuals. Here, we investigated the impact of OMV immunization and the HI state on...

  14. Meningococcal Outer Membrane Protein NhhA Is Essential for Colonization and Disease by Preventing Phagocytosis and Complement Attack▿

    OpenAIRE

    Sjölinder, Hong; Eriksson, Jens; Maudsdotter, Lisa; Aro, Helena; Jonsson, Ann-Beth

    2008-01-01

    Neisseria meningitidis is a leading cause of meningitis and septicemia worldwide, with a rapid onset of disease and a high morbidity and mortality. NhhA is a meningococcal outer membrane protein included in the family of trimeric autotransporter adhesins. The protein binds to the extracellular matrix proteins heparan sulfate and laminin and facilitates attachment to host epithelial cells. In this study, we show that NhhA is essential for bacterial colonization of the nasopharyngeal mucosa in ...

  15. Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice.

    Science.gov (United States)

    Liu, Qiong; Liu, Qing; Zhao, Xinxin; Liu, Tian; Yi, Jie; Liang, Kang; Kong, Qingke

    2016-03-22

    Lipopolysaccharide (LPS) is a major virulence factor present in the outer membrane of Salmonella enterica serovar Typhimurium (S. Typhimurium). Outer membrane proteins (OMPs) from Salmonella show high immunogenicity and provide protection against Salmonella infection, and truncated LPS alters the outer membrane composition of the cell wall. In our previous study, we demonstrated that Salmonella mutants carrying truncated LPS failed to induce strong immune responses and cross-reaction to other enteric bacteria, due to their high attenuation and low colonization in the host. Therefore, we plan to investigate whether outer membrane proteins from Salmonella mutants with truncated LPS resulting from a series of nonpolar mutations, including ∆waaC12, ∆waaF15, ∆waaG42, ∆rfaH49, ∆waaI43, ∆waaJ44, ∆waaL46, ∆wbaP45 and ∆wzy-48, affect immunogenicity and provide protection against diverse Salmonella challenge. In this study, the immunogenicity and cross-protection efficiency of purified OMPs from all mutants were investigated to explore a potential OMP vaccine to protect against homologous or heterologous serotype Salmonella challenge. The results demonstrated that OMPs from three Salmonella mutants (∆waaC12, ∆waaJ44 and ∆waaL46) induced higher immune responses and provided good protection against homologous S. Typhimurium. The OMPs from these three mutants were also selected to determine the cross-protective efficacy against homologous and heterologous serotype Salmonella. Our results indicated that the mutant ∆waaC12 can elicit higher cross-reactivity and can provide good protection against S. Choleraesuis and S. Enteritidis infection and that the cross-reactivity may be ascribed to an antigen of approximately 18.4-30 kDa.

  16. Critical role of quorum sensing-dependent glutamate metabolism in homeostatic osmolality and outer membrane vesiculation in Burkholderia glumae

    Science.gov (United States)

    Kang, Yongsung; Goo, Eunhye; Kim, Jinwoo; Hwang, Ingyu

    2017-01-01

    Metabolic homeostasis in cooperative bacteria is achieved by modulating primary metabolism in a quorum sensing (QS)-dependent manner. A perturbed metabolism in QS mutants causes physiological stress in the rice bacterial pathogen Burkholderia glumae. Here, we show that increased bacterial osmolality in B. glumae is caused by unusually high cellular concentrations of glutamate and betaine generated by QS deficiencies. QS negatively controls glutamate uptake and the expression of genes involved in the glutamine synthetase and glutamine oxoglutarate aminotransferase cycles. Thus, cellular glutamate levels were significantly higher in the QS mutants than in the wild type, and they caused hyperosmotic cellular conditions. Under the hypotonic conditions of the periplasm in the QS mutants, outer membrane bulging and vesiculation were observed, although these changes were rescued by knocking out the gltI gene, which encodes a glutamate transporter. Outer membrane modifications were not detected in the wild type. These results suggest that QS-dependent glutamate metabolism is critical for homeostatic osmolality. We suggest that outer membrane bulging and vesiculation might be the outcome of a physiological adaptation to relieve hypotonic osmotic stress in QS mutants. Our findings reveal how QS functions to maintain bacterial osmolality in a cooperative population. PMID:28272446

  17. Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp.

    Science.gov (United States)

    Maiti, Biswajit; Shetty, Mahesh; Shekar, Malathi; Karunasagar, Iddya; Karunasagar, Indrani

    2011-12-20

    Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His(6) residues was estimated to have a molecular weight of ~38kDa. In Western blot the native protein showed expression at ~36kDa molecular weight which was within the range of major outer membrane proteins (36-44kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.

  18. Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

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    B Josh Lane

    2008-03-01

    Full Text Available Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein. TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs, Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K, appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

  19. Species-specificity of the BamA component of the bacterial outer membrane protein-assembly machinery.

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    Elena B Volokhina

    Full Text Available The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner in vitro. In this work, we further studied species specificity in vivo by testing the functioning of BamA homologs of the proteobacteria Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Burkholderia mallei, and Escherichia coli in E. coli and in N. meningitidis. We found that no BamA functioned in another species than the authentic one, except for N. gonorrhoeae BamA, which fully complemented a N. meningitidis bamA mutant. E. coli BamA was not assembled into the N. meningitidis outer membrane. In contrast, the N. meningitidis BamA protein was assembled into the outer membrane of E. coli to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of N. meningitidis and E. coli BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded β-barrel conformation of the membrane-embedded domain of BamA.

  20. Rapid Diagnosis of Chlamydial Conjunctivitis in Laboratory

    Institute of Scientific and Technical Information of China (English)

    LiuJX; LiYP

    1999-01-01

    Purpose:To evaluate the techniques of rapid and accurate diagnosis of chlamydial conjunctivitis.Methods:Total 100 conjunctivitis patients(200 eyes) were studied.Twenty-tw of 100 cases were diagnosed as chlamydial conjunctivitis.The infected epithelia were scraped from tarsal conjunctive of both eyes and stained separately with Giemsa(100 cases)and immunofluorescnce (anti-chlamydial antigen monoclonal antibody,100 cases)Result:In immunofluorescent staining,38 cases were seen positive staining and 62 were negative.In giemsa staining,29 were positive,and 71 were negative.In 22 cases with clinical diagnosis of chlamydial conjunctivitis,13 cases were confirmed,and 9 were excluded by immunofluorescent staining,Technically,immunofluorescent and Gemsa stain takes 45 and 40 minutes,respectively.Conclusion:Comparing to Giemsa stain,38 of 100 scraping specimens were positive (38%) by immunofluorescent stainign,29 of 100 per cent were psitive by Giemsa staining,Giemsa staining takes 5 minutes less than immunofluorescent stainign (40 versus 45 minutes),however,the positive staining in immunofluorescent staining is much easier to be recognized than Giemsa staining.

  1. Progress of Study on Chlamydial Pathogenesis

    Institute of Scientific and Technical Information of China (English)

    刘全中; 傅宜志; 陈锦英

    2001-01-01

    Chlamydia, a human pathogen that mediates chronic, persistent and repeated infection progress, is capable of inflicting permanent scar formation. A typical reticular body is found in persistent infections. Chlamydial Hsp 60, interferon(IFN) -gamma and other cytokines function in the course of repeated infection. Immunopathological factors mediate chronic infection.

  2. Laboratory diagnosis of persistent human chlamydial infection

    Directory of Open Access Journals (Sweden)

    Mirja ePuolakkainen

    2013-12-01

    Full Text Available Diagnostic assays for persistent chlamydial infection are much needed to conduct high-quality, large-scale studies investigating the persistent state in vivo, its disease associations and the response to therapy. Yet in most studies the distinction between acute and persistent infection is based on the interpretation of the data obtained by the assays developed to diagnose acute infections or on complex assays available for research only and/or difficult to establish for clinical use. Novel biomarkers for detection of persistent chlamydial infection are urgently needed. Chlamydial whole genome proteome arrays are now available and they can identify chlamydial antigens that are differentially expressed between acute infection and persistent infection. Utilizing these data will lead to the development of novel diagnostic assays. Carefully selected specimens from well-studied patient populations are clearly needed in the process of translating the proteomic data into assays useful for clinical practice. Before such antigens are identified and validated assays become available, we face a challenge of deciding whether the persistent infection truly induced appearance of the proposed marker or do we just base our diagnosis of persistent infection on the presence of the suggested markers. Consequently, we must bear this in mind when interpreting the available data.

  3. Outer membrane β-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA.

    Science.gov (United States)

    Gessmann, Dennis; Chung, Yong Hee; Danoff, Emily J; Plummer, Ashlee M; Sandlin, Clifford W; Zaccai, Nathan R; Fleming, Karen G

    2014-04-22

    Outer membrane β-barrel proteins (OMPs) are crucial for numerous cellular processes in prokaryotes and eukaryotes. Despite extensive studies on OMP biogenesis, it is unclear why OMPs require assembly machineries to fold into their native outer membranes, as they are capable of folding quickly and efficiently through an intrinsic folding pathway in vitro. By investigating the folding of several bacterial OMPs using membranes with naturally occurring Escherichia coli lipids, we show that phosphoethanolamine and phosphoglycerol head groups impose a kinetic barrier to OMP folding. The kinetic retardation of OMP folding places a strong negative pressure against spontaneous incorporation of OMPs into inner bacterial membranes, which would dissipate the proton motive force and undoubtedly kill bacteria. We further show that prefolded β-barrel assembly machinery subunit A (BamA), the evolutionarily conserved, central subunit of the BAM complex, accelerates OMP folding by lowering the kinetic barrier imposed by phosphoethanolamine head groups. Our results suggest that OMP assembly machineries are required in vivo to enable physical control over the spontaneously occurring OMP folding reaction in the periplasm. Mechanistic studies further allowed us to derive a model for BamA function, which explains how OMP assembly can be conserved between prokaryotes and eukaryotes.

  4. Outer-selective pressure-retarded osmosis hollow fiber membranes from vacuum-assisted interfacial polymerization for osmotic power generation

    KAUST Repository

    Sun, Shipeng

    2013-11-19

    In this paper, we report the technical breakthroughs to synthesize outer-selective thin-film composite (TFC) hollow fiber membranes, which is in an urgent need for osmotic power generation with the pressure-retarded osmosis (PRO) process. In the first step, a defect-free thin-film composite membrane module is achieved by vacuum-assisted interfacial polymerization. The PRO performance is further enhanced by optimizing the support in terms of pore size and mechanical strength and the TFC layer with polydopamine coating and molecular engineering of the interfacial polymerization solution. The newly developed membranes can stand over 20 bar with a peak power density of 7.63 W/m2, which is equivalent to 13.72 W/m2 of its inner-selective hollow fiber counterpart with the same module size, packing density, and fiber dimensions. The study may provide insightful guidelines for optimizing the interfacial polymerization procedures and scaling up of the outer-selective TFC hollow fiber membrane modules for PRO power generation. © 2013 American Chemical Society.

  5. Outer-selective pressure-retarded osmosis hollow fiber membranes from vacuum-assisted interfacial polymerization for osmotic power generation.

    Science.gov (United States)

    Sun, Shi-Peng; Chung, Tai-Shung

    2013-11-19

    In this paper, we report the technical breakthroughs to synthesize outer-selective thin-film composite (TFC) hollow fiber membranes, which is in an urgent need for osmotic power generation with the pressure-retarded osmosis (PRO) process. In the first step, a defect-free thin-film composite membrane module is achieved by vacuum-assisted interfacial polymerization. The PRO performance is further enhanced by optimizing the support in terms of pore size and mechanical strength and the TFC layer with polydopamine coating and molecular engineering of the interfacial polymerization solution. The newly developed membranes can stand over 20 bar with a peak power density of 7.63 W/m(2), which is equivalent to 13.72 W/m(2) of its inner-selective hollow fiber counterpart with the same module size, packing density, and fiber dimensions. The study may provide insightful guidelines for optimizing the interfacial polymerization procedures and scaling up of the outer-selective TFC hollow fiber membrane modules for PRO power generation.

  6. In silico evolutionary analysis of Helicobacter pylori outer membrane phospholipase A (OMPLA

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    Vollan Hilde S

    2012-09-01

    Full Text Available Abstract Background In the past decade, researchers have proposed that the pldA gene for outer membrane phospholipase A (OMPLA is important for bacterial colonization of the human gastric ventricle. Several conserved Helicobacter pylori genes have distinct genotypes in different parts of the world, biogeographic patterns that can be analyzed through phylogenetic trees. The current study will shed light on the importance of the pldA gene in H. pylori. In silico sequence analysis will be used to investigate whether the bacteria are in the process of preserving, optimizing, or rejecting the pldA gene. The pldA gene will be phylogenetically compared to other housekeeping (HK genes, and a possible origin via horizontal gene transfer (HGT will be evaluated through both intra- and inter-species evolutionary analyses. Results In this study, pldA gene sequences were phylogenetically analyzed and compared with a large reference set of concatenated HK gene sequences. A total of 246 pldA nucleotide sequences were used; 207 were from Norwegian isolates, 20 were from Korean isolates, and 19 were from the NCBI database. Best-fit evolutionary models were determined with MEGA5 ModelTest for the pldA (K80 + I + G and HK (GTR + I + G sequences, and maximum likelihood trees were constructed. Both HK and pldA genes showed biogeographic clustering. Horizontal gene transfer was inferred based on significantly different GC contents, the codon adaptation index, and a phylogenetic conflict between a tree of OMPLA protein sequences representing 171 species and a tree of the AtpA HK protein for 169 species. Although a vast majority of the residues in OMPLA were predicted to be under purifying selection, sites undergoing positive selection were also found. Conclusions Our findings indicate that the pldA gene could have been more recently acquired than seven of the HK genes found in H. pylori. However, the common biogeographic patterns of both the HK and pldA sequences

  7. Helicobacter pylori outer membrane protein Q allele distribution is associated with distinct pathologies in Pakistan.

    Science.gov (United States)

    Yakoob, Javed; Abbas, Zaigham; Khan, Rustam; Salim, Saima Azhar; Awan, Safia; Abrar, Ambar; Jafri, Wasim

    2016-01-01

    Helicobacter pylori (H. pylori) strains expressing outer membrane protein Q (HopQ) promote adherence to the gastric epithelial cell. We characterized HopQ alleles in relation to H. pylori-related disease, histology and virulence markers. Gastric biopsies were obtained at esophagogastroduodenoscopy from patients with upper gastrointestinal symptoms. H. pylori culture, histology and polymerase chain reaction (PCR) for HopQ types, cagA, cagA-promoter and vacA alleles were performed. DNA extracted was used for PCR. Sequencing of PCR products of HopQ types 1 and 2 was followed by BLAST query. We examined 241 H. pylori isolates. HopQ type 1 was positive in 70 (29%) isolates, type 2 in 60 (25%) isolates, while both type 1 and type 2 in 111 (46%) H. pylori isolates, respectively. Nonulcer dyspepsia (NUD) was associated with HopQ type 2 in 48 (41%) isolates, while gastric carcinoma (GC) in 37 (53%) (P<0.001) with type 1 isolates. Gastric ulcers (GU) were 39 (46%) (P<0.001) in H. pylori infection with multiple HopQ alleles compared to 6 (23%) in HopQ type 1. Multivariate analysis demonstrated that multiple HopQ alleles were associated with GU OR 2.9 (1.07-7.8) (P=0.03). HopQ type 1 was associated with cagA 58 (84%) (P<0.001) and cagA-promoter 58 (83%) (P<0.001) compared to 14 (23%) and 17 (28%) respectively, in type 2. VacAs1a was associated with HopQ type 1 in 59 (84%) isolates compared to HopQ type 2 in 35 (58%) (P=0.002) isolates. VacAm1 was associated with HopQ type 1 in 53 (76%) isolates compared to HopQ type 2 in 32 (53%) (P=0.004) isolates. H. pylori infection with multiple HopQ alleles was predominant. H. pylori infection with single HopQ type 1 was associated with GC in the presence of other H. pylori virulence markers.

  8. Overexpression of an outer membrane protein associated with decreased susceptibility to carbapenems in Proteus mirabilis.

    Science.gov (United States)

    Tsai, Yi-Lin; Wang, Min-Cheng; Hsueh, Po-Ren; Liu, Ming-Che; Hu, Rouh-Mei; Wu, Yue-Jin; Liaw, Shwu-Jen

    2015-01-01

    Proteus mirabilis isolates commonly have decreased susceptibility to imipenem. Previously, we found P. mirabilis hfq mutant was more resistant to imipenem and an outer membrane protein (OMP) could be involved. Therefore, we investigated the role of this OMP in carbapenem susceptibility. By SDS-PAGE we found this OMP (named ImpR) was increased in hfq mutant and LC-MS/MS revealed it to be the homologue of Salmonella YbfM, which is a porin for chitobiose and subject to MicM (a small RNA) regulation. We demonstrated that ImpR overexpression resulted in increased carbapenem MICs in the laboratory strain and clinical isolates. Chitobiose induced expression of chb (a chitobiose utilization operon). Real-time RT-PCR and SDS-PAGE were performed to elucidate the relationship of hfq, impR, chb and MicM in P. mirabilis. We found MicM RNA was decreased in hfq mutant and chbBC-intergenic region (chbBC-IGR) overexpression strain (chbIGRov), while impR mRNA was increased in hfq mutant, micM mutant and chbIGRov strain. In addition, mutation of hfq or micM and overexpression of chbBC-IGR increased ImpR protein level. Accordingly, chitobiose made wild-type have higher levels of ImpR protein and are more resistant to carbapenems. Hfq- and MicM-complemented strains restored wild-type MICs. Mutation of both impR and hfq eliminated the increase in carbapenem MICs observed in hfq mutant and ImpR-complementation of hfq/impR double mutant resulted in MICs as hfq mutant, indicating that the ImpR-dependent decreased carbapenem susceptibility of hfq mutant. These indicate MicM was antisense to impR mRNA and was negatively-regulated by chbBC-IGR. Together, overexpression of ImpR contributed to the decreased carbapenem susceptibility in P. mirabilis.

  9. Energetics of primary processes in visula escitation: photocalorimetry of rhodopsin in rod outer segment membranes.

    Science.gov (United States)

    Cooper, A; Converse, C A

    1976-07-13

    A sensitive technique for the direct calorimetric determination of the energetics of photochemical reactions under low levels of illumination, and its application to the study of primary processes in visula excitation, are described. Enthlpies are reported for various steps in the bleaching of rhodopsin in intact rod outer segment membranes, together with the heats of appropriate model reactions. Protonation changes are also determined calorimetrically by use of buffers with differing heats of proton ionization. Bleaching of rhodopsin is accompanied by significant uptake of heat energy, vastly in excess of the energy required for simple isomerization of the retinal chromophore. Metarhodopsin I formation involves the uptake of about 17 kcal/mol and no net change in proton ionization of the system. Formation of metarhodopsin II requires an additional energy of about 10 kcal/mol and involves the uptake on one hydrogen ion from solution. The energetics of the overall photolysis reaction, rhodopsin leads to opsin + all-trans-retinal, are pH dependent and involve the exposure of an additional titrating group on opsin. This group has a heat of proton ionization of about 12 kcal/mal, characteristic of a primary amine, but a pKa in the region of neutrality. We suggest that this group is the Schiff base lysine of the chromophore binding site of rhodopsin which becomes exposed on photolysis. The low pKa for this active lysine would result in a more stable retinal-opsin linkage, and might be induced by a nearby positively charged group on the protein (either arginine or a second lysine residue). This leads to a model involving intramolecular protonation of the Schiff base nitrogen in the retinal-opsin linkage of rhodopsin, which is consistent with the thermodynamic and spectroscopic properties of the system. We further propose that the metarhodopsin I leads to metarhodopsin II step in the bleaching sequence involves reversible hydrolysis of the Schiff base linkage in the

  10. All subtypes of the Pmp adhesin family are implicated in chlamydial virulence and show species-specific function.

    Science.gov (United States)

    Becker, Elisabeth; Hegemann, Johannes H

    2014-08-01

    The bacterial pathogens Chlamydia trachomatis and C. pneumoniae are obligate intracellular parasites, cause a number of serious diseases, and can infect various cell types in humans. Chlamydial infections are probably initiated by binding of the bacterial outer membrane protein OmcB to host cell glycosaminoglycans (GAGs). Here, we show that all nine members of the polymorphic membrane protein (Pmp) family of C. trachomatis mediate adhesion to human epithelial and endothelial cells. Importantly, exposure of infectious particles to soluble recombinant Pmps blocks subsequent infection, thus implicating an important function of the entire protein family in the infection process. Analogous experiments with pairs of recombinant Pmps or a combination of Pmp and OmcB revealed that all Pmps probably act in an adhesion pathway that is distinct from the OmcB-GAG pathway. Finally, we provide evidence that the Pmps of C. trachomatis and C. pneumoniae exhibit species and tissue specificity. These findings argue for the involvement of C. trachomatis Pmps in the initial phase of infection and suggest that they may interact with a receptor other than the epidermal growth factor receptor recently identified for their counterparts in C. pneumoniae.

  11. Comparison of dot-ELISA and standard ELISA for detection of Neisseria meningitidis outer membrane complex-specific antibodies

    Directory of Open Access Journals (Sweden)

    Elza FT Belo

    2010-02-01

    Full Text Available Dot-ELISA using the outer membrane complex antigens of Neisseria meningitidis as a target was standardized for rapid detection of meningococcal-specific antibodies in human serum. We investigated the level of meningococcal-specific IgG, IgA, and IgM in serum using dot-ELISA with outer membrane antigens prepared from Neisseria meningitidis serotype B:4.19:P1.15,3,7,9 (a strain isolated from a Brazilian epidemic. The dot-ELISA is based on the same principles as the standard ELISA and is useful for detection of anti-N. meningitidis B antibodies in serum of patients with meningococcal infections. For the assay, outer membrane complexes (OMCs were absorbed by nitrocellulose membrane and blocked with a 5% skim milk solution. Serum samples were drawn upon hospital admission and during convalescence from patients with meningococcal septicemia, and single samples were drawn from uninfected controls. We retrospectively examined a total of 57 serum samples: 35 from patients infected with N. meningitidis B, 12 from patients infected with Haemophilus influenzae b, and 10 from health individuals. When performed at room temperature, dot-ELISA took approximately four hours to perform, and the optimum antigen concentration was 0.42 µg per dot. The specificity of IgG, IgM, and IgA demonstrates that dot-ELISA using OMCs from N. meningitidis B as a target is suitable for serologic verification of clinically suspected meningococcal disease in patients and for titer determination of antibodies produced during different phases of natural infection. Furthermore, the sensitivity of dot-ELISA was comparable to that of standard ELISA. Overall, dot-ELISA is simple to perform, rapid, and low cost. Further validation of the test as a screening tool is required.

  12. Degraded and stable phosphatidylglycerol in Escherichia coli inner and outer membranes, and recycling of fatty acyl residues.

    Science.gov (United States)

    Joseleau-Petit, D; Kepes, A

    1982-04-15

    The metabolic fate of membrane phospholipids in exponentially growing Escherichia coli was reexamined by incorporation and chase of labeled precursors: [32P]phosphate, [2-3H]glycerol and 3H-labeled fatty acids. It was found that the well-known turnover of phosphatidylglycerol lasted only about two generation times; after which period, the remaining labeled phosphatidylglycerol, approximately one-third of the total, was stable for at least the subsequent two generation times. The location of the stable phosphatidylglycerol pool remaining after the turnover in the outer and inner membrane was investigated. Both envelopes were found to contain stable phosphatidylglycerol so that the existence of a stable portion cannot be ascribed to its exclusive location in one leaflet. In some experiments, a small loss of labeled phosphatidylethanolamine was also observed, and upon fractionation this was found to occur exclusively in the outer membrane. [32P]Phosphate and [2-3H]glycerol labels of the degraded phospholipids were lost from lipid-soluble material, whereas labeled fatty acid, palmitate or oleate was reincorporated into newly synthesized phosphatidylethanolamine and phosphatidylglycerol, so that total fatty acid label remained constant in (membrane) phospholipid during chase. In view of the amount of glycerol lost, the recycling of the fatty acids under the form of diacylglycerols to phosphatidic acid does not appear to be the predominant pathway of reincorporation. After double labeling with [32P]phosphate and [3H]palmitate, followed by chase, a complete balance sheet of loss and reincorporation of fatty acid, in the three phospholipids, in the two envelopes could be established. Results indicate that fatty acid was reincorporated essentially in the inner membrane phospholipids. Movements of phospholipids and of fatty acids from one membrane to another and in the plane of each layer are discussed in the light of the results.

  13. Modification of the biosynthesis and composition of polyglycerophosphatides in outer and inner mitochondrial membranes by cytidine liponucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Stuhne-Sekalec, L.; Stanacev, N.Z. (Univ. of Toronto (Canada))

    1989-01-01

    The biosynthesis of (3H)polyglycerophosphatides ((3H)phosphatidylglycerophosphate and (3H)phosphatidylglycerol) in mitochondrial and submitochondrial (outer and inner) membranes isolated from guinea pig liver was examined. Experimental results have established that the amount of biosynthesized (3H)polyglycerophosphatides and the relative amounts of biosynthesized (3H)phosphatidylglycerol and (3H)phosphatidylglycerolphosphate can be influenced by varying the composition of fatty acids in CDP-diglycerides and by altering the incubation time of the mixture containing CDP-diglycerides (obligatory precursor), sn-(2-3H)glycerol-3-phosphate and mitochondria or submitochondrial membranes. The changes thus obtained in respect to the amount and composition of biosynthesized (3H)polyglycerophosphatides are different in mitochondria and submitochondrial membranes. The highest amount of biosynthesized (3H)polyglycerophosphatides was obtained with CDP-didecanoin and inner mitochondrial membranes. The greatest accumulation of (3H)phosphatidylglycerol with CDP-didecanoin was obtained in mitochondria and outer mitochondrial membranes, while in inner mitochondrial membranes the amounts of (3H)phosphatidylglycerol and (3H)phosphatidylglycerolphosphate accumulated were approximately the same. In general, prolongation of the incubation time decreased the relative amounts of (3H)phosphatidylglycerolphosphate and increased the amount of accumulated (3H)phosphatidylglycerol, but the absolute amounts of these (3H)polyglycerophosphatides were more dependent on fatty acid composition of CDP-diglycerides tested. The following cytidine liponucleotides were tested: CDP-didecanoin, CDP-dipalmitin, CDP-diolein, and CDP-diglycerides containing saturated and unsaturated fatty acids similar to those in egg yolk lecithin.

  14. Two outer membrane proteins contribute to cellular fitness in Caulobacter crescentus by preventing intracellular S-layer protein accumulation.

    Science.gov (United States)

    Overton, K Wesley; Park, Dan M; Yung, Mimi C; Dohnalkova, Alice C; Smit, John; Jiao, Yongqin

    2016-09-23

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport S-layer protein from the cytoplasm to the cell exterior. Caulobacter crescentus is unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFa and RsaFb, that, together with other components, form a type I protein translocation pathway for S-layer export. These proteins have homology to E. coli TolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFa and RsaFb are not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFa and RsaFb are required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFa and RsaFb led to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and RNA-seq, we show that loss of both RsaFa and RsaFb led to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein mis-folding and degradation pathways. These findings provide new insight into the requirement for RsaFa and RsaFb in cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in C. crescentus IMPORTANCE: Decreased growth rate and reduced cell fitness are common side effects of protein production in overexpression systems. Inclusion bodies typically form inside the cell largely due to lack of sufficient export machinery to transport the overexpressed proteins to the extracellular environment. This phenomenon can conceivably also occur in natural

  15. Shewanella oneidensis MR-1 Nanowires are Outer Membrane and Periplasmic Extensions of the Extracellular Electron Transport Components

    Energy Technology Data Exchange (ETDEWEB)

    Pirbadian, S.; Barchinger, S. E.; Leung, K. M.; Byun, H. S.; Jangir, Y.; Bouhenni, Rachida; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad; Shi, Liang; Gorby, Yuri A.; Golbeck, J. H.; El-Naggar, Mohamed Y.

    2014-08-20

    Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella neidensis MR-1. Using live fluorescence measurements, immunolabeling, and quantitative gene expression analysis, we report that S. oneidensis MR-1 nanowires are extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures, as previously thought. These bacterial nanowires were also associated with outer membrane vesicles and vesicle chains, structures ubiquitous in gram-negative bacteria. Redoxfunctionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

  16. Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

    Science.gov (United States)

    Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

    2014-04-11

    The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.

  17. Murein lipoprotein is a critical outer membrane component involved in Salmonella enterica serovar typhimurium systemic infection.

    Science.gov (United States)

    Fadl, A A; Sha, J; Klimpel, G R; Olano, J P; Niesel, D W; Chopra, A K

    2005-02-01

    Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppA and lppB) located on the chromosome of Salmonella enterica serovar Typhimurium contributed to bacterial virulence. In this study, we characterized lppA and lppB single-knockout (SKO) mutants and compared them with an lpp double-knockout (DKO) mutant using in vitro and in vivo models. Compared to the lpp DKO mutant, which was nonmotile, the motility of the lpp SKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT) S. enterica serovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with the lpp SKO mutants compared to the cytotoxicity when cells were infected with the lpp DKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with the lppB SKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WT S. enterica serovar Typhimurium, than the level in host cells infected with the lppA SKO mutant. The lpp DKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with the lppB SKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-alpha and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in the lppA SKO mutant-infected mice than in animals challenged with the lpp DKO mutant. The increased cytokine levels observed with the lppB SKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with the lppA SKO and lpp DKO mutants. Moreover, the lppB SKO mutant-infected mice had increased

  18. Evaluation of methods for predicting the topology of β-barrel outer membrane proteins and a consensus prediction method

    Directory of Open Access Journals (Sweden)

    Hamodrakas Stavros J

    2005-01-01

    Full Text Available Abstract Background Prediction of the transmembrane strands and topology of β-barrel outer membrane proteins is of interest in current bioinformatics research. Several methods have been applied so far for this task, utilizing different algorithmic techniques and a number of freely available predictors exist. The methods can be grossly divided to those based on Hidden Markov Models (HMMs, on Neural Networks (NNs and on Support Vector Machines (SVMs. In this work, we compare the different available methods for topology prediction of β-barrel outer membrane proteins. We evaluate their performance on a non-redundant dataset of 20 β-barrel outer membrane proteins of gram-negative bacteria, with structures known at atomic resolution. Also, we describe, for the first time, an effective way to combine the individual predictors, at will, to a single consensus prediction method. Results We assess the statistical significance of the performance of each prediction scheme and conclude that Hidden Markov Model based methods, HMM-B2TMR, ProfTMB and PRED-TMBB, are currently the best predictors, according to either the per-residue accuracy, the segments overlap measure (SOV or the total number of proteins with correctly predicted topologies in the test set. Furthermore, we show that the available predictors perform better when only transmembrane β-barrel domains are used for prediction, rather than the precursor full-length sequences, even though the HMM-based predictors are not influenced significantly. The consensus prediction method performs significantly better than each individual available predictor, since it increases the accuracy up to 4% regarding SOV and up to 15% in correctly predicted topologies. Conclusions The consensus prediction method described in this work, optimizes the predicted topology with a dynamic programming algorithm and is implemented in a web-based application freely available to non-commercial users at http://bioinformatics.biol.uoa.gr/ConBBPRED.

  19. Key Residues of Outer Membrane Protein OprI Involved in Hexamer Formation and Bacterial Susceptibility to Cationic Antimicrobial Peptides

    OpenAIRE

    Chang, Ting-Wei; Wang, Chiu-Feng; Huang, Hsin-Jye; Wang, Iren; Hsu, Shang-Te Danny; Liao, You-Di

    2015-01-01

    Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI from Pseudomonas aeruginosa or its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985–8994, 2010, http://dx.doi.org/10.1074...

  20. Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

    OpenAIRE

    2005-01-01

    It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (Mw and pI). ...

  1. Outer membrane protein profiling to distinguish between Haemophilus aegyptius and non-capsulate Haemophilus influenzae biotype III.

    Science.gov (United States)

    Leaves, N I; Anderson, E C; Toy, S J

    1994-12-01

    Outer membrane protein profiling was used to assist in determining the identity of an isolate of Haemophilus spp. that was presumptively identified as non-capsulate Haemophilus influenzae biotype III. The possibility that this strain was in fact Haemophilus aegyptius was queried because of clinical information and the source of the isolate. Sodium dodecyl-sulphate polyacrylamide gel electrophoresis was used to establish the identity of the isolate as non-capsulate H. influenzae biotype III and no H. aegyptius. Generally, protein profiling compared very favourably with other standard tests for identifying H. aegyptius: the method was easily and rapidly performed and gave an unequivocal result.

  2. Disruption the Outer Membrane of Enteropathogenic and Enterotoxigenic Escherichia coli using Proanthocyanidins

    Science.gov (United States)

    American cranberry (Vaccinium macrocarpon) proanthocyanidins (PACs) have been reported as a natural antibacterial agent to suppress the growth of pathogenic Escherichia coli. The objective of this study was to investigate the efficacy of cranberry-derived proanthocyanidins on destabilizing the outer...

  3. Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes

    Science.gov (United States)

    Shen, Hsin-Hui; Belousoff, Matthew J.; Noinaj, Nicholas; Lu, Jingxiong; Holt, Stephen A.; Tan, Khershing; Selkrig, Joel; Webb, Chaille T.; Buchanan, Susan K.; Martin, Lisandra L.; Lithgow, Trevor

    2015-01-01

    In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation assembly module (the TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by Quartz Crystal Microbalance with Dissipation (QCM-D) and Magnetic Contrast Neutron Reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here, we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines. PMID:25341963

  4. Detecting the form of selection in the outer membrane protein C of Enterobacter aerogenes strains and Salmonella species.

    Science.gov (United States)

    Padhi, Abinash; Verghese, Bindhu; Otta, Subhendu K

    2009-01-01

    The types of selective pressure operating on the outer membrane protein C (ompC) of Enterobacter aerogenes strains, the causative agent for nosocomial infections, and Salmonella sp., the hazardous pathogen are investigated using the maximum likelihood-based codon substitution models. Although the rate of amino acid replacement to the silent substitution (omega) across the entire codon sites of ompC of E. aerogenes (omega=0.3194) and Salmonella sp. (omega=0.2047) indicate that the gene is subjected to purifying selection (i.e. omega1). Such contrast in the intensity of selective pressures in both pathogens could be associated with the differential response to the adverse environmental changes. In E. aerogenes, majority of the positively selected sites are located in the hypervariable cell-surface-exposed domains whereas the trans-membrane domains are functionally highly constrained.

  5. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M., E-mail: fmarassi@sbmri.org [Sanford-Burnham Medical Research Institute (United States)

    2015-04-15

    Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales.

  6. LytM Proteins Play a Crucial Role in Cell Separation, Outer Membrane Composition, and Pathogenesis in Nontypeable Haemophilus influenzae

    Science.gov (United States)

    Ercoli, Giuseppe; Tani, Chiara; Pezzicoli, Alfredo; Vacca, Irene; Martinelli, Manuele; Pecetta, Simone; Petracca, Roberto; Rappuoli, Rino; Pizza, Mariagrazia; Soriani, Marco

    2015-01-01

    ABSTRACT LytM proteins belong to a family of bacterial metalloproteases. In Gram-negative bacteria, LytM factors are mainly reported to have a direct effect on cell division by influencing cleavage and remodeling of peptidoglycan. In this study, mining nontypeable Haemophilus influenzae (NTHI) genomes, three highly conserved open reading frames (ORFs) containing a LytM domain were identified, and the proteins encoded by the ORFs were named YebA, EnvC, and NlpD on the basis of their homology with the Escherichia coli proteins. Immunoblotting and confocal analysis showed that while NTHI NlpD is exposed on the bacterial surface, YebA and EnvC reside in the periplasm. NTHI ΔyebA and ΔnlpD deletion mutants revealed an aberrant division phenotype characterized by an altered cell architecture and extensive membrane blebbing. The morphology of the ΔenvC deletion mutant was identical to that of the wild-type strain, but it showed a drastic reduction of periplasmic proteins, including the chaperones HtrA, SurA, and Skp, and an accumulation of β-barrel-containing outer membrane proteins comprising the autotransporters Hap, IgA serine protease, and HMW2A, as observed by proteomic analysis. These data suggest that EnvC may influence the bacterial surface protein repertoire by facilitating the passage of the periplasmic chaperones through the peptidoglycan layer to the close vicinity of the inner face of the outer membrane. This hypothesis was further corroborated by the fact that an NTHI envC defective strain had an impaired capacity to adhere to epithelial cells and to form biofilm. Notably, this strain also showed a reduced serum resistance. These results suggest that LytM factors are not only important components of cell division but they may also influence NTHI physiology and pathogenesis by affecting membrane composition. PMID:25714719

  7. Chlamydia genomics: providing novel insights into chlamydial biology.

    Science.gov (United States)

    Bachmann, Nathan L; Polkinghorne, Adam; Timms, Peter

    2014-08-01

    Chlamydiaceae are obligate intracellular pathogens that have successfully evolved to colonize a diverse range of hosts. There are currently 11 described species of Chlamydia, most of which have a significant impact on the health of humans or animals. Expanding chlamydial genome sequence information has revolutionized our understanding of chlamydial biology, including aspects of their unique lifecycle, host-pathogen interactions, and genetic differences between Chlamydia strains associated with different host and tissue tropisms. This review summarizes the major highlights of chlamydial genomics and reflects on the considerable impact these have had on understanding the biology of chlamydial pathogens and the changing nature of genomics tools in the 'post-genomics' era.

  8. Fusion between perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB.

    Science.gov (United States)

    Wright, Catherine C; Wisner, Todd W; Hannah, Brian P; Eisenberg, Roselyn J; Cohen, Gary H; Johnson, David C

    2009-11-01

    Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic "fusion loops." These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.

  9. Undressing of Waddlia chondrophila to enrich its outer membrane proteins to develop a new species-specific ELISA

    Directory of Open Access Journals (Sweden)

    J. Lienard

    2014-01-01

    Full Text Available Waddlia chondrophila, an obligate intracellular bacterium of the Chlamydiales order, is considered as an agent of bovine abortion and a likely cause of miscarriage in humans. Its role in respiratory diseases was questioned after the detection of its DNA in clinical samples taken from patients suffering from pneumonia or bronchiolitis. To better define the role of Waddlia in both miscarriage and pneumonia, a tool allowing large-scale serological investigations of Waddlia seropositivity is needed. Therefore, enriched outer membrane proteins of W. chondrophila were used as antigens to develop a specific ELISA. After thorough analytical optimization, the ELISA was validated by comparison with micro-immunofluorescence and it showed a sensitivity above 85% with 100% specificity. The ELISA was subsequently applied to human sera to specify the role of W. chondrophila in pneumonia. Overall, 3.6% of children showed antibody reactivity against W. chondrophila but no significant difference was observed between children with and without pneumonia. Proteomic analyses were then performed using mass spectrometry, highlighting members of the outer membrane protein family as the dominant proteins. The major Waddlia putative immunogenic proteins were identified by immunoblot using positive and negative human sera. The new ELISA represents an efficient tool with high throughput applications. Although no association with pneumonia and Waddlia seropositivity was observed, this ELISA could be used to specify the role of W. chondrophila in miscarriage and in other diseases.

  10. Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum.

    Science.gov (United States)

    Hellman, J; Loiselle, P M; Tehan, M M; Allaire, J E; Boyle, L A; Kurnick, J T; Andrews, D M; Sik Kim, K; Warren, H S

    2000-05-01

    Complexes containing lipopolysaccharide (LPS) and three outer membrane proteins (OMPs) are released by gram-negative bacteria incubated in human serum and into the circulation in an experimental model of sepsis. The same OMPs are bound by immunoglobulin G (IgG) in the cross-protective antiserum raised to Escherichia coli J5 (anti-J5 IgG). This study was performed to identify the three OMPs. The 35-kDa OMP was identified as outer membrane protein A (OmpA) by immunoblotting studies using OmpA-deficient bacteria and recombinant OmpA protein. The 18-kDa OMP was identified as peptidoglycan-associated lipoprotein (PAL) based on peptide sequences from the purified protein and immunoblotting studies using PAL-deficient bacteria. The 5- to 9-kDa OMP was identified as murein lipoprotein (MLP) based on immunoblotting studies using MLP-deficient bacteria. The studies identify the OMPs released into human serum and into the circulation in an experimental model of sepsis as OmpA, PAL, and MLP.

  11. Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

    Science.gov (United States)

    Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing

    2016-11-01

    Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

  12. Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein

    DEFF Research Database (Denmark)

    Gram, Trine; Ahrens, Peter

    1998-01-01

    The gene (omlA) coding for an outer membrane protein of Actinobacillus pleuropneumoniae serotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay, The corresponding regions of all 12 A. pleuropneumoniae reference strains of biovar 1 were sequenc...... and sensitivity of this PCR compared to those of culture suggest the use of this PCR for routine identification of A. pleuropneumoniae.......The gene (omlA) coding for an outer membrane protein of Actinobacillus pleuropneumoniae serotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay, The corresponding regions of all 12 A. pleuropneumoniae reference strains of biovar 1 were sequenced...... species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of an A. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 10(2) CFU/PCR test tube. The specificity...

  13. Association of spin-labeled lipids with beta-barrel proteins from the outer membrane of Escherichia coli.

    Science.gov (United States)

    Ramakrishnan, Muthu; Pocanschi, Cosmin L; Kleinschmidt, Jörg H; Marsh, Derek

    2004-09-21

    The interaction of spin-labeled lipids with beta-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for alpha-helical integral proteins. The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of dimyristoylphosphatidylglycerol. The ESR spectra from phosphatidylglycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the beta-barrel, in addition to that from the fluid bilayer lipids. The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio. It is proportional to the number of transmembrane beta-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein. Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins. The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the beta-sheet and shorter interconnecting loops on the extracellular side of FhuA.

  14. Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses.

    Science.gov (United States)

    Kim, Oh Youn; Hong, Bok Sil; Park, Kyong-Su; Yoon, Yae Jin; Choi, Seng Jin; Lee, Won Hee; Roh, Tae-Young; Lötvall, Jan; Kim, Yoon-Keun; Gho, Yong Song

    2013-04-15

    Outer membrane vesicles (OMVs), secreted from Gram-negative bacteria, are spherical nanometer-sized proteolipids enriched with outer membrane proteins. OMVs, also known as extracellular vesicles, have gained interests for use as nonliving complex vaccines and have been examined for immune-stimulating effects. However, the detailed mechanism on how OMVs elicit the vaccination effect has not been studied extensively. In this study, we investigated the immunological mechanism governing the protective immune response of OMV vaccines. Immunization with Escherichia coli-derived OMVs prevented bacteria-induced lethality and OMV-induced systemic inflammatory response syndrome. As verified by adoptive transfer and gene-knockout studies, the protective effect of OMV immunization was found to be primarily by the stimulation of T cell immunity rather than B cell immunity, especially by the OMV-Ag-specific production of IFN-γ and IL-17 from T cells. By testing the bacteria-killing ability of macrophages, we also demonstrated that IFN-γ and IL-17 production is the main factor promoting bacterial clearances. Our findings reveal that E. coli-derived OMV immunization effectively protects bacteria-induced lethality and OMV-induced systemic inflammatory response syndrome primarily via Th1 and Th17 cell responses. This study therefore provides a new perspective on the immunological detail regarding OMV vaccination.

  15. Development of new cloning vectors for the production of immunogenic outer membrane fusion proteins in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Cornelis, P.; Sierra, J.C.; Lim, A. Jr.; Malur, A. [Vrije Universiteit Brussel, Paardenstraat (Belgium)] [and others

    1996-02-01

    The Pseudomonas aeruginosa lipoprotein gene (oprI) was modified by cloning an in-frame polylinker in both orientations at the end of oprI. The resulting plasmids pVUBI and pVUB2 allow high lipoprotein production in E. coli after IPTG induction. The modified lipoproteins are present in the outer membrane and surface-exposed. Outer membrane-bound fusion proteins of different sizes were produced and used to generate antibodies without use of adjuvant. An 87 bp DNA fragment from the vp72 capsid protein gene of African Swine Fever virus (ASFV) and the entire Leishmania major glycoprotein gp63 gene were expressed in this system. Finally, a fusion lipoprotein containing a 16 amino acid epitope from the preS2b region of Hepatitis B virus (HBV) was presented by an antigen-presenting cell line to a T-cell hybridoma while the corresponding cross-linked S2b peptide was not. The results suggest that OprI-based fusion proteins can be used to generate both humoral and cellular immune responses. 44 refs., 7 figs.

  16. Characterization and pathogenic role of outer membrane vesicles produced by the fish pathogen Piscirickettsia salmonis under in vitro conditions.

    Science.gov (United States)

    Oliver, Cristian; Valenzuela, Karla; Hernández, Mauricio; Sandoval, Rodrigo; Haro, Ronie E; Avendaño-Herrera, Ruben; Cárcamo, Juan G; Villar, Maite T; Artigues, Antonio; Garduño, Rafael; Yáñez, Alejandro J

    2016-02-29

    Piscirickettsia salmonis is one of the major fish pathogens affecting Chilean aquaculture. This Gram-negative bacterium is highly infectious and is the etiological agent of Piscirickettsiosis. Little is currently known about how the virulence factors expressed by P. salmonis are delivered to host cells. However, it is known that several Gram-negative microorganisms constitutively release outer membrane vesicles (OMVs), which have been implicated in the delivery of virulence factors to host cells. In this study, OMVs production by P. salmonis was observed during infection in CHSE-214 cells and during normal growth in liquid media. The OMVs were spherical vesicles ranging in size between 25 and 145 nm. SDS-PAGE analysis demonstrated that the protein profile of the OMVs was similar to the outer membrane protein profile of P. salmonis. Importantly, the bacterial chaperonin Hsp60 was found in the OMVs of P. salmonis by Western-blot and LC-MS/MS analyses. Finally, in vitro infection assays showed that purified OMVs generated a cytopathic effect on CHSE-214 cells, suggesting a role in pathogenesis. Therefore, OMVs might be an important vehicle for delivering effector molecules to host cells during P. salmonis infection.

  17. Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement.

    Science.gov (United States)

    Oikawa, Kazusato; Yamasato, Akihiro; Kong, Sam-Geun; Kasahara, Masahiro; Nakai, Masato; Takahashi, Fumio; Ogura, Yasunobu; Kagawa, Takatoshi; Wada, Masamitsu

    2008-10-01

    Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.

  18. A novel Geobacteraceae-specific outer membrane protein J (OmpJ is essential for electron transport to Fe (III and Mn (IV oxides in Geobacter sulfurreducens

    Directory of Open Access Journals (Sweden)

    Schiffer Marianne

    2005-07-01

    Full Text Available Abstract Background Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III reduction in the Geobacter species that are the predominant Fe(III reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. Results When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J, was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe (III and insoluble Mn (IV oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal

  19. Insertion of Cecropin A and reconstitution of bacterial outer membrane protein FhuA variants in polymeric membranes

    OpenAIRE

    Muhammad, Noor

    2011-01-01

    Polymer based nanocompartments (polymersomes) have potential applications in synthetic biology (pathway engineering), medicine (drug release), and industrial biotechnology (chiral nanoreactors, multistep synthesis, bioconversions in non-aqueous environments, and selective product recovery). The aforementioned goals can be accomplished by polymer membrane functionalization through covalent bonding or inclusion of proteins/peptides, to obtain specific properties like recognition, catalytic acti...

  20. Chlamydial genes shed light on the evolution of photoautotrophic eukaryotes

    Directory of Open Access Journals (Sweden)

    Melkonian Michael

    2008-07-01

    Full Text Available Abstract Background Chlamydiae are obligate intracellular bacteria of protists, invertebrates and vertebrates, but have not been found to date in photosynthetic eukaryotes (algae and embryophytes. Genes of putative chlamydial origin, however, are present in significant numbers in sequenced genomes of photosynthetic eukaryotes. It has been suggested that such genes were acquired by an ancient horizontal gene transfer from Chlamydiae to the ancestor of photosynthetic eukaryotes. To further test this hypothesis, an extensive search for proteins of chlamydial origin was performed using several recently sequenced algal genomes and EST databases, and the proteins subjected to phylogenetic analyses. Results A total of 39 proteins of chlamydial origin were retrieved from the photosynthetic eukaryotes analyzed and their identity verified through phylogenetic analyses. The distribution of the chlamydial proteins among four groups of photosynthetic eukaryotes (Viridiplantae, Rhodoplantae, Glaucoplantae, Bacillariophyta was complex suggesting multiple acquisitions and losses. Evidence is presented that all except one of the chlamydial genes originated from an ancient endosymbiosis of a chlamydial bacterium into the ancestor of the Plantae before their divergence into Viridiplantae, Rhodoplantae and Glaucoplantae, i.e. more than 1.1 BYA. The chlamydial proteins subsequently spread through secondary plastid endosymbioses to other eukaryotes. Of 20 chlamydial proteins recovered from the genomes of two Bacillariophyta, 10 were of rhodoplant, and 10 of viridiplant origin suggesting that they were acquired by two different secondary endosymbioses. Phylogenetic analyses of concatenated sequences demonstrated that the viridiplant secondary endosymbiosis likely occurred before the divergence of Chlorophyta and Streptophyta. Conclusion We identified 39 proteins of chlamydial origin in photosynthetic eukaryotes signaling an ancient invasion of the ancestor of the

  1. Engineering of the E. coli Outer Membrane Protein FhuA to overcome the Hydrophobic Mismatch in Thick Polymeric Membranes

    Directory of Open Access Journals (Sweden)

    Fioroni Marco

    2011-03-01

    Full Text Available Abstract Background Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. Results To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext. The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB1000-PEG6000-PIB1000 (PIB = polyisobutylene, PEG = polyethyleneglycol has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine. Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB1000-PEG6000-PIB1000 membrane. Furthermore labeling of the Lys-NH2 groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. Conclusion Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

  2. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice C.; Smit, John; Jiao, Yongqin; Parales, R. E.

    2016-09-23

    ABSTRACT

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in

  3. Role of Tim50 in the transfer of precursor proteins from the outer to the inner membrane of mitochondria.

    Science.gov (United States)

    Mokranjac, Dejana; Sichting, Martin; Popov-Celeketić, Dusan; Mapa, Koyeli; Gevorkyan-Airapetov, Lada; Zohary, Keren; Hell, Kai; Azem, Abdussalam; Neupert, Walter

    2009-03-01

    Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.

  4. Proteome-scale identification of outer membrane proteins in Mycobacterium avium subspecies paratuberculosis using a structure based combined hierarchical approach.

    Science.gov (United States)

    Rana, Aarti; Rub, Abdur; Akhter, Yusuf

    2014-07-29

    Outer membrane proteins (OMPs) in eubacteria have several important roles, which range from membrane transport to the host-pathogen interactions. These are directly involved in pathogen attachment, entry and activation of several pathogen-induced signaling cascades in the cell. The cardinal structural features of OMPs include the presence of a β-barrel, a signal peptide and the absence of the transmembrane helix. This is the first report on proteome-wide identification of OMPs of ruminant pathogen, Mycobacterium avium subsp. paratuberculosis (MAP). The complete proteome of MAP was analyzed using a pipeline of algorithms, which screens the amino acid sequences and structural features shared by OMPs in other bacteria. Secondary structure of these proteins is also analyzed and scores are calculated for amphiphilic β-strands. From the set of 588 exported proteins, 264 proteins are predicted to be inner membrane proteins while 83 proteins are identified as potential OMPs in MAP. Finally, this study identified 57 proteins as top candidates, on the basis of computed isoelectric points, as the core set of OMPs. Significantly, the resulting data for OMPs are not only useful in designing novel vaccines but may also open avenues for the development of early serodiagnostic tools for MAP.

  5. Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice.

    Science.gov (United States)

    Karch, Jason; Kwong, Jennifer Q; Burr, Adam R; Sargent, Michelle A; Elrod, John W; Peixoto, Pablo M; Martinez-Caballero, Sonia; Osinska, Hanna; Cheng, Emily H-Y; Robbins, Jeffrey; Kinnally, Kathleen W; Molkentin, Jeffery D

    2013-08-27

    A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death. DOI:http://dx.doi.org/10.7554/eLife.00772.001.

  6. Charged anaesthetics alter LM-fibroblast plasma-membrane enzymes by selective fluidization of inner or outer membrane leaflets.

    Science.gov (United States)

    Sweet, W D; Schroeder, F

    1986-10-15

    The functional consequences of the differences in lipid composition and structure between the two leaflets of the plasma membrane were investigated. Fluorescence of 1,6-diphenylhexa-1,3,5-triene(DPH), quenching, and differential polarized phase fluorimetry demonstrated selective fluidization by local anaesthetics of individual leaflets in isolated LM-cell plasma membranes. As measured by decreased limiting anisotropy of DPH fluorescence, cationic (prilocaine) and anionic (phenobarbital and pentobarbital) amphipaths preferentially fluidized the cytofacial and exofacial leaflets respectively. Unlike prilocaine, procaine, also a cation, fluidized both leaflets of these membranes equally. Pentobarbital stimulated 5'-nucleotidase between 0.1 and 5 mM and inhibited at higher concentrations, whereas phenobarbital only inhibited, at higher concentrations. Cationic drugs were ineffective. Two maxima of (Na+ + K+)-ATPase activation were obtained with both anionic drugs. Only one activation maximum was obtained with both cationic drugs. The maximum in activity below 1 mM for all four drugs clustered about a single limiting anisotropy value in the cytofacial leaflet, whereas there was no correlation between activity and limiting anisotropy in the exofacial leaflets. Therefore, although phenobarbital and pentobarbital below 1 mM fluidized the exofacial leaflet more than the cytofacial leaflet, the smaller fluidization in the cytofacial leaflet was functionally significant for (Na+ + K+)-ATPase. Mg2+-ATPase was stimulated at 1 mM-phenobarbital, unaffected by pentobarbital and slightly stimulated by both cationic drugs at concentrations fluidizing both leaflets. Thus the activity of (Na+ + K+)-ATPase was highly sensitive to selective fluidization of the leaflet containing its active site, whereas the other enzymes examined were little affected by fluidization of either leaflet.

  7. Protection against Chlamydia trachomatis infection and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of the major outer membrane protein

    DEFF Research Database (Denmark)

    Olsen, Anja W.; Follmann, Frank; Erneholm, Karin Susanne

    2015-01-01

    The VD4 region from the Chlamydia trachomatis major outer membrane protein contains important neutralizing B-cell epitopes of relevance for antibody-mediated protection against genital tract infection. We developed a multivalent vaccine construct based on VD4s and their surrounding constant segme...... of the major outer membrane protein resulted in a protective and broadly neutralizing vaccine. Our findings emphasize the important role of antibodies in protection against Chlamydia trachomatis.......The VD4 region from the Chlamydia trachomatis major outer membrane protein contains important neutralizing B-cell epitopes of relevance for antibody-mediated protection against genital tract infection. We developed a multivalent vaccine construct based on VD4s and their surrounding constant...... segments from serovars D, E, and F. Adjuvanted with cationic liposomes, this construct promoted strong immune responses to serovar-specific epitopes, the conserved LNPTIAG epitope and neutralized serovars D, E, and F. Vaccinated mice were protected against challenge, with protection defined as reduced...

  8. VDAC electronics: 3. VDAC-Creatine kinase-dependent generation of the outer membrane potential in respiring mitochondria.

    Science.gov (United States)

    Lemeshko, Victor V

    2016-07-01

    Mitochondrial energy in cardiac cells has been reported to be channeled into the cytosol through the intermembrane contact sites formed by the adenine nucleotide translocator, creatine kinase and VDAC. Computational analysis performed in this study showed a high probability of the outer membrane potential (OMP) generation coupled to such a mechanism of energy channeling in respiring mitochondria. OMPs, positive inside, calculated at elevated concentrations of creatine are high enough to restrict ATP release from mitochondria, to significantly decrease the apparent K(m,ADP) for state 3 respiration and to maintain low concentrations of Ca(2+) in the mitochondrial intermembrane space. An inhibition by creatine of Ca(2+)-induced swelling of isolated mitochondria and other protective effects of creatine reported in the literature might be explained by generated positive OMP. We suggest that VDAC-creatine kinase-dependent generation of OMP represents a novel physiological factor controlling metabolic state of mitochondria, cell energy channeling and resistance to death.

  9. Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant.

    Science.gov (United States)

    Chu, Chun-Yen; Liu, Chia-Hui; Liou, Jhong-Jie; Lee, Jai-Wei; Cheng, Li-Ting

    2015-01-01

    Riemerella anatipestifer, a Gram-negative bacillus, causes septicemia that can result in high mortality for ducklings. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant outer membrane protein A (rOmpA) and plasmid constructs containing CpG oligodeoxynucleotides (ODN). Results showed that CpG ODN enhanced both humoral and cell-mediated immunity elicited by rOmpA as early as two weeks after primary immunization. When compared to ducks immunized with rOmpA, ducks immunized with rOmpA+CpG ODN showed higher levels (pvaccine reduced the pathological score by 90% in comparison with the saline control. In conclusion, our study found that CpG ODN can enhance both humoral and cellular immunity elicited by a rOmpA vaccine. The rOmpA+CpG ODN vaccine can be further developed as a subunit vaccine against R. anatipestifer.

  10. Investigations of hemadsorption characteristics of glycoprotein HN and F antigen of parainfluenza 3 virus outer membrane in vitro

    Directory of Open Access Journals (Sweden)

    Nišavić Jakov

    2006-01-01

    Full Text Available The objective of our investigations was to examine the hemadsorption characteristics of glycoprotein HN and F antigens of the outer membrane of the parainfluenza 3 virus. Six hours following inoculation of the cell line with the Vero virus PI3, previously activated with 0.025 g/dl trypsinversen, the appearance of less intense hemadsorption was established in antigen dilutions of 1:2 and 1:4. Hemadsorption was established in the mentioned cell lines also 12 h after inoculation in virus dilutions of 1:8 and 1:16, while 24 h following inoculation, hemadsorption was present also in a virus dilution of 1:64. Inhibition of hemadsorption in inoculated cells with immune serum dilutions of 1:16 was established 24 h after inoculation of the cell line with Vero samples of the virus PI3, activated with trypsinversen and with the addition of specific immune serum. .

  11. Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?

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    Paramasivam Nagarajan

    2012-09-01

    Full Text Available Abstract Background In Gram-negative bacteria, the outer membrane is composed of an asymmetric lipid bilayer of phopspholipids and lipopolysaccharides, and the transmembrane proteins that reside in this membrane are almost exclusively β-barrel proteins. These proteins are inserted into the membrane by a highly conserved and essential machinery, the BAM complex. It recognizes its substrates, unfolded outer membrane proteins (OMPs, through a C-terminal motif that has been speculated to be species-specific, based on theoretical and experimental results from only two species, Escherichia coli and Neisseria meningitidis, where it was shown on the basis of individual sequences and motifs that OMPs from the one cannot easily be over expressed in the other, unless the C-terminal motif was adapted. In order to determine whether this species specificity is a general phenomenon, we undertook a large-scale bioinformatics study on all predicted OMPs from 437 fully sequenced proteobacterial strains. Results We were able to verify the incompatibility reported between Escherichia coli and Neisseria meningitidis, using clustering techniques based on the pairwise Hellinger distance between sequence spaces for the C-terminal motifs of individual organisms. We noticed that the amino acid position reported to be responsible for this incompatibility between Escherichia coli and Neisseria meningitidis does not play a major role for determining species specificity of OMP recognition by the BAM complex. Instead, we found that the signal is more diffuse, and that for most organism pairs, the difference between the signals is hard to detect. Notable exceptions are the Neisseriales, and Helicobacter spp. For both of these organism groups, we describe the specific sequence requirements that are at the basis of the observed difference. Conclusions Based on the finding that the differences between the recognition motifs of almost all organisms are small, we assume that

  12. Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach.

    Directory of Open Access Journals (Sweden)

    Gabriel Gomez

    Full Text Available Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.

  13. Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach.

    Science.gov (United States)

    Gomez, Gabriel; Pei, Jianwu; Mwangi, Waithaka; Adams, L Garry; Rice-Ficht, Allison; Ficht, Thomas A

    2013-01-01

    Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign) to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.

  14. Cloning and expression of Brucella outer membrane protein 36kDa (OMP2b in E. coli

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    Nazanin Behshti

    2011-09-01

    Full Text Available Background & Objective: Brucellosis is an important zoonotic disease of economic significance. Brucella species are gram-negative, facultative intracellular bacteria, and are capable of replicating in the phagosomes of macrophages. They cause infection in several animal species and humans. Prevention of new diseases and diagnosis of cases infected with the organism are both essential for eradication of the disease. Characterization and evaluation of different antigens of Brucella cells has a key role in progression of prevention and diagnosis programs. Here, we report the production and purification of recombinant 31kDa outer membrane protein Brucella abortus (Omp2b. Materials & Methods: Brucella abortus 36kDa outer membrane protein gene was amplified with PrimeSTAR® HS DNA polymerase, cloned in pJET1.2. The target gene was subcloned in pET28a (+. Recombinant pET28a vectors were transformed into E coli BL21 (DE3. Expression of recombinant protein was induced with 1mM IPTG. Proteins were absorbed to Ni-NTA agarose resins and Recombinant proteins were eluted with 250mM imidazol. Imidazol removed by dialysis. Proteins were assayed by Western-blotting and rOmp2b was probed by Brucella rabbit anti serum. Result: Appearance of a golden brown band at the site of reaction, in Western blotting confirmed successfully clone and expression. We purified Omp2b by affinity chromatography and this method prepared refolds proteins on the column. Conclusion: Omp2b were successfully cloned, expressed and purified. The recombinant proteins were recognized by polyclonal antiserum which suggests the accuracy of procedure.

  15. Leptospira interrogans endostatin-like outer membrane proteins bind host fibronectin, laminin and regulators of complement.

    Directory of Open Access Journals (Sweden)

    Brian Stevenson

    Full Text Available The pathogenic spirochete Leptospira interrogans disseminates throughout its hosts via the bloodstream, then invades and colonizes a variety of host tissues. Infectious leptospires are resistant to killing by their hosts' alternative pathway of complement-mediated killing, and interact with various host extracellular matrix (ECM components. The LenA outer surface protein (formerly called LfhA and Lsa24 was previously shown to bind the host ECM component laminin and the complement regulators factor H and factor H-related protein-1. We now demonstrate that infectious L. interrogans contain five additional paralogs of lenA, which we designated lenB, lenC, lenD, lenE and lenF. All six genes encode domains predicted to bear structural and functional similarities with mammalian endostatins. Sequence analyses of genes from seven infectious L. interrogans serovars indicated development of sequence diversity through recombination and intragenic duplication. LenB was found to bind human factor H, and all of the newly-described Len proteins bound laminin. In addition, LenB, LenC, LenD, LenE and LenF all exhibited affinities for fibronectin, a distinct host extracellular matrix protein. These characteristics suggest that Len proteins together facilitate invasion and colonization of host tissues, and protect against host immune responses during mammalian infection.

  16. Fusion of the endoplasmic reticulum and mitochondrial outer membrane in rats brown adipose tissue: activation of thermogenesis by Ca2+.

    Directory of Open Access Journals (Sweden)

    Leopoldo de Meis

    Full Text Available Brown adipose tissue (BAT mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1, GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER membrane with the mitochondrial outer membrane of rats BAT. Ca(2+-ATPase (SERCA 1 was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca(2+ effect in BAT mitochondria thermogenesis. We found that Ca(2+ increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca(2+ concentration needed for half-maximal activation varied between 0.08 and 0.11 microM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN. Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca(2+ strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca(2+ activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver.

  17. Robust outer-selective thin-film composite polyethersulfone hollow fiber membranes with low reverse salt flux for renewable salinity-gradient energy generation

    KAUST Repository

    Cheng, Zhen Lei

    2016-01-08

    This study reports outer-selective thin-film composite (TFC) hollow fiber membranes with extremely low reverse salt fluxes and robustness for harvesting salinity-gradient energy from pressure retarded osmosis (PRO) processes. Almost defect-free polyamide layers with impressive low salt permeabilities were synthesized on top of robust polyethersulfone porous supports. The newly developed TFC-II membrane shows a maximum power density of 7.81 W m−2 using 1 M NaCl and DI water as feeds at 20 bar. Reproducible data obtained in the 2nd and 3rd runs confirm its stability under high hydraulic pressure differences. Comparing to other PRO membranes reported in the literature, the newly developed membrane exhibits not only the smallest slope between water flux decline and ΔPΔP increase but also the lowest ratio of reverse salt flux to water flux. Thus, the effective osmotic driving force could be well maintained even under high pressure operations. For the first time, the effect of feed pressure buildup induced by feed flowrate was evaluated towards PRO performance. A slight increment in feed pressure buildup was found to be beneficial to water flux and power density up to 10.06 W m−2 without comprising the reverse salt flux. We believe this study may open up new perspectives on outer-selective PRO hollow fiber membranes and provide useful insights to understand and design next-generation outer-selective TFC hollow fiber membranes for osmotic power generation.

  18. Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

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    Norkin Leonard C

    2004-07-01

    Full Text Available Abstract Background Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function. Methods We utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions. Results We show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp, C. caviae (GPIC, and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi. Conclusion Caveolin-2 associates with the chlamydial

  19. Host cell interactions of outer membrane vesicle-associated virulence factors of Enterohemorrhagic Escherichia coli O157: intracellular delivery, trafficking and mechanisms of cell injury

    Science.gov (United States)

    Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, confocal laser...

  20. Health economics of a hexavalent meningococcal outer-membrane vesicle vaccine in children : potential impact of introduction in the Dutch vaccination program

    NARCIS (Netherlands)

    Bos, JM; Rumke, HC; Welte, R; Postma, MJ; Jager, JC

    2001-01-01

    The cost-effectiveness of universal vaccination of infants with a new hexavalent meningococcal B outer-membrane vesicle vaccine is projected for The Netherlands by applying decision analysis. The societal perspective is taken and direct and productivity costs (friction costs method) are considered.

  1. Identification of two novel genes encoding 97- to 99-kilodalton outer membrane proteins of Chlamydia pneumoniae.Infect Immun. 1999 Jan;67(1):375-83

    DEFF Research Database (Denmark)

    Knudsen, K; Madsen, AS; Mygind, P

    1999-01-01

    of putative outer membrane proteins encoded by the Chlamydia psittaci and Chlamydia trachomatis gene families. By use of a monospecific polyclonal antibody against purified recombinant Omp4, it was shown that without heating, the protein migrated at 65 to 75 kDa in sodium dodecyl sulfate-polyacrylamide gel...

  2. Role of the Helicobacter pylori outer-membrane proteins AlpA and AlpB in colonization of the guinea pig stomach

    NARCIS (Netherlands)

    R. de Jonge (Robert); Z. Durrani; S.G. Rijpkema; E.J. Kuipers (Ernst); A.H.M. van Vliet (Arnoud); J.G. Kusters (Johannes)

    2004-01-01

    textabstractThe human gastric pathogen Helicobacter pylori expresses several putative outer-membrane proteins (OMPs), but the role of individual OMPs in colonization of the stomach by H. pylori is still poorly understood. The role of four such OMPs (AlpA, AlpB, OipA and HopZ) in a

  3. Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C.

    Directory of Open Access Journals (Sweden)

    Kalyan C Nallaparaju

    Full Text Available Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918 of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida FTN_0444 (homolog of FTT0918 fopC mutant to study the virulence-associated mechanism(s of FTT0918.The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO mice, including MHC I, MHC II, and µmT (B cell deficient, but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity facilitating the activity and localization of acid phosphatases and other F. novicida cell components.

  4. Functional characterization of ExFadLO, an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa.

    Science.gov (United States)

    Martínez, Eriel; Estupiñán, Mónica; Pastor, F I Javier; Busquets, Montserrat; Díaz, Pilar; Manresa, Angeles

    2013-02-01

    Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance.

  5. Localization of the outer membrane protein OmpA2 in Caulobacter crescentus depends on the position of the gene in the chromosome.

    Science.gov (United States)

    Ginez, Luis David; Osorio, Aurora; Poggio, Sebastian

    2014-08-01

    The outer membrane of Gram-negative bacteria is an essential structure involved in nutrient uptake, protection against harmful substances, and cell growth. Different proteins keep the outer membrane from blebbing out by simultaneously interacting with it and with the cell wall. These proteins have been mainly studied in enterobacteria, where OmpA and the Braun and Pal lipoproteins stabilize the outer membrane. Some degree of functional redundancy exists between these proteins, since none of them is essential but the absence of two of them results in a severe phenotype. Caulobacter crescentus has a different strategy to maintain its outer membrane, since it lacks the Braun lipoprotein and Pal is essential. In this work, we characterized OmpA2, an OmpA-like protein, in this bacterium. Our results showed that this protein is required for normal stalk growth and that it plays a minor role in the stability of the outer membrane. An OmpA2 fluorescent fusion protein showed that the concentration of this protein decreases from the stalk to the new pole. This localization pattern is important for its function, and it depends on the position of the gene locus in the chromosome and, as a consequence, in the cell. This result suggests that little diffusion occurs from the moment that the gene is transcribed until the mature protein attaches to the cell wall in the periplasm. This mechanism reveals the integration of different levels of information from protein function down to genome arrangement that allows the cell to self-organize.

  6. Treponema pallidum in Gel Microdroplets: A Method for Topological Analysis of BamA (TP0326) and Localization of Rare Outer Membrane Proteins.

    Science.gov (United States)

    Luthra, Amit; Anand, Arvind; Radolf, Justin D

    2015-01-01

    The noncultivable spirochete Treponema pallidum subspecies pallidum (T. pallidum) is the etiological agent of venereal syphilis. In contrast to the outer membranes (OMs) of gram-negative bacteria, the OM of T. pallidum lacks lipopolysaccharide, contains a paucity of integral membrane proteins, and is extremely labile. The lability of the T. pallidum OM greatly hinders efforts to localize the bacterium's rare outer membrane proteins (OMPs). To circumvent this problem, we developed the gel microdroplet method in which treponemes are encapsulated in porous agarose beads and then probed with specific antibodies in the absence or presence of low concentrations of the non-ionic detergent Triton X-100. To demonstrate the general utility of this method for surface localization of any T. pallidum antigen, herein we describe a protocol for immunolabeling of encapsulated treponemes using antibodies directed against the β-barrel and POTRA domains of TP0326, the spirochete's BamA ortholog.

  7. Study finds OC users protected against onset of chlamydial PID.

    Science.gov (United States)

    1987-09-01

    Preliminary results from a large case-controlled study on the relationship between oral contraceptives and chlamydial infections refute the suspicion that pills increase the chance of contracting chlamydia, and clearly show that pills inhibit progression of an existing infection into chlamydial pelvic inflammatory disease (PID). The research was conducted in Seattle at Washington University. Subjects recruited from a sexually transmitted disease clinic included 700 patients, 100 with clinically verified PID, classified into those with chlamydia, gonorrhea, neither, and PID or no PID. Compared to 762 controls, oral contraceptive users had 1/8 the risk of chlamydial PID; compared to women using no contraception, their risk was 1/11. Oral contraception was safer than other methods with respect to chlamydial PID. No such pattern was evident when gonorrhea incidence was examined. It should be emphasized that pills do not protect against other types of PID. Nor do they prevent the start of a chlamydia infection.

  8. Monogalactosyldiacylglycerol synthesis in the outer envelope membrane of chloroplasts is required for enhanced growth under sucrose supplementation

    Directory of Open Access Journals (Sweden)

    Masato eMurakawa

    2014-06-01

    Full Text Available Plant galactolipid synthesis on the outer envelope membranes of chloroplasts is an important biosynthetic pathway for sustained growth under conditions of phosphate (Pi depletion. During Pi starvation, the amount of digalactosyldiacylglycerol (DGDG is increased to substitute for the phospholipids that are degraded for supplying Pi. An increase in DGDG concentration depends on an adequate supply of monogalactosyldiacylglycerol (MGDG, which is a substrate for DGDG synthesis and is synthesized by a type-B MGDG synthase, MGD3. Recently, sucrose was suggested to be a global regulator of plant responses to Pi starvation. Thus, we analyzed expression levels of several genes involved in lipid remodeling during Pi starvation in Arabidopsis thaliana and found that the abundance of MGD3 mRNA increased when sucrose was exogenously supplied to the growth medium. Sucrose supplementation retarded the growth of the Arabidopsis MGD3 knockout mutant mgd3 but enhanced the growth of transgenic Arabidopsis plants overexpressing MGD3 compared with wild type, indicating the involvement of MGD3 in plant growth under sucrose-replete conditions. Although most features such as chlorophyll content, photosynthetic activity, and Pi content were comparable between wild-type and the transgenic plants overexpressing MGD3, sucrose content in shoot tissues decreased and incorporation of exogenously supplied carbon to DGDG was enhanced in the MGD3-overexpressing plants compared with wild type. Our results suggest that MGD3 plays an important role in supplying DGDG as a component of extraplastidial membranes to support enhanced plant growth under conditions of carbon excess.

  9. Multiple conformational states and gate opening of outer membrane protein TolC revealed by molecular dynamics simulations.

    Science.gov (United States)

    Wang, Beibei; Weng, Jingwei; Wang, Wenning

    2014-09-01

    Outer membrane protein TolC serves as an exit duct for exporting substances out of cell. The occluded periplasmic entrance of TolC is required to open for substrate transport, although the opening mechanism remains elusive. In this study, systematic molecular dynamics (MD) simulations for wild type TolC and six mutants were performed to explore the conformational dynamics of TolC. The periplasmic gate was shown to sample multiple conformational states with various degrees of gating opening. The gate opening was facilitated by all mutations except Y362F, which adopts an even more closed state than wild type TolC. The interprotomer salt-bridge R367-D153 is turned out to be crucial for periplasmic gate opening. The mutations that disrupt the interactions at the periplasmic tip may affect the stability of the trimeric assembly of TolC. Structural asymmetry of the periplasmic gate was observed to be opening size dependent. Asymmetric conformations are found in moderately opening states, while the most and the least opening states are often more symmetric. Finally, it is shown that lowering pH can remarkably stabilize the closed state of the periplasmic gate.

  10. The identification of global patterns and unique signatures of proteins across 14 environments using outer membrane proteomics of bacteria.

    Science.gov (United States)

    Schliep, Martin; Ryall, Ben; Ferenci, Thomas

    2012-11-01

    We test the hypothesis that organisms sourced from different environments exhibit unique fingerprints in macromolecular composition. Experimentally, we followed proteomic changes with 14 different sub-lethal environmental stimuli in Escherichia coli at controlled growth rates. The focus was on the outer membrane sub-proteome, which is known to be extremely sensitive to environmental controls. The analyses surprisingly revealed that pairs of proteins belonging to very different regulons, such as Slp and OmpX or FadL and OmpF, have the closest patterns of change with the 14 conditions. Fe-limited and cold-cultured bacteria have the most distinct global patterns of spot changes, but the patterns with fast growth and oxygen limitation are the closest amongst the 14 environments. These unexpected but statistically robust results suggest that we have an incomplete picture of bacterial regulation across different stress responses; baseline choices and growth-rate influences are probably underestimated factors in such systems-level analysis. In terms of our aim of getting a unique profile for each of the 14 investigated environments, we find that it is unnecessary to compare all the proteins in a proteome and that a panel of five proteins is sufficient for identification of environmental fingerprints. This demonstrates the future feasibility of tracing the history of contaminating bacteria in hospitals, foods or industrial settings as well as for released organisms and biosecurity purposes.

  11. MitoNEET Is a Uniquely Folded 2Fe-2S Outer Mitochondrial Membrane Protein Stabilized By Pioglitazone

    Energy Technology Data Exchange (ETDEWEB)

    Paddock, M.L.; Wiley, S.E.; Axelrod, H.L.; Cohen, A.E.; Roy, M.; Abresch, E.C.; Capraro, D.; Murphy, A.N.; Nechushtai, R.; Dixon, J.E.; Jennings, P.A.; /UC, San Diego /SLAC, SSRL /Hebrew U.

    2007-10-19

    Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Angstrom x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the {approx}650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a {beta}-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.

  12. Edwardsiella tarda Outer Membrane Protein C: An Immunogenic Protein Induces Highly Protective Effects in Flounder (Paralichthys olivaceus against Edwardsiellosis

    Directory of Open Access Journals (Sweden)

    Fuguo Liu

    2016-07-01

    Full Text Available Outer membrane protein C of Edwardsiella tarda is a major cell surface antigen and it was identified to be an immunogenic protein by Western blot using flounder (Paralichthys olivaceus anti-recombinant OmpC (rOmpC, and anti-E. tarda antibodies. rOmpC tested the immune protective effect against E. tarda challenge in a flounder model and produced a relative percentage of survival rate of 85%. The immune response of flounder induced by rOmpC was investigated, and the results showed that: (1 the levels of specific serum antibodies induced by rOmpC were significantly higher than the control group after the second week after immunization, and the peak level occurred at week five after immunization; (2 rOmpC could induce the proliferation of sIg+ lymphocytes, and the peak levels of sIg+ lymphocytes in blood, spleen, and pronephros occurred at 4–5 weeks after immunization; and (3 the MHCIIα, CD4-1, IL-1β, IL-6 and TNF-α genes were significantly induced after being injected with rOmpC. Taken together, these results demonstrated that rOmpC could evoke highly protective effects against E. tarda challenge and induce strong innate immune response and humoral immune response of flounder, which indicated that OmpC was a promising vaccine candidate against E. tarda infection.

  13. Phase I Safety and Immunogenicity Study of a Candidate Meningococcal Disease Vaccine Based on Neisseria lactamica Outer Membrane Vesicles▿

    Science.gov (United States)

    Gorringe, Andrew R.; Taylor, Stephen; Brookes, Charlotte; Matheson, Mary; Finney, Michelle; Kerr, Moyra; Hudson, Michael; Findlow, Jamie; Borrow, Ray; Andrews, Nick; Kafatos, George; Evans, Cariad M.; Read, Robert C.

    2009-01-01

    Natural immunity to meningococcal disease in young children is associated epidemiologically with carriage of commensal Neisseria species, including Neisseria lactamica. We have previously demonstrated that outer membrane vesicles (OMVs) from N. lactamica provide protection against lethal challenge in a mouse model of meningococcal septicemia. We evaluated the safety and immunogenicity of an N. lactamica OMV vaccine in a phase I placebo-controlled, double-blinded clinical trial. Ninety-seven healthy young adult male volunteers were randomized to receive three doses of either an OMV vaccine or an Alhydrogel control. Subsequently, some subjects who had received the OMV vaccine also received a fourth dose of OMV vaccine, 6 months after the third dose. Injection site reactions were more frequent in the OMV-receiving group, but all reactions were mild or moderate in intensity. The OMV vaccine was immunogenic, eliciting rises in titers of immunoglobulin G (IgG) against the vaccine OMVs, together with a significant booster response, as determined by an enzyme-linked immunosorbent assay. Additionally, the vaccine induced modest cross-reactive immunity to six diverse strains of serogroup B Neisseria meningitidis, including IgG against meningococcal OMVs, serum bactericidal antibodies, and opsonophagocytic activity. The percentages of subjects showing ≥4-fold rises in bactericidal antibody titer obtained were similar to those previously reported for the Norwegian meningococcal OMV vaccine against the same heterologous meningococcal strain panel. In conclusion, this N. lactamica OMV vaccine is safe and induces a weak but broad humoral immune response to N. meningitidis. PMID:19553555

  14. Phase I safety and immunogenicity study of a candidate meningococcal disease vaccine based on Neisseria lactamica outer membrane vesicles.

    Science.gov (United States)

    Gorringe, Andrew R; Taylor, Stephen; Brookes, Charlotte; Matheson, Mary; Finney, Michelle; Kerr, Moyra; Hudson, Michael; Findlow, Jamie; Borrow, Ray; Andrews, Nick; Kafatos, George; Evans, Cariad M; Read, Robert C

    2009-08-01

    Natural immunity to meningococcal disease in young children is associated epidemiologically with carriage of commensal Neisseria species, including Neisseria lactamica. We have previously demonstrated that outer membrane vesicles (OMVs) from N. lactamica provide protection against lethal challenge in a mouse model of meningococcal septicemia. We evaluated the safety and immunogenicity of an N. lactamica OMV vaccine in a phase I placebo-controlled, double-blinded clinical trial. Ninety-seven healthy young adult male volunteers were randomized to receive three doses of either an OMV vaccine or an Alhydrogel control. Subsequently, some subjects who had received the OMV vaccine also received a fourth dose of OMV vaccine, 6 months after the third dose. Injection site reactions were more frequent in the OMV-receiving group, but all reactions were mild or moderate in intensity. The OMV vaccine was immunogenic, eliciting rises in titers of immunoglobulin G (IgG) against the vaccine OMVs, together with a significant booster response, as determined by an enzyme-linked immunosorbent assay. Additionally, the vaccine induced modest cross-reactive immunity to six diverse strains of serogroup B Neisseria meningitidis, including IgG against meningococcal OMVs, serum bactericidal antibodies, and opsonophagocytic activity. The percentages of subjects showing > or =4-fold rises in bactericidal antibody titer obtained were similar to those previously reported for the Norwegian meningococcal OMV vaccine against the same heterologous meningococcal strain panel. In conclusion, this N. lactamica OMV vaccine is safe and induces a weak but broad humoral immune response to N. meningitidis.

  15. TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein.

    Science.gov (United States)

    Desrosiers, Daniel C; Anand, Arvind; Luthra, Amit; Dunham-Ems, Star M; LeDoyt, Morgan; Cummings, Michael A D; Eshghi, Azad; Cameron, Caroline E; Cruz, Adriana R; Salazar, Juan C; Caimano, Melissa J; Radolf, Justin D

    2011-06-01

    Definitive identification of Treponema pallidum rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in T. pallidum with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modelling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic β-barrel. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the β-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in T. pallidum larger than that of Escherichia coli. Non-orthologous ancillary factors and self-association of TP0326 via its β-barrel may both contribute to the Bam complex. T. pallidum-infected rabbits mount a vigorous antibody response to both POTRA and β-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochaete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.

  16. Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli.

    Science.gov (United States)

    Yoo, Gu; Saenger, Thorsten; Bong, Ji-Hong; Jose, Joachim; Kang, Min-Jung; Pyun, Jae-Chul

    2015-12-01

    In this work, two proteins, Z-domains and bovine casein, were auto-displayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different auto-display vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-auto-displayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-auto-displayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-auto-displayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-auto-displayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with auto-displayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.

  17. Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

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    Marlena M Wilson

    Full Text Available Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

  18. Isolation and Identification of Outer Membrane Proteins of Helicobacter Pylori of Iranian Patient by SDS-PAGE

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    M. Doosty

    1998-04-01

    Full Text Available The function of Helicobacter pylori (H.pylori is confirmed as one of the factors which motivates gastric and duodenal ulcer and gastritis. Various methods are used to diagnose the infection. Serological tests are the easiest and most harmless for the patients. Probably, H.pylori strains in Iran are different from the strains in other countries. Hence, it seems neccessary to design a specific serological test to recognize and identify different strains of bacterial antigenic proteins of Iranian patients."nSince the most manifest and specific to these bacterial antigens are the "Outer Membrane Protein" (OMP, therefore, the first necessary step is to separate and purify H.pylori OMP and then to identify antigenic proteins."nIn this study, we received bacteria colony that belonged to 15 patients with gastric or duodenal ulcer, which had been growed in blood agar or brucella broth. After processing such as washing, freezing and defreezing, sonicating, centrifugation with high speed (10,000 g and treatment with sarcosyl, the sarcosyl insoluble fraction was extracted. Sodium Dodecyl Sulfate - Poly Acrylamide Gel Electrophoresis (SDS-PAGE was preformed. From all 15 OMP specimens, we isolated protein bands."nThe first two bands with higher MW, were major bands and the two lighter bands were the minor bands. Approximate MW of these 4 proteins are equal to 67000, 61000, 30000 and 17000 dalton

  19. Immunity Provided by an Outer Membrane Vesicle Cholera Vaccine Is Due to O-Antigen-Specific Antibodies Inhibiting Bacterial Motility.

    Science.gov (United States)

    Wang, Zhu; Lazinski, David W; Camilli, Andrew

    2017-01-01

    An outer membrane vesicle (OMV)-based cholera vaccine is highly efficacious in preventing intestinal colonization in the suckling mouse model. Immunity from OMVs comes from immunoglobulin (Ig), particularly IgG, in the milk of mucosally immunized dams. Anti-OMV IgG renders Vibrio cholerae organisms immotile, thus they pass through the small intestine without colonizing. However, the importance of motility inhibition for protection and the mechanism by which motility is inhibited remain unclear. By using both in vitro and in vivo experiments, we found that IgG inhibits motility by specifically binding to the O-antigen of V. cholerae We demonstrate that the bivalent structure of IgG, although not required for binding to the O-antigen, is required for motility inhibition. Finally, we show using competition assays in suckling mice that inhibition of motility appears to be responsible for most, if not all, of the protection engendered by OMV vaccination, thus providing insight into the mechanism of immune protection.

  20. Large-scale preparation of the homogeneous LolA–lipoprotein complex and efficient in vitro transfer of lipoproteins to the outer membrane in a LolB-dependent manner

    OpenAIRE

    Watanabe, Shoji; Oguchi, Yuki; Yokota, Naoko; Tokuda, Hajime

    2007-01-01

    An ATP-binding cassette transporter LolCDE complex of Escherichia coli releases lipoproteins destined to the outer membrane from the inner membrane as a complex with a periplasmic chaperone, LolA. Interaction of the LolA–lipoprotein complex with an outer membrane receptor, LolB, then causes localization of lipoproteins to the outer membrane. As far as examined, formation of the LolA–lipoprotein complex strictly depends on ATP hydrolysis by the LolCDE complex in the presence of LolA. It has be...

  1. 羊流产衣原体主要外膜蛋白基因的克隆与序列分析%CLONING AND SEQUENCE ANALYSIS OF THE MAJOR OUTER MEMBRANE PROTEIN GENE OF AN OVINE ABORTION STRAIN OF CHLAMYDIA PSITTACI

    Institute of Scientific and Technical Information of China (English)

    苗振川; 哈斯阿古拉; 赵亚芳; 张宝发; 张鹤龄

    2001-01-01

    In order to clone the major outer membrane protein(MOMP) gene of a goat abortion strain of Chlamydia psittaci isolated from Inner Mongolia area,a pair of primers were synthesized according to the published sequences of Chlamydial MOMP gene,and the genomic DNA was extracted from the purified Chlamydial particles and employed as templat,then polymerase chain reaction was performed.As a result,a 1.17 Kb DNA fragment was amplified.The PCR product was recovered from low melting point agarose gel and underwent cleavage by restriction enzymes whose recognition sites had been designed precedingly on the ends of the primers,then ligated to the corresponding site of plasmid pUC19.The ligation reaction mixture was used to trans form E.coli DH5α.The recombinant was screened by PCR detection or restriction enzymes analysis.The DNA sequencing was performed.It showed that this recombinant fragment covered the whole coding region of the MOMP gene whice was 1170 bp long.It also revealed that this MOMP gene of Chlamydial strain was just the same as that of England ovine abortion strain S26/3,and there was only one nucleotide synonymous mutation in comparison with that of strain B577.%将自行分离、传代培养的内蒙古地区山羊流产衣原体按常规方法分离纯化,提取衣原体基因组DNA作为模板,按照国外发表的衣原体主要外膜蛋白(MOMP)基因两端序列设计合成一对引物,用PCR方法扩增出一1.17 Kb的DNA片段。利用引物上预先设计的限制性内切酶位点,将扩增片段经限制性内切酶切割后连接到pUC19质粒相应位点上,转化大肠杆菌DH5α,筛选重组子。经PCR检测和内切酶分析鉴定含MOMP基因的重组子质粒。对克隆片段进行全序列分析,结果证明得到MOMP全编码序列的基因克隆。本株衣原体MOMP编码区由1170个核苷酸组成。序列比较发现本株衣原体的MOMP基因与国外的羊流产衣原体S26

  2. Functional recruitment of human complement inhibitor C4B-binding protein to outer membrane protein Rck of Salmonella.

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    Derek K Ho

    Full Text Available Resistance to complement mediated killing, or serum resistance, is a common trait of pathogenic bacteria. Rck is a 17 kDa outer membrane protein encoded on the virulence plasmid of Salmonella enterica serovars Typhimurium and Enteritidis. When expressed in either E. coli or S. enterica Typhimurium, Rck confers LPS-independent serum resistance as well as the ability to bind to and invade mammalian cells. Having recently shown that Rck binds the inhibitor of the alternative pathway of complement, factor H (fH, we hypothesized that Rck can also bind the inhibitor of the classical and lectin pathways, C4b-binding protein (C4BP. Using flow cytometry and direct binding assays, we demonstrate that E. coli expressing Rck binds C4BP from heat-inactivated serum and by using the purified protein. No binding was detected in the absence of Rck expression. C4BP bound to Rck is functional, as we observed factor I-mediated cleavage of C4b in cofactor assays. In competition assays, binding of radiolabeled C4BP to Rck was reduced by increasing concentrations of unlabeled protein. No effect was observed by increasing heparin or salt concentrations, suggesting mainly non-ionic interactions. Reduced binding of C4BP mutants lacking complement control protein domains (CCPs 7 or 8 was observed compared to wt C4BP, suggesting that these CCPs are involved in Rck binding. While these findings are restricted to Rck expression in E. coli, these data suggest that C4BP binding may be an additional mechanism of Rck-mediated complement resistance.

  3. Effects of outer membrane protein TolC on the transport of Escherichia coli within saturated quartz sands.

    Science.gov (United States)

    Feriancikova, Lucia; Bardy, Sonia L; Wang, Lixia; Li, Jin; Xu, Shangping

    2013-06-04

    The outer membrane protein (OMP) TolC is the cell surface component of several drug efflux pumps that are responsible for bacterial resistance against a variety of antibiotics. In this research, we investigated the effects of OMP TolC on E. coli transport within saturated sands through column experiments using a wild-type E. coli K12 strain (with OMP TolC), as well as the corresponding transposon mutant (tolC::kan) and the markerless deletion mutant (ΔtolC). Our results showed OMP TolC could significantly enhance the transport of E. coli when the ionic strength was 20 mM NaCl or higher. The deposition rate coefficients for the wild-type E. coli strain (with OMP TolC) was usually >50% lower than those of the tolC-negative mutants. The measurements of contact angles using three probe liquids suggested that TolC altered the surface tension components of E. coli cells and lead to lower Hamaker constants for the cell-water-sand system. The interaction energy calculations using the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory suggested that the deposition of the E. coli cell primarily occurred at the secondary energy minimum. The depth of the secondary energy minimum increased with ionic strength, and was greater for the TolC-deletion strains under high ionic strength conditions. Overall, the transport behavior of three E. coli strains within saturated sands could be explained by the XDLVO calculations. Results from this research suggested that antibiotic resistant bacteria expressing OMP TolC could spread more widely within sandy aquifers.

  4. Outer membrane protein complex of Meningococcus enhances the antipolysaccharide antibody response to pneumococcal polysaccharide-CRM₁₉₇ conjugate vaccine.

    Science.gov (United States)

    Lai, Zengzu; Schreiber, John R

    2011-05-01

    Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM₁₉₇ conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM₁₉₇ conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands.

  5. Recombinant M2e outer membrane vesicle vaccines protect against lethal influenza A challenge in BALB/c mice.

    Science.gov (United States)

    Rappazzo, C Garrett; Watkins, Hannah C; Guarino, Cassandra M; Chau, Annie; Lopez, Jody L; DeLisa, Matthew P; Leifer, Cynthia A; Whittaker, Gary R; Putnam, David

    2016-03-04

    Currently approved influenza vaccines predominantly protect through antibodies directed against the highly variable glycoprotein hemagglutinin (HA), necessitating annual redesign and formulation based on epidemiological prediction of predominant circulating strains. More conserved influenza protein sequences, such as the ectodomain of the influenza M2 protein, or M2e, show promise as a component of a universal influenza A vaccine, but require a Th1-biased immune response for activity. Recently, recombinant, bacterially derived outer membrane vesicles (OMVs) demonstrated potential as a platform to promote a Th1-biased immune response to subunit antigens. Here, we engineer three M2e-OMV vaccines and show that all elicit strong IgG titers, with high IgG2a:IgG1 ratios, in BALB/c mice. Additionally, the administration of one M2e-OMV construct containing tandem heterologous M2e peptides (M2e4xHet-OMV) resulted in 100% survival against lethal doses of the mouse-adapted H1N1 influenza strain PR8. Passive transfer of antibodies from M2e4xHet-OMV vaccinated mice to unvaccinated mice also resulted in 100% survival to challenge, indicating that protection is driven largely via antibody-mediated immunity. The potential mechanism through which M2e-OMVs initiated the immune response was explored and it was found that the constructs triggered TLR1/2, TLR4, and TLR5. Our data indicate that OMVs have potential as a platform for influenza A vaccine development due to their unique adjuvant profile and intrinsic pathogen-mimetic nature.

  6. Helicobacter pylori outer membrane protein, HomC, shows geographic dependent polymorphism that is influenced by the Bab family.

    Science.gov (United States)

    Kim, Aeryun; Servetas, Stephanie L; Kang, Jieun; Kim, Jinmoon; Jang, Sungil; Choi, Yun Hui; Su, Hanfu; Jeon, Yeong-Eui; Hong, Youngmin A; Yoo, Yun-Jung; Merrell, D Scott; Cha, Jeong-Heon

    2016-12-01

    The array of outer membrane proteins (OMPs) found in Helicobacter pylori provides a crucial component for persistent colonization within the gastric niche. Not only does H. pylori harbor a wide number of OMPs, but these OMPs often vary across strains; this likely contributes to immune evasion, adaptation during long term colonization, and potentially differential disease progression. Previous work from our group described OMP differences among the Bab family (babA, babB, and babC) and Hom family (homA and homB) from 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). In the current study, we expanded our investigation to include the less well characterized Hom family member, HomC.Overall, we identified and genotyped three homC variants: homC (S) , homC (L) , and homC (M) , in both populations. Similar to other polymorphic genes, the KH group showed less overall diversity, with 97.5% of strains harboring homC (L) . In contrast, a more heterogeneous profile was observed in strains derived from an American population; we found nearly equal distribution of homC (S) and homC (L) . Further analysis of the AH group identified associations between homC polymorphism and bab genotype; in AH strains, there was a significant association between homC (L) and carriage of babA at locus A. Since babA is an important virulence factor for the development of severe gastric disease, these data may suggest that homC polymorphism plays a role in H. pylori pathogenesis.

  7. Rickettsia rickettsii outer membrane protein YbgF induces protective immunity in C3H/HeN mice.

    Science.gov (United States)

    Gong, Wenping; Qi, Yong; Xiong, Xiaolu; Jiao, Jun; Duan, Changsong; Wen, Bohai

    2015-01-01

    Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever (RMSF). YbgF and TolC are outer membrane-associated proteins of R. rickettsii that play important roles in its interaction with host cells. We investigated the immunogenicity of YbgF and TolC for protection against RMSF. We immunized C3H/HeN mice with recombinant R. rickettsii YbgF (rYbgF) or TolC (rTolC). Rickettsial burden and impairment in the lungs, spleens, and livers of rYbgF-immunized mice were significantly lower than in rTolC-immunized mice. The ratio of IgG2a to IgG1 in rYbgF-immunized mice continued to increase over the course of our experiments, while that in rTolC-immunized mice was reduced. The proliferation and cytokine secretion of CD4(+) and CD8(+) T cells isolated from R. rickettsii-infected mice were analyzed following antigen stimulation. The results indicated that proliferation and interferon (IFN)-γ secretion of CD4(+) or CD8(+) T cells in R. rickettsii-infected mice were significantly greater than in uninfected mice after stimulation with rYbgF. YbgF is a novel protective antigen of R. rickettsii. Protection conferred by YbgF is dependent upon IFN-γ-producing CD4(+) and CD8(+) T cells and IgG2a, which act in synergy to control R. rickettsii infection.

  8. Helicobacter pylori Outer Membrane Protein 18 (Hp1125 Is Involved in Persistent Colonization by Evading Interferon-γ Signaling

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    Yuqun Shan

    2015-01-01

    Full Text Available Outer membrane proteins (OMPs can induce an immune response. Omp18 (HP1125 of H. pylori is a powerful antigen that can induce significant interferon-γ (IFN-γ levels. Previous studies have suggested that IFN-γ plays an important role in H. pylori clearance. However, H. pylori has multiple mechanisms to avoid host immune surveillance for persistent colonization. We generated an omp18 mutant (H. pylori 26695 and H. pylori SS1 strain to examine whether Omp18 interacts with IFN-γ and is involved in H. pylori colonization. qRT-PCR revealed that IFN-γ induced Omp18 expression. qRT-PCR and western blot analysis revealed reduced expressions of virulence factors CagA and NapA in H. pylori 26695 with IFN-γ treatment, but they were induced in the Δomp18 strain. In C57BL/6 mice infected with H. pylori SS1 and the Δomp18 strain, the Δomp18 strain conferred defective colonization and activated a stronger inflammatory response. Signal transducer phosphorylation and transcription 1 (STAT1 activator was downregulated by the wild-type strain but not the Δomp18 strain in IFN-γ-treated macrophages. Furthermore, Δomp18 strain survival rates were poor in macrophages compared to the wild-type strain. We concluded that H. pylori Omp18 has an important function influencing IFN-γ-mediated immune response to participate in persistent colonization.

  9. Amelioration of the Fitness Costs of Antibiotic Resistance Due To Reduced Outer Membrane Permeability by Upregulation of Alternative Porins.

    Science.gov (United States)

    Knopp, Michael; Andersson, Dan I

    2015-12-01

    The fitness cost of antibiotic resistance is a key parameter in determining the evolutionary success of resistant bacteria. Studies of the effect of antibiotic resistance on bacterial fitness are heavily biased toward target alterations. Here we investigated how the costs in the form of a severely impaired growth rate associated with resistance due to absence of two major outer membrane porins can be genetically compensated. We performed an evolution experiment with 16 lineages of a double mutant of Escherichia coli with the ompCF genes deleted, and reduced fitness and increased resistance to different classes of antibiotics, including the carbapenems ertapenem and meropenem. After serial passage for only 250 generations, the relative growth rate increased from 0.85 to 0.99 (susceptible wild type set to 1.0). Compensation of the costs followed two different adaptive pathways where upregulation of expression of alternative porins bypassed the need for functional OmpCF porins. The first compensatory mechanism involved mutations in the phoR and pstS genes, causing constitutive high-level expression of the PhoE porin. The second mechanism involved mutations in the hfq and chiX genes that disrupted Hfq-dependent small RNA regulation, causing overexpression of the ChiP porin. Although susceptibility was restored in compensated mutants with PhoE overexpression, evolved mutants with high ChiP expression maintained the resistance phenotype. Our findings may explain why porin composition is often altered in resistant clinical isolates and provide new insights into how bypass mechanisms may allow genetic adaptation to a common multidrug resistance mechanism.

  10. Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

    Science.gov (United States)

    Luo, Zhang; Liu, Zhixin; Fu, Jianping; Zhang, Qiusheng; Huang, Bei; Nie, Pin

    2016-11-01

    Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes ( IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon ( IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.

  11. Outer membrane vesicles from Brucella abortus promote bacterial internalization by human monocytes and modulate their innate immune response.

    Directory of Open Access Journals (Sweden)

    Cora N Pollak

    Full Text Available Outer membrane vesicles (OMVs released by some gram-negative bacteria have been shown to exert immunomodulatory effects that favor the establishment of the infection. The aim of the present study was to assess the interaction of OMVs from Brucella abortus with human epithelial cells (HeLa and monocytes (THP-1, and the potential immunomodulatory effects they may exert. Using confocal microscopy and flow cytometry, FITC-labeled OMVs were shown to be internalized by both cell types. Internalization was shown to be partially mediated by clathrin-mediated endocytosis. Pretreatment of THP-1 cells with Brucella OMVs inhibited some cytokine responses (TNF-α and IL-8 to E. coli LPS, Pam3Cys or flagellin (TLR4, TLR2 and TLR5 agonists, respectively. Similarly, pretreatment with Brucella OMVs inhibited the cytokine response of THP-1 cells to B. abortus infection. Treatment of THP-1 cells with OMVs during IFN-γ stimulation reduced significantly the inducing effect of this cytokine on MHC-II expression. OMVs induced a dose-dependent increase of ICAM-1 expression on THP-1 cells and an increased adhesion of these cells to human endothelial cells. The addition of OMVs to THP-1 cultures before the incubation with live B. abortus resulted in increased numbers of adhered and internalized bacteria as compared to cells not treated with OMVs. Overall, these results suggest that OMVs from B. abortus exert cellular effects that promote the internalization of these bacteria by human monocytes, but also downregulate the innate immune response of these cells to Brucella infection. These effects may favor the persistence of Brucella within host cells.

  12. Proteomic study via a non-gel based approach of meningococcal outer membrane vesicle vaccine obtained from strain CU385: a road map for discovering new antigens.

    Science.gov (United States)

    Gil, Jeovanis; Betancourt, L Zaro H; Sardiñas, Gretel; Yero, Daniel; Niebla, Olivia; Delgado, Maité; García, Darien; Pajón, Rolando; Sánchez, Aniel; González, Luis J; Padrón, Gabriel; Campa, Concepción; Sotolongo, Franklin; Barberó, Ramón; Guillén, Gerardo; Herrera, Luis; Besada, Vladimir

    2009-05-01

    This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatographymass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.

  13. Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif.

    Science.gov (United States)

    He, Ming-Xiong; Feng, Hong; Zhang, Yi-Zheng

    2008-12-01

    A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed beta-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant beta-amylase showed to significantly increase hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol.

  14. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

    Science.gov (United States)

    Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

    2015-01-01

    The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  15. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM.

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    Daniel O Frank

    Full Text Available The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM. In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20 by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  16. Pivotal roles of the outer membrane polysaccharide export and polysaccharide copolymerase protein families in export of extracellular polysaccharides in gram-negative bacteria.

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    Cuthbertson, Leslie; Mainprize, Iain L; Naismith, James H; Whitfield, Chris

    2009-03-01

    Many bacteria export extracellular polysaccharides (EPS) and capsular polysaccharides (CPS). These polymers exhibit remarkably diverse structures and play important roles in the biology of free-living, commensal, and pathogenic bacteria. EPS and CPS production represents a major challenge because these high-molecular-weight hydrophilic polymers must be assembled and exported in a process spanning the envelope, without compromising the essential barrier properties of the envelope. Emerging evidence points to the existence of molecular scaffolds that perform these critical polymer-trafficking functions. Two major pathways with different polymer biosynthesis strategies are involved in the assembly of most EPS/CPS: the Wzy-dependent and ATP-binding cassette (ABC) transporter-dependent pathways. They converge in an outer membrane export step mediated by a member of the outer membrane auxiliary (OMA) protein family. OMA proteins form outer membrane efflux channels for the polymers, and here we propose the revised name outer membrane polysaccharide export (OPX) proteins. Proteins in the polysaccharide copolymerase (PCP) family have been implicated in several aspects of polymer biogenesis, but there is unequivocal evidence for some systems that PCP and OPX proteins interact to form a trans-envelope scaffold for polymer export. Understanding of the precise functions of the OPX and PCP proteins has been advanced by recent findings from biochemistry and structural biology approaches and by parallel studies of other macromolecular trafficking events. Phylogenetic analyses reported here also contribute important new insight into the distribution, structural relationships, and function of the OPX and PCP proteins. This review is intended as an update on progress in this important area of microbial cell biology.

  17. The outer membrane protein TolC is required for phytoalexin resistance and virulence of the fire blight pathogen Erwinia amylovora

    OpenAIRE

    2009-01-01

    Summary Erwinia amylovora causes fire blight on several plant species such as apple and pear, which produce diverse phytoalexins as defence mechanisms. An evolutionary successful pathogen thus must develop resistance mechanisms towards these toxic compounds. The E. amylovora outer membrane protein, TolC, might mediate phytoalexin resistance through its interaction with the multidrug efflux pump, AcrAB. To prove this, a tolC mutant and an acrB/tolC double mutant were constructed. The minimal i...

  18. Pentavalent outer membrane vesicles of Vibrio cholerae induce adaptive immune response and protective efficacy in both adult and passive suckling mice models.

    Science.gov (United States)

    Sinha, Ritam; Koley, Hemanta; Nag, Dhrubajyoti; Mitra, Soma; Mukhopadhyay, Asish K; Chattopadhyay, Brajadulal

    2015-03-01

    Recently, we demonstrated oral immunizations with single serotype outer membrane vesicles of Vibrio cholerae induced serogroup specific protective immunity in the RITARD model. In our present study, we advanced our research by formulating multi-serotype outer membrane vesicles, mixing the OMVs of five virulent V. cholerae strains. Four doses of oral immunization with cholera pentavalent outer membrane vesicles (CPMVs) induced V. cholerae specific B and T cell responses. CPMVs-immunized mice generated long lasting serum IgG, IgA, IgM as well as mucosal sIgA and also elicited a higher percentage of CD4+ T cell distribution in spleen. Our study revealed that in vitro CPMVs-activated dendritic cells were secreting T cell polarizing cytokines, IL-12p40, IL-4, IL-6 and IL-1β. Moreover, purified splenic CD4+ T cells of immunized mice also secreted IL-4, IL-13 and IL-17 cytokines, indicating the initiation of Th2 and Th17 cell mediated immune responses. CPMVs immunized adult female mice and their offspring were significantly protected from heterologous challenge with wild type V. cholerae. CPMVs could be exploited for the development of a novel non-living vaccine against circulating cholera in near future.

  19. Do bacterial vaginosis and chlamydial infection affect serum cytokine level?

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    Bogavac Mirjana

    2010-01-01

    Full Text Available Introduction. Serbia is the country with extremely low birth rate and a relatively high percentage of preterm deliveries (8%. With this in mind, discovering new diagnostic methods that could be used for the prediction of preterm delivery is of great importance. In this study we tried to determine whether bacterial vaginosis and chlamydial infection could provoke preterm delivery by activation of systemic cytokine network. Objective. The aim of this study was to determine serum levels of proinflammatory cytokines (IL-1β, IL-8, IFN-γ, IL-6 and TNF-α in pregnant women with symptoms of preterm delivery and to make correlation between these parameters and the presence of bacterial vaginosis or chlamydial infection. Method. In the serum of 35 pregnant women, which were divided in groups according to the presence or absence of bacterial vaginosis and chlamydial infection, commercial ELISA tests for proinflammatory cytokines were performed. Results. The serum level of IFN-γ was significantly increased in pregnant women having chlamydial infection, as well as the level of IL-1β in women with bacterial vaginosis. The levels of TNF-α, IL-6 and IL-8 were not significantly different between the investigated groups. Conclusion. The preliminary results obtained in this research point out the possibility that not only intrauterine or systemic infections, but also bacterial vaginosis and chlamydial infection can cause a partial activation of systemic cytokine network and contribute to the occurrence of preterm delivery.

  20. Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex

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    Gidda Satinder K

    2008-09-01

    Full Text Available Abstract Background Carnation Italian ringspot virus (CIRV is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36, are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined. Results Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM, but not with the sorting and assembly machinery (SAM. Conclusion Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer

  1. Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I: OUTER MEMBRANE INSERTION, TRIMERIZATION, AND PORIN FUNCTION REQUIRE A C-TERMINAL β-BARREL DOMAIN.

    Science.gov (United States)

    Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D

    2015-05-08

    We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP.

  2. Chlamydial infection induces host cytokinesis failure at abscission.

    Science.gov (United States)

    Brown, Heather M; Knowlton, Andrea E; Grieshaber, Scott S

    2012-10-01

    Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.

  3. The Antitoxin Protein of a Toxin-Antitoxin System from Xylella fastidiosa Is Secreted via Outer Membrane Vesicles

    Science.gov (United States)

    Santiago, André da Silva; Mendes, Juliano S.; dos Santos, Clelton A.; de Toledo, Marcelo A. S.; Beloti, Lilian L.; Crucello, Aline; Horta, Maria A. C.; Favaro, Marianna T. de Pinho; Munar, Duber M. M.; de Souza, Alessandra A.; Cotta, Mônica A.; de Souza, Anete P.

    2016-01-01

    The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible

  4. Disease association with two Helicobacter pylori duplicate outer membrane protein genes, homB and homA

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    Oleastro Monica

    2009-06-01

    Full Text Available Abstract Background homB encodes a Helicobacter pylori outer membrane protein. This gene was previously associated with peptic ulcer disease (PUD and was shown to induce activation of interleukin-8 secretion in vitro, as well as contributing to bacterial adherence. Its 90%-similar gene, homA, was previously correlated with gastritis. The present study aimed to evaluate the gastric disease association with homB and homA, as well as with the H. pylori virulence factors cagA, babA and vacA, in 415 H. pylori strains isolated from patients from East Asian and Western countries. The correlation among these genotypes was also evaluated. Results Both homB and homA genes were heterogeneously distributed worldwide, with a marked difference between East Asian and Western strains. In Western strains (n = 234, 124 PUD and 110 non-ulcer dyspepsia (NUD, homB, cagA and vacA s1 were all significantly associated with PUD (p = 0.025, p = 0.014, p = 0.039, respectively, and homA was closely correlated with NUD (p = 0.072. In East Asian strains (n = 138, 73 PUD and 65 NUD, homB was found more frequently than homA, and none of these genes was associated with the clinical outcome. Overall, homB was associated with the presence of cagA (p = 0.043 and vacA s1 (p homA was found more frequently in cagA-negative (p = 0.062 and vacA s2 (p Polymorphisms in homB and homA copy number were observed, with a clear geographical specificity, suggesting an involvement of these genes in host adaptation. A correlation between the homB two-copy genotype and PUD was also observed, emphasizing the role of homB in the virulence of the strain. Conclusion The global results suggest that homB and homA contribute to the determination of clinical outcome.

  5. The efficacy of oral vaccination of mice with alginate encapsulated outer membrane proteins of Pasteurella haemolytica and One-Shot.

    Science.gov (United States)

    Kidane, A; Guimond, P; Ju, T R; Sanchez, M; Gibson, J; Bowersock, T L

    2001-03-21

    The goal of this study was to examine the efficacy of oral delivery of alginate encapsulated outer membrane proteins (OMP) of Pasteurella haemolytica and a commercial One-Shot vaccine in inducing protection in mice against lethal challenge with virulent P. haemolytica. We examined two alginate microsphere formulations and compared them with oral unencapsulated and subcutaneously administered vaccines. Alginate microspheres were made by the emulsion-cross-linking technique. They were examined for size, hydrophobicity, and antigen loading efficiency before they were used in the study. Mice were vaccinated by administering 200 microg of antigens in 200 microl of microspheres suspension orally or subcutaneously. One group of mice received blank microspheres and a second group was given unencapsulated antigen orally. A third and a fourth group received different formulations of alginate encapsulated antigens by oral administration. Three groups received subcutaneous inoculations (alginate encapsulated, non-adjuvanted and unencapsulated antigens, and adjuvanted One-Shot), and one group received water (naïve group). Mice were vaccinated orally for four consecutive days and challenged with P. haemolytica 5 weeks after the first vaccination. Weekly serum and feces samples were assayed for antigen specific antibodies. The number of dead mice in each group 4 days post challenge was used to compare the efficacy of the various vaccination groups. The mean volume sizes of blank alginate microsphere formulations A, and AA were 15.9, 16 and 9.2 microm, respectively. Hydrophobicity of the microspheres was evaluated by measuring contact angle on a glass slide coated with the microspheres. The contact angles on A and AA were 37.8 and 74.3 degrees, respectively. Antigen concentration in a 1:1 w/w suspension of microspheres in water was 0.9 mg/ml. Rate of death for the blank group was 42.8% whereas for groups vaccinated with antigens encapsulated in A and AA the death rates were 40

  6. CCL5 regulation of mucosal chlamydial immunity and infection

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    Igietseme Joseph U

    2008-08-01

    Full Text Available Abstract Background Following genital chlamydial infection, an early T helper type 1 (Th1-associated immune response precedes the activation and recruitment of specific Th1 cells bearing distinct chemokine receptors, subsequently leading to the clearance of Chlamydia. We have shown that CCR5, a receptor for CCL5, is crucial for protective chlamydial immunity. Our laboratory and others have also demonstrated that CCL5 deficiencies found in man and animals can increase the susceptibility and progression of infectious diseases by modulating mucosal immunity. These findings suggest the CCR5-CCL5 axis is necessary for optimal chlamydial immunity. We hypothesized CCL5 is required for protective humoral and cellular immunity against Chlamydia. Results The present study revealed that CCR5 and CCL5 mRNAs are elevated in the spleen, iliac lymph nodes (ILNs, and genital mucosa following Chlamydia muriduram challenge. Antibody (Ab-mediated inhibition of CCL5 during genital chlamydial infection suppressed humoral and Th1 > Th2 cellular responses by splenic-, ILN-, and genital mucosa-derived lymphocytes. Antigen (Ag-specific proliferative responses of CD4+ T cells from spleen, ILNs, and genital organs also declined after CCL5 inhibition. Conclusion The suppression of these responses correlated with delayed clearance of C. muriduram, which indicate chlamydial immunity is mediated by Th1 immune responses driven in part by CCL5. Taken together with other studies, the data show that CCL5 mediates the temporal recruitment and activation of leukocytes to mitigate chlamydial infection through enhancing adaptive mucosal humoral and cellular immunity.

  7. MOLECULAR MODELING STUDY OF THE CONTRIBUTIONS OF SIDE AMINO ACID RESIDUES OF POLYMYXIN B3 TO ITS BINDING WITH E.COLI OUTER MEMBRANE LIPOPOLYSACCHARIDE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2014-12-01

    Full Text Available Last decades, antimicrobial peptides (AMPs are the subject of intense investigations aimed to develop effective drugs against extremely resistant nosocomial bacterial pathogens (especially Gram-negative bacteria. In particular, there has been greatly renewed interest to polymyxins, the representatives of AMPs which are specific and highly potent against Gram-negative bacteria, but have potential nephrotoxic side effect. A prerequisite of purposeful enhancement of therapeutic properties of polymyxins is a detailed knowledge of the molecular mechanisms of their interactions with cell targets. Lipopolysaccharide (LPS, the main component of the outer leaflet of outer membrane of gram-negative bacteria, is a primary cell target of polymyxins. The aim of the paper was to study the peculiarities of molecular interactions of polymyxin В3 with lipopolysaccharide of the outer membrane of gram-negative bacterium. Materials and methods The complexes of polymyxin В3 (PmВ3 and its alaninederivatives with E. coli outer membrane lipopolysaccharide were built and studied by molecular modeling methods (minimization, simulated annealing, docking. Atom coordinates of polymyxin В3 and LPS structures were taken from nuclear magnetic resonance and X-ray crystallography experiments, respectively. The AMBER03 force field was used with a 1.05 nm force cutoff. Longrange electrostatic interactions were treated by the Particle Mesh Ewald method. Results and discussion Alanine scanning of PmВ3 molecule has been carried out and the role of its side amino acid residues in the formation of complex with lipopolysaccharide has been investigated. It has been shown that substitutions of polymyxin’s Dab residues in positions 1, 3, 5, 8 and 9 for alanine markedly reduce the binding energy of PmB3-LPS complex, where as the similar substitutions of residues in positions 2, 6, 7 and 10 leave the binding energy virtually unchanged. Structural aspects of antimicrobial action of

  8. Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

    Science.gov (United States)

    Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

    2007-06-12

    Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

  9. Chlamydial virulence determinants in atherogenesis: the role of chlamydial lipopolysaccharide and heat shock protein 60 in macrophage-lipoprotein interactions.

    Science.gov (United States)

    Kalayoglu, M V; Indrawati; Morrison, R P; Morrison, S G; Yuan, Y; Byrne, G I

    2000-06-01

    Data from a spectrum of epidemiologic, pathologic, and animal model studies show that Chlamydia pneumoniae infection is associated with coronary artery disease, but it is not clear how the organism may initiate or promote atherosclerosis. It is postulated that C. pneumoniae triggers key atherogenic events through specific virulence determinants. C. pneumoniae induces mononuclear phagocyte foam cell formation by chlamydial lipopolysaccharide (cLPS) and low-density lipoprotein oxidation by chlamydial hsp60 (chsp60). Thus, different chlamydial components may promote distinct events implicated in the development of atherosclerosis. Data implicating cLPS and chsp60 in the pathogenesis of atherosclerosis are discussed and novel approaches are presented for attempting to elucidate how these putative virulence determinants signal mononuclear phagocytes to modulate lipoprotein influx and modification.

  10. Establishing homology between mitochondrial calcium uniporters, prokaryotic magnesium channels and chlamydial IncA proteins.

    Science.gov (United States)

    Lee, Andre; Vastermark, Ake; Saier, Milton H

    2014-08-01

    Mitochondrial calcium uniporters (MCUs) (TC no. 1.A.77) are oligomeric channel proteins found in the mitochondrial inner membrane. MCUs have two well-conserved transmembrane segments (TMSs), connected by a linker, similar to bacterial MCU homologues. These proteins and chlamydial IncA proteins (of unknown function; TC no. 9.B.159) are homologous to prokaryotic Mg(2+) transporters, AtpI and AtpZ, based on comparison scores of up to 14.5 sds. A phylogenetic tree containing all of these proteins showed that the AtpZ proteins cluster coherently as a subset within the large and diverse AtpI cluster, which branches separately from the MCUs and IncAs, both of which cluster coherently. The MCUs and AtpZs share the same two TMS topology, but the AtpIs have four TMSs, and IncAs can have either two (most frequent) or four (less frequent) TMSs. Binary alignments, comparison scores and motif analyses showed that TMSs 1 and 2 align with TMSs 3 and 4 of the AtpIs, suggesting that the four TMS AtpI proteins arose via an intragenic duplication event. These findings establish an evolutionary link interconnecting eukaryotic and prokaryotic Ca(2+) and Mg(2+) transporters with chlamydial IncAs, and lead us to suggest that all members of the MCU superfamily, including IncAs, function as divalent cation channels.

  11. Neisserial outer membrane vesicles bind the coinhibitory receptor carcinoembryonic antigen-related cellular adhesion molecule 1 and suppress CD4+ T lymphocyte function.

    Science.gov (United States)

    Lee, Hannah S W; Boulton, Ian C; Reddin, Karen; Wong, Henry; Halliwell, Denise; Mandelboim, Ofer; Gorringe, Andrew R; Gray-Owen, Scott D

    2007-09-01

    Pathogenic Neisseria bacteria naturally liberate outer membrane "blebs," which are presumed to contribute to pathology, and the detergent-extracted outer membrane vesicles (OMVs) from Neisseria meningitidis are currently employed as meningococcal vaccines in humans. While the composition of these vesicles reflects the bacteria from which they are derived, the functions of many of their constituent proteins remain unexplored. The neisserial colony opacity-associated Opa proteins function as adhesins, the majority of which mediate bacterial attachment to human carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs). Herein, we demonstrate that the Opa proteins within OMV preparations retain the capacity to bind the immunoreceptor tyrosine-based inhibitory motif-containing coinhibitory receptor CEACAM1. When CD4(+) T lymphocytes were exposed to OMVs from Opa-expressing bacteria, their activation and proliferation in response to a variety of stimuli were effectively halted. This potent immunosuppressive effect suggests that localized infection will generate a "zone of inhibition" resulting from the diffusion of membrane blebs into the surrounding tissues. Moreover, it demonstrates that OMV-based vaccines must be developed from strains that lack CEACAM1-binding Opa variants.

  12. A new iron-oxidizing/O2-reducing supercomplex spanning both inner and outer membranes, isolated from the extreme acidophile Acidithiobacillus ferrooxidans.

    Science.gov (United States)

    Castelle, Cindy; Guiral, Marianne; Malarte, Guillaume; Ledgham, Fouzia; Leroy, Gisèle; Brugna, Myriam; Giudici-Orticoni, Marie-Thérèse

    2008-09-19

    The iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans involves various metalloenzymes. Here we demonstrate that the oxygen reduction pathway from ferrous iron (named downhill pathway) is organized as a supercomplex constituted of proteins located in the outer and inner membranes as well as in the periplasm. For the first time, the outer membrane-bound cytochrome c Cyc2 was purified, and we showed that it is responsible for iron oxidation and determined that its redox potential is the highest measured to date for a cytochrome c. The organization of metalloproteins inside the supramolecular structure was specified by protein-protein interaction experiments. The isolated complex spanning the two membranes had iron oxidase as well as oxygen reductase activities, indicating functional electron transfer between the first iron electron acceptor, Cyc2, and the Cu(A) center of cytochrome c oxidase aa(3). This is the first characterization of a respirasome from an acidophilic bacterium. In Acidithiobacillus ferrooxidans,O(2) reduction from ferrous iron must be coupled to the energy-consuming reduction of NAD(+)(P) from ferrous iron (uphill pathway) required for CO(2) fixation and other anabolic processes. Besides the proteins involved in the O(2) reduction, there were additional proteins in the supercomplex, involved in uphill pathway (bc complex and cytochrome Cyc(42)), suggesting a possible physical link between these two pathways.

  13. Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation.

    Science.gov (United States)

    Burdman, S; Dulguerova, G; Okon, Y; Jurkevitch, E

    2001-04-01

    The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

  14. Diagnosis of Helicobacter pylori infection and diseases associated with Helicobacter pylori by Helicobacter pylori outer membrane proteins

    Institute of Scientific and Technical Information of China (English)

    Zheng Jiang; Ai-Long Huang; Xiao-Hong Tao; Pi-Long Wang

    2004-01-01

    AIM: To examine the serological response of patients with upper gastrointestinal diseases and Helicobocter pylori(Hpylori)infection to two H pylori outer membrane proteins (OMPs)(Mr18 000 and Mr26 000) acquired by gene recombinanttechnique, and to determine the diagnostic significance of serological tests derived from these OMPs.METHODS: Recombinant vectors encoding the two H pylori OMPs were used to transform and express in BL21 (DE3)E. coli. After purification with Ni2+-NTA agarose resin, colloid gold kits were prepared with purified recombinant proteins to detect H pylori infection and H pylori-associated diseases by the immunity-marker technology. We selected 150 patients with H pyloriinfection and digestive symptoms without previous treatment, induding chronic gastritis (n = 60), duodenal ulcer (n = 30), gastric ulcer (n = 30), and gastric cancer (n = 30).As controls, 33 H pylori-negative healthy volunteers were also recruited. Serum samples were collected from all subjects, and the antibodies to specific proteins of H pylori were tested with the colloid gold test kits. The sensitivity,specificity and accuracy of the colloid gold tests were evaluated, by using the combination of standard diagnostic methods (13C urea breath test and bacteria culture) and classic enzyme-linked immunosorbent assay (ELISA) as reference.RESULTS: After purification with Ni2+-NTA agarose resin,the purity of recombinant fusion proteins was about 95%.The recombinant fusion proteins were recognized by the specific monoclonal antibodies against the two H pylori OMPs,as demonstrated by the ELISA. Of the 150 serum samples from patients infected with H pylori 141 (94.0%) responded positively to the recombinant protein with Mr26 000, while the seropositive rates were 95.0%, 96.7%, 96.7% and 90.0%for patients with H pylori-associated chronic gastritis,duodenal ulcer, gastric ulcer, and gastric cancer respectively.The sensitivity, specificity, and accuracy of the colloid gold kit with Mr26 000

  15. The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H

    OpenAIRE

    Rauna eRiva; Korhonen, Timo K.; Seppo eMeri

    2015-01-01

    The virulence factor PgtE is an outer membrane protease (omptin) of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e., rough LPS, as observed e.g., in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B) and H (H), key proteins controlling formation and inactivation of the complement pro...

  16. Identification of a TcpC-TcpQ Outer Membrane Complex Involved in the Biogenesis of the Toxin-Coregulated Pilus of Vibrio cholerae

    OpenAIRE

    Bose, Niranjan; Taylor, Ronald K.

    2005-01-01

    The toxin-coregulated pilus (TCP) of Vibrio cholerae and the soluble TcpF protein that is secreted via the TCP biogenesis apparatus are essential for intestinal colonization. The TCP biogenesis apparatus is composed of at least nine proteins but is largely uncharacterized. TcpC is an outer membrane lipoprotein required for TCP biogenesis that is a member of the secretin protein superfamily. In the present study, analysis of TcpC in a series of strains deficient in each of the TCP biogenesis p...

  17. Distribution of Salmonella paratyphi A outer membrane protein X gene and immune-protective effect related to its recombinant expressed products

    Institute of Scientific and Technical Information of China (English)

    李明雷

    2014-01-01

    Objective To determine the distribution and sequence conservation of outer membrane protein X(ompX)gene in Salmonella paratyphi A isolates as well as the immunogenicity and immono-protection of ompX gene products.Methods OmpX gene in Salmonella paratyphi A isolates was detected by PCR and the amplification products were sequenced after the T-A cloning process.OmpX gene product was expressed with E.coli expression system and the expressed rOmpX was extracted by Ni-NTA affinity

  18. From Evolution to Pathogenesis: The Link Between β-Barrel Assembly Machineries in the Outer Membrane of Mitochondria and Gram-Negative Bacteria

    Directory of Open Access Journals (Sweden)

    Jhih-Hang Jiang

    2012-06-01

    Full Text Available β-barrel proteins are the highly abundant in the outer membranes of Gram-negative bacteria and the mitochondria in eukaryotes. The assembly of β-barrels is mediated by two evolutionary conserved machineries; the β-barrel Assembly Machinery (BAM in Gram-negative bacteria; and the Sorting and Assembly Machinery (SAM in mitochondria. Although the BAM and SAM have functionally conserved roles in the membrane integration and folding of β-barrel proteins, apart from the central BamA and Sam50 proteins, the remaining components of each of the complexes have diverged remarkably. For example all of the accessory components of the BAM complex characterized to date are located in the bacterial periplasm, on the same side as the N-terminal domain of BamA. This is the same side of the membrane as the substrates that are delivered to the BAM. On the other hand, all of the accessory components of the SAM complex are located on the cytosolic side of the membrane, the opposite side of the membrane to the N-terminus of Sam50 and the substrate receiving side of the membrane. Despite the accessory subunits being located on opposite sides of the membrane in each system, it is clear that each system is functionally equivalent with bacterial proteins having the ability to use the eukaryotic SAM and vice versa. In this review, we summarize the similarities and differences between the BAM and SAM complexes, highlighting the possible selecting pressures on bacteria and eukaryotes during evolution. It is also now emerging that bacterial pathogens utilize the SAM to target toxins and effector proteins to host mitochondria and this will also be discussed from an evolutionary perspective.

  19. Lipopolysaccharide (LPS) inner-core phosphates are required for complete LPS synthesis and transport to the outer membrane in Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Delucia, Angela M; Six, David A; Caughlan, Ruth E; Gee, Patricia; Hunt, Ian; Lam, Joseph S; Dean, Charles R

    2011-01-01

    Gram-negative outer membrane (OM) integrity is maintained in part by Mg(2+) cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria, waaP, encoding an inner-core kinase, could not be inactivated in Pseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential in P. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), two l-glycero-d-manno-heptoses (Hep), and one hexose but completely devoid of phosphates, indicating that phosphorylation by WaaP is necessary for subsequent core phosphorylations. MS analysis of lipid A from WaaP-depleted cells revealed extensive 4-amino-4-deoxy-l-arabinose modification. OM prepared from these cells by Sarkosyl extraction of total membranes or by sucrose density gradient centrifugation lacked truncated LPS. Instead, truncated LPS was detected in the inner membrane fractions, consistent with impaired transport/assembly of this species into the OM. IMPORTANCE Gram-negative bacteria have an outer membrane (OM) comprised of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The OM protects cells from toxic molecules and is important for survival during infection. The LPS core kinase gene waaP can be deleted in several Gram-negative bacteria but not in Pseudomonas aeruginosa. We used a controlled-expression system to deplete WaaP directly in P. aeruginosa cells, which halted growth. WaaP depletion

  20. Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamy- dia-induced upper genital tract gross pathology and histopathological characterization were also de- tected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were sig- nificantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vacci- nated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against patho- logical consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immu- nization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.

  1. Cloning, expression of Vibrio alginolyticus outer membrane protein-OmpU gene and its potential application as vaccine in crimson snapper, Lutjanus erythropterus Bloch.

    Science.gov (United States)

    Cai, S H; Lu, Y S; Wu, Z H; Jian, J C

    2013-08-01

    The outer membrane proteins of the marine aquatic animal pathogen, Vibrio alginolyticus, play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein-OmpU was cloned and expressed in Escherichia coli. Polyclonal antibodies were raised in rabbits against the purified recombinant OmpU, and the reaction of the antibody was confirmed by Western blotting using the isolated OmpU and the recombinant OmpU of V. alginolyticus. To analyze the immunogenicity of the recombinant OmpU, crimson snapper, Lutjanus erythropterus Bloch, were immunized by intraperitoneal injection, and antibody response was assessed by the enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the recombinant OmpU produced an observable antibody response in all sera of the vaccinated fish. The vaccinated fish were challenged by virulent V. alginolyticus and observed to have high resistance to infection. These results indicate that the recombinant OmpU is an effective vaccine candidate against V. alginolyticus in L. erythropterus.

  2. Small-Molecule Transport by CarO, an Abundant Eight-Stranded β-Barrel Outer Membrane Protein from Acinetobacter baumannii.

    Science.gov (United States)

    Zahn, Michael; D'Agostino, Tommaso; Eren, Elif; Baslé, Arnaud; Ceccarelli, Matteo; van den Berg, Bert

    2015-07-17

    Outer membrane (OM) β-barrel proteins composed of 12-18 β-strands mediate cellular entry of small molecules in Gram-negative bacteria. Small OM proteins with barrels of 10 strands or less are not known to transport small molecules. CarO (carbapenem-associated outer membrane protein) from Acinetobacter baumannii is a small OM protein that has been implicated in the uptake of ornithine and carbapenem antibiotics. Here we report crystal structures of three isoforms of CarO. The structures are very similar and show a monomeric eight-stranded barrel lacking an open channel. CarO has a substantial extracellular domain resembling a glove that contains all the divergent residues between the different isoforms. Liposome swelling experiments demonstrate that full-length CarO and a "loop-less" truncation mutant mediate small-molecule uptake at low levels but that they are unlikely to mediate passage of carbapenem antibiotics. These results are confirmed by biased molecular dynamics simulations that allowed us to quantitatively model the transport of selected small molecules.

  3. Naturally Occurring IgG Antibodies Provide Innate Protection against Vibrio cholerae Bacteremia by Recognition of the Outer Membrane Protein U.

    Science.gov (United States)

    Aung, Kyaw Min; Sjöström, Annika E; von Pawel-Rammingen, Ulrich; Riesbeck, Kristian; Uhlin, Bernt Eric; Wai, Sun Nyunt

    2016-01-01

    Cholera epidemics are caused by Vibrio cholerae serogroups O1 and O139, whereas strains collectively known as non-O1/non-O139 V. cholerae are found in cases of extraintestinal infections and bacteremia. The mechanisms and factors influencing the occurrence of bacteremia and survival of V. cholerae in normal human serum have remained unclear. We found that naturally occurring IgG recognizing V. cholerae outer membrane protein U (OmpU) mediates a serum-killing effect in a complement C1q-dependent manner. Moreover, outer membrane vesicles (OMVs) containing OmpU caused enhanced survival of highly serum-sensitive classical V. cholerae in a dose-dependent manner. OMVs from wild-type and ompU mutant V. cholerae thereby provided a novel means to verify by extracellular transcomplementation the involvement of OmpU. Our data conclusively indicate that loss, or reduced expression, of OmpU imparts resistance to V. cholerae towards serum killing. We propose that the difference in OmpU protein levels is a plausible reason for differences in serum resistance and the ability to cause bacteremia observed among V. cholerae biotypes. Our findings provide a new perspective on how naturally occurring antibodies, perhaps induced by members of the microbiome, may play a role in the recognition of pathogens and the provocation of innate immune defense against bacteremia.

  4. The β-barrel assembly machinery (BAM) is required for the assembly of a primitive S-layer protein in the ancient outer membrane of Thermus thermophilus.

    Science.gov (United States)

    Acosta, Federico; Ferreras, Eloy; Berenguer, José

    2012-11-01

    The ancient bacterial lineage Thermus spp has a primitive form of outer membrane attached to the cell wall through SlpA, a protein that shows intermediate properties between S-layer proteins and outer membrane (OM) porins. In E. coli and related Proteobacteria, porins are secreted through the BAM (β-barrel assembly machinery) pathway, whose main component is BamA. A homologue to this protein is encoded in all the Thermus spp so far sequenced, so we wondered if this pathway could be responsible for SlpA secretion in this ancient bacterial model. To analyse this hypothesis, we attempted to get mutants on this BamA(th) of T. thermophilus HB27. Knockout and deletion mutants lacking the last 10 amino acids were not viable, whereas its depletion by means of a BamA antisense RNA lead defective attachment to the cell wall of its OM-like envelope. Such defects were related to defective folding of the SlpA protein that was more sensitive to proteases than in a wild-type strain. A similar phenotype was found in mutants lacking the terminal Phe of SlpA. Further protein-protein interaction assays confirmed the existence of specific binding between SlpA and BamA(th). Taking together, these data suggest that SlpA is secreted through a BAM-like pathway in this ancestral bacterial lineage, supporting an ancient origin of this pathway before the evolution of the Proteobacteria.

  5. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

    Science.gov (United States)

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-11-13

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

  6. The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

    Directory of Open Access Journals (Sweden)

    Ofir Bahar

    2014-01-01

    Full Text Available Pattern recognition receptors (PRRs play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo. In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity.

  7. Quantification of lipopolysaccharides in outer membrane vesicle vaccines against meningococcal disease. High-performance liquid chromatographic determination of the constituent 3-hydroxy-lauric acid.

    Science.gov (United States)

    Lyngby, Janne; Olsen, Linda H; Eidem, Tove; Lundanes, Elsa; Jantzen, Erik

    2002-03-01

    A high-performance liquid chromatographic (HPLC) assay for quantification of lipopolysaccharides (LPSs, endotoxins) in outer membrane vesicle vaccines against meningococcal disease has been developed. The LPS constituent, 3-hydroxy-lauric acid, served as marker substance for the quantification. LPS from the vaccine was precipitated by ethanol and the fatty acid constituents, including 3-hydroxy-lauric acid, were released by acidic hydrolysis, collected and purified by solid phase extraction on C18 disc-cartridges and converted into phenacyl esters for UV detection at 240 nm. Quantification of the derivatized 3-hydroxy-lauric acid was achieved by HPLC using a Brownlee RP-18 reversed phase column with acetonitrile/water (68:32, v/v) as mobile phase. The method was found to be linear over the range 3-49 microg LPS/ml with a sensitivity of 1.6 (microg/ml)(-1). The repeatability (within-day precision) of the method at three levels (3-49 microg LPS/ml) was 6-14% relative standard deviation and the intermediate (between-day) precision was 7% relative standard deviation (at level 15 microg LPS/ml). The method has been successfully used in the quality control of a meningococcal B outer membrane vesicle vaccine, containing 4-8% LPS relative to protein (w/w), in our laboratory for three years.

  8. Crystal structures of OprN and OprJ, outer membrane factors of multidrug tripartite efflux pumps of Pseudomonas aeruginosa.

    Science.gov (United States)

    Yonehara, Ryo; Yamashita, Eiki; Nakagawa, Atsushi

    2016-06-01

    The genome of Pseudomonas aeruginosa encodes tripartite efflux pumps that extrude functionally and structurally dissimilar antibiotics from the bacterial cell. MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM are the main tripartite efflux pumps responsible for multidrug resistance in P. aeruginosa. The outer membrane factors OprN, OprJ, and OprM are essential components of functional tripartite efflux pumps. To elucidate the structural basis of multidrug resistance, we determined the crystal structures of OprN and OprJ. These structures revealed several features, including tri-acylation of the N-terminal cysteine, a small pore in the β-barrel domain, and a tightly sealed gate in the α-barrel domain. Despite the overall similarity of OprN, OprJ, and OprM, a comparison of their structures and electrostatic distributions revealed subtle differences at the periplasmic end of the α-barrel domain. These results suggested that the overall structures of these outer membrane factors are specifically optimized for particular tripartite efflux pumps. Proteins 2016; 84:759-769. © 2016 Wiley Periodicals, Inc.

  9. Improvement of outer membrane-permeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification.

    Science.gov (United States)

    Piers, K L; Brown, M H; Hancock, R E

    1994-10-01

    Antimicrobial cationic peptides have been discovered in many different organisms and often possess a broad range of activity. In this study, we investigated the mechanisms of actions of melittin and two synthetic peptides, CEME (a cecropin-melittin hybrid) and CEMA, against gram-negative bacteria. CEMA was produced by recombinant DNA procedures and is an analog of CEME with a modified C terminus resulting in two additional positive charges. All three peptides showed good antimicrobial activity against four different gram-negative bacteria, but only CEMA was able to somewhat augment the activity of some conventional antibiotics in synergy studies. Studies using the bacteria Pseudomonas aeruginosa and Enterobacter cloacae showed that the peptides all possessed the ability to permeabilize bacterial outer membranes to the hydrophobic fluorophor 1-N-phenylnaphthylamine and the protein lysozyme, with CEMA being the most active. CEMA also had the strongest relative binding affinity for bacterial endotoxin (lipopolysaccharide). These data collectively indicated that these peptides all cross the outer membrane by the self-promoted uptake pathway and that CEMA is the peptide most effective at accessing this pathway.

  10. Structural analysis and cross-protective efficacy of recombinant 87 kDa outer membrane protein (Omp87) of Pasteurella multocida serogroup B:2.

    Science.gov (United States)

    Kumar, Abhinendra; Yogisharadhya, Revanaiah; Ramakrishnan, Muthannan A; Viswas, K N; Shivachandra, Sathish B

    2013-12-01

    Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic septicaemia (HS) in cattle and buffalo especially in tropical regions of Asian and African countries, is known to possess several outer membrane proteins (OMPs) as immunogenic antigens. In the present study, omp87 gene encoding for 87 kDa OMP (Omp87) protein of P. multocida serogroup B:2 strain P52, has been amplified (∼2304 bp), cloned in to pET32a vector and over-expressed in recombinant Escherichia coli as fusion protein. The recombinant Omp87 protein (∼102 kDa) including N-terminus hexa-histidine tag was purified under denaturing condition. Immunization of mice with rOmp87 resulted in increased antigen specific IgG titres in serum and provided protection of 66.6 and 83.3% following homologous (B:2) and heterologous (A:1) challenge, respectively. A homology model of Omp87 revealed the presence of two distinct domains; N-terminal domain with four POTRA repeats in the periplasmic space and a pore forming C-terminal β-barrel domain (β1- β16) in the outer membrane of P. multocida, which belong to Omp85-TpsB transporter superfamily of OMPs. The study indicated the potential possibilities to use rOmp87 protein along with suitable adjuvant in developing subunit vaccine for haemorrhagic septicaemia and pasteurellosis in livestock.

  11. Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida A:3 in cattle: molecular characterization of the immunodominant heme acquisition system receptor (HasR) protein.

    Science.gov (United States)

    Prado, M E; Dabo, S M; Confer, A W

    2005-02-25

    The iron-regulated outer membrane proteins (IROMPs) of Pasteurella multocida A:3 strain 232 (Pm232), a bovine isolate, were investigated as potential immunogens in cattle. We addressed the ability of P. multocida IROMP-enriched fractions to induce antibody responses in cattle by different vaccination strategies and the protective efficacy of these antibodies using a P. multocida-induced pneumonia challenge model. Vaccination of cattle with outer membrane-enriched fractions derived from Pm232 grown on either iron-depleted (IROMPs) or iron-sufficient (OMPs) conditions induced significant antibody responses; however, the correlation with lung lesion scores was not significant (P = 0.01 and P multocida (strain Pm70) and was confirmed by DNA sequence analysis of the cloned Pm232 hasR gene. Further analyses indicated that Pm232 HasR is a surface-exposed OMP and conserved among most P. multocida isolates investigated. In addition, cattle vaccinated with live Pm232 or IROMPs had significantly higher antibody responses to the 96 kDa protein band and the correlation with lung lesion scores approached significance (P = 0.056). These results indicate that antibody responses in cattle are induced by P. multocida IROMPs, and that the 96 kDa HasR protein is an immunodominant IROMP.

  12. Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

    Science.gov (United States)

    Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C

    2009-04-01

    Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

  13. Protective effect of indoleamines on in vitro ascorbate-Fe2+ dependent lipid peroxidation of rod outer segment membranes of bovine retina.

    Science.gov (United States)

    Guajardo, Margarita H; Terrasa, Ana M; Catalá, Angel

    2003-11-01

    Rod outer segment membranes (ROS) are highly vulnerable to autooxidation because of their high content of long chain polyunsaturated fatty acids (PUFAs). Melatonin and N-acetylserotonin are indoleamines synthesized in the pineal gland, retina and other tissues. These compounds are free radical scavengers and indirect antioxidants because of their stimulatory effect on antioxidative enzymes. We compared the in vitro protective effect of melatonin and N-acetylserotonin on the ascorbate-Fe2+ induced lipid peroxidation of PUFAs located in ROS membranes. This process was measured by chemiluminescence and fatty acid composition of total lipids of ROS. We assayed increasing concentrations of melatonin (0-10 mm) and N-acetylserotonin (0-2 mm). In both cases the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of either melatonin or N-acetylserotonin; this decreased proportional to the concentration of the indole. Thus, 10 mm melatonin and 2 mm N-acetylserotonin produced a reduction of 51 +/- 6 and 100% in the total chemiluminescene (lipid peroxidation), respectively. We also noticed a PUFAs protection: the docosahexaenoic acid content decreased considerably when the membranes were submitted to oxidative damage. This reduction was from 37.6 +/- 2.1% in the native membranes to 6.2 +/- 0.8% in those which were peroxidized. These changes were less pronounced in treated ROS membranes; as an example in the presence of 10 mm melatonin or 2 mm N-acetylserotonin we observed a content preservation of 22:6 n-3 (23.6 +/- 1.2 and 39.1 +/- 1.2% respectively). The concentration of each compound required to inhibit 50% of the lipid peroxidation (IC50) was 9.82 mm for melatonin and 0.43 mm for N-acetylserotonin, respectively. N-acetylserotonin shows a protective effect about 20 times higher than that of melatonin.

  14. In-vivo identification of direct electron transfer from Shewanella oneidensis MR-1 to electrodes via outer-membrane OmcA-MtrCAB protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Okamoto, Akihiro [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Nakamura, Ryuhei, E-mail: nakamura@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Hashimoto, Kazuhito, E-mail: hashimoto@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); ERATO/JST, HASHIMOTO Light Energy Conversion Project (Japan)

    2011-06-30

    Graphical abstract: . Display Omitted Highlights: > Monolayer biofilm of Shewanella cells was prepared on an ITO electrode. > Extracellular electron transfer (EET) process was examined with series of mutants. > Direct ET was confirmed with outer-membrane-bound OmcA-MtrCAB complex. > The EET process was not prominently influenced by capsular polysaccharide. - Abstract: The direct electron-transfer (DET) property of Shewanella bacteria has not been resolved in detail due to the complexity of in vivo electrochemistry in whole-cell systems. Here, we report the in vivo assignment of the redox signal indicative of the DET property in biofilms of Shewanella oneidensis MR-1 by cyclic voltammetry (CV) with a series of mutants and a chemical marking technique. The CV measurements of monolayer biofilms formed by deletion mutants of c-type cytochromes ({Delta}mtrA, {Delta}mtrB, {Delta}mtrC/{Delta}omcA, and {Delta}cymA), and pilin ({Delta}pilD), capsular polysaccharide ({Delta}SO3177) and menaquinone ({Delta}menD) biosynthetic proteins demonstrated that the electrochemical redox signal with a midpoint potential at 50 mV (vs. SHE) was due to an outer-membrane-bound OmcA-MtrCAB protein complex of decaheme cytochromes, and did not involve either inner-membrane-bound CymA protein or secreted menaquinone. Using the specific binding affinity of nitric monoxide for the heme groups of c-type cytochromes, we further confirmed this conclusion. The heterogeneous standard rate constant for the DET process was estimated to be 300 {+-} 10 s{sup -1}, which was two orders of magnitude higher than that previously reported for the electron shuttling process via riboflavin. Experiments using a mutant unable to produce capsular polysaccharide ({Delta}SO3177) revealed that the DET property of the OmcA-MtrCAB complex was not influenced by insulating and hydrophilic extracellular polysaccharide. Accordingly, under physiological conditions, S. oneidensis MR-1 utilizes a high density of outer-membrane

  15. Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins.

    Science.gov (United States)

    Zaccai, Nathan R; Sandlin, Clifford W; Hoopes, James T; Curtis, Joseph E; Fleming, Patrick J; Fleming, Karen G; Krueger, Susan

    2016-01-01

    In Gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archaeal prefoldins and the mitochondrial Tim chaperones, that is the α-helical "tentacles" extend from a β-strand "body" to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis, and structure modeling.

  16. Molecular mimicry and horror autotoxicus: do chlamydial infections elicit autoimmunity?

    Science.gov (United States)

    Swanborg, Robert H; Boros, Dov L; Whittum-Hudson, Judith A; Hudson, Alan P

    2006-11-30

    All species of the order Chlamydiales are obligate intracellular eubacterial pathogens of their various hosts. Two chlamydial species, Chlamydia trachomatis and Chlamydia pneumoniae, are primarily human pathogens, and each is known to cause important diseases. Some strains of C. trachomatis are sexually transmitted and frequently cause severe reproductive problems, primarily in women. Other strains of the organism serve as the aetiological agents for blinding trachoma, still the leading cause of preventable blindness in underdeveloped nations. C. pneumoniae is a respiratory pathogen known to cause community-acquired pneumonia. Importantly, both organisms engender an immunopathogenic response in the human host, and both have been associated with widely diverse, relatively common and currently idiopathic chronic diseases, most of which include an important autoimmune component. In this article, we explore the available experimental data regarding the possible elicitation of autoimmunity in various contexts by chlamydial infection, and we suggest several avenues for research to explore this potentially important issue further.

  17. Chlamydiaceae and chlamydial infections in sheep or goats.

    Science.gov (United States)

    Rodolakis, A; Laroucau, K

    2015-12-14

    Chlamydiae induce a range of pathological syndromes in small ruminants. Abortion is the most common clinical expression of the infection that causes important economic losses and presents a risk to human health, particularly in pregnant women. The present paper gives an overview of chlamydial infections in sheep and goats, focusing specifically on abortion and on recent data brought by cellular and genomic approaches regarding genotyping, virulence of strains, epidemiology, diagnosis, pathogenesis and control of the disease.

  18. Chlamydial genes shed light on the evolution of photoautotrophic eukaryotes

    OpenAIRE

    2008-01-01

    Abstract Background Chlamydiae are obligate intracellular bacteria of protists, invertebrates and vertebrates, but have not been found to date in photosynthetic eukaryotes (algae and embryophytes). Genes of putative chlamydial origin, however, are present in significant numbers in sequenced genomes of photosynthetic eukaryotes. It has been suggested that such genes were acquired by an ancient horizontal gene transfer from Chlamydiae to the ancestor of photosynthetic eukaryotes. To further tes...

  19. Epidemiological and clinical correlates of chlamydial infection of the cervix.

    OpenAIRE

    Arya, O P; Mallinson, H; Goddard, A D

    1981-01-01

    Of 474 women studied to identify epidemiological and clinical correlates of chlamydial infection of the cervix, Chlamydia trachomatis was isolated from 158 (33.3%) of all women, from 48.3% of those infected with Neisseria gonorrhoeae, from 43% of the sexual consorts of men with nongonococcal urethritis, and from 74% of those whose consorts were also infected with C trachomatis. C trachomatis was the sole pathogen found in 58 women. Age, marital state, occupation, past history of gonorrhoea, m...

  20. Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors.

    Science.gov (United States)

    Veith, Paul D; Chen, Yu-Yen; Chen, Dina; O'Brien-Simpson, Neil M; Cecil, Jessica D; Holden, James A; Lenzo, Jason C; Reynolds, Eric C

    2015-12-04

    Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC-MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.

  1. Protection against Chlamydia trachomatis infection and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of the major outer membrane protein

    DEFF Research Database (Denmark)

    Olsen, Anja W.; Follmann, Frank; Erneholm, Karin Susanne;

    2015-01-01

    The VD4 region from the Chlamydia trachomatis major outer membrane protein contains important neutralizing B-cell epitopes of relevance for antibody-mediated protection against genital tract infection. We developed a multivalent vaccine construct based on VD4s and their surrounding constant...... segments from serovars D, E, and F. Adjuvanted with cationic liposomes, this construct promoted strong immune responses to serovar-specific epitopes, the conserved LNPTIAG epitope and neutralized serovars D, E, and F. Vaccinated mice were protected against challenge, with protection defined as reduced...... bacterial numbers in vagina and prevention of pathological changes in the upper genital tract. Adoptive transfer of serumand T-cell depletion experiments demonstrated a dominant role for antibodies and CD4+ T cells in the protective immune response. Integrating a multivalent VD4 construct into the sequence...

  2. Efficacy of bacterin-, outer membrane protein- and fimbriae extract-based vaccines for the control of Salmonella Enteritidis experimental infection in chickens

    Directory of Open Access Journals (Sweden)

    Márcia C. Menão

    2013-03-01

    Full Text Available The efficacy of three vaccines was evaluated in chickens for the control of experimental infection with Salmonella Enteritidis (SE phage type 4. The vaccines were produced with bacterin, outer membrane proteins (OMP and fimbriae crude extract (FE. The chickens were vaccinated intramuscularly with two doses of each vaccine at 12 and 15 weeks of age. The chickens were then orally challenged with 10(9 CFU/chicken Salmonella Enteritidis phage type 4 at 18 weeks of age. Fecal swabs were performed for the recovery of shedding SE, and SE was recovered from the liver and spleen. Additionally, antibody titers were measured in the serum by micro-agglutination test. The results indicated that the vaccine produced with bacterin yielded better results and resulted in reduction of fecal shedding and organ invasion by SE after oral challenge, although no vaccine was 100% effective for the control of SE experimental infection.

  3. The mechanisms of complement activation in normal bovine serum and normal horse serum against Yersinia enterocolitica O:9 strains with different outer membrane proteins content.

    Science.gov (United States)

    Miętka, K; Brzostek, K; Guz-Regner, K; Bugla-Płoskońska, G

    2016-01-01

    Yersinia enterocolitica is a common zoonotic pathogen and facultative intracellular bacterium which can survive within blood cells. Cattle and horses are considered a reservoir of Y. enterocolitica which often causes several serious syndromes associated with yersiniosis such as abortions, premature births or infertility. The aim of our investigation was to determine the vitality of Y. enterocolitica O:9 strains (Ye9) in bovine and horse sera (NBS and NHrS) and explain the role of outer membrane proteins (OMPs) in serum resistance of these bacteria. Our previous studies demonstrated moderate human serum (NHS) resistance of the wild type Ye9 strain, whereas mutants lacking YadA, Ail or OmpC remained sensitive to the bactericidal activity of NHS. The present study showed that the wild type of Ye9 strain was resistant to the bactericidal activity of both NHrS and NBS, while Ye9 mutants lacking the YadA, Ail and OmpC proteins were sensitive to NHrS and NBS as well as to NHS. The mechanisms of complement activation against Ye9 strains lacking Ail and YadA were distinguished, i.e. activation of the classical/lectin pathways decisive in the bactericidal mechanism of complement activation of NBS, parallel activation of the classical/lectin and alternative pathways of NHrS. In this research the mechanism of independent activation of the classical/lectin or the alternative pathway of NBS and NHrS against Ye9 lacking OmpC porin was also established. The results indicate that serum resistance of Ye9 is multifactorial, in which extracellular structures, i.e. outer membrane proteins (OMPs) such as Ail, OmpC or YadA, play the main role.

  4. Calcineurin B-like Protein CBL10 Directly Interacts with TOC34 (Translocon of the Outer Membrane of the Chloroplasts) and Decreases Its GTPase Activity in Arabidopsis

    Science.gov (United States)

    Cho, Joo Hyuk; Lee, Jeong Hwan; Park, Yoon Kook; Choi, Mi Na; Kim, Kyung-Nam

    2016-01-01

    As calcium sensor relays in plants, calcineurin B-like (CBL) proteins provide an important contribution to decoding Ca2+ signatures elicited by a variety of abiotic stresses. Currently, it is well known that CBLs perceive and transmit the Ca2+ signals mainly to a group of serine/threonine protein kinases called CBL-interacting protein kinases (CIPKs). In this study, we report that the CBL10 member of this family has a novel interaction partner besides the CIPK proteins. Yeast two-hybrid screening with CBL10 as bait identified an Arabidopsis cDNA clone encoding a TOC34 protein, which is a member of the TOC (Translocon of the Outer membrane of the Chloroplasts) complex and possesses the GTPase activity. Further analyses showed that in addition to CBL10, CBL7 also interacts with TOC34 at much lower strength in the yeast two-hybrid system. However, the rest of the CBL family members failed to interact with TOC34. Bimolecular fluorescence complementation (BiFC) analysis verified that the CBL10-TOC34 interaction occurs at the outer membrane of chloroplasts in vivo. In addition, we also demonstrated that CBL10 physically associates with TOC34 in vitro, resulting in a significant decrease in the GTPase activity of the TOC34 protein. Taken together, our findings clearly indicate that a member of the CBL family, CBL10, can modulate not only the CIPK members but also TOC34, allowing the CBL family to relay the Ca2+ signals in more diverse ways than currently known. PMID:28018422

  5. Evolutionary Implication of Outer Membrane Lipoprotein-Encoding Genes ompL1, lipL32 and lipL41 of Pathogenic Leptospira Species

    Institute of Scientific and Technical Information of China (English)

    K. Vedhagiri; K. Natarajaseenivasan; P. Chellapandi; S.G. Prabhalaran; Joseph Selvin; S. Sharma; P. Vijayachari

    2009-01-01

    Leptospirosis is recognized as the most widespread zoonosis with a global distribu-tion. In this study, the antigenic variation in Leptospira interrogans and Leptospira borgpetersenii isolated from human urine and field rat kidney was preliminarily confirmed by microscopic agglutination test using monoclonal antibodies, and was further subjected to amplification and identification of outer membrane lipopro-teins with structural gene variation. Sequence similarity analysis revealed that these protein sequences, namely OmpL1, LipL32 and LipL41, showed no more ho-mologies to outer membrane lipoproteins of non-pathogenic Leptospira and other closely related Spirochetes, but showed a strong identity within L. interrogans, suggesting intra-specific phylogenetic lineages that might be originated from a common pathogenic leptospiral origin. Moreover, the ompL1 gene showed more antigenic variation than lipL32 and lipL41 due to less conservation in secondary structural evolution within closely related species. Phylogenetically, ompL1 and lipL41 of these strains gave a considerable proximity to L. weilii and L. santaro-sai. The ompL1 gene of L. interrogans clustered distinctly from other pathogenic and non-pathogenic leptospiral species. The diversity of ompL genes has been an-alyzed and it envisaged that sequence-specific variations at antigenic determinant sites would result in slow evolutionary changes along with new serovar origina-tion within closely related species. Thus, a crucial work on effective recombinant vaccine development and engineered antibodies will hopefully meet to solve the therapeutic challenges.

  6. SDS-PAGE Analysis of the Outer Membrane Proteins of Uropathogenic Escherichia coli Isolated from Patients in Different Wards of Nemazee Hospital, Shiraz, Iran

    Directory of Open Access Journals (Sweden)

    Behzad Dehghani

    2016-09-01

    Full Text Available Background: Outer membrane proteins (OMPs constitute the main structure and about half of the cell wall of Gram-negative bacteria. The OMPs of Escherichia coli (E. coli play an important role in its drug resistance. Previous studies have shown that the OMPs of E. coli enhance its pathogenic effects by helping the bacterium to evade the immune defense and promote its adsorption to host cells. We sought to compare E. coli isolates collected from different hospital wards and to perform a primary investigation of the association between the serotypes and profiles of their OMPs. We also aimed to detect the diversity of the E. coli isolates from the hospitalized patients. Methods: A total of 115 isolates of E. coli were collected from patients hospitalized in Nemazee Hospital, Shiraz, Iran. After biochemical and serological tests, OMPs were extracted by using glass beads and N-Lauroylsarcosine sodium. OMP typing was done by 10% SDS-PAGE and Coomassie brilliant blue staining. In terms of the number of protein bands, OMP-I was detected with 2 bands, OMP-α with 3 bands, and OMP-β with1 band. Results: Of the 115 isolates, 103 were OMP-I and 12 were OMP-α; none of the isolates belonged to OMP-β. Our statistical analyses showed a relationship between OMP patterns and other factors, including hospital wards and source of samples. Serotyping showed that the majority of the isolates were O128. Conclusion: Our results demonstrated some similarities between the OMP band patterns of the analyzed groups of E. coli. Of all the OMPs in the isolates from the hospitalized and outpatient department patients, OmpA and OmpC were the most prevalent proteins in the outer membrane of the studied uropathogenic E. coli.

  7. Particle size effect and the mechanism of hematite reduction by the outer membrane cytochrome OmcA of Shewanella oneidensis MR-1

    Science.gov (United States)

    Liu, Juan; Pearce, Carolyn I.; Shi, Liang; Wang, Zheming; Shi, Zhi; Arenholz, Elke; Rosso, Kevin M.

    2016-11-01

    The cycling of iron at the Earth's near surface is profoundly influenced by dissimilatory metal reducing microorganisms, and many studies have focused on unraveling electron transfer mechanisms between these bacteria and Fe(III)-(oxyhydr)oxides. However, these efforts have been complicated by the fact that these minerals often occur in the micro- to nanosize regime, and in relevant natural environments as well as in the laboratory are subject to aggregation. The nature of the physical interface between the cellular envelope, the outer-membrane cytochromes responsible for facilitating the interfacial electron transfer step, and these complex mineral particulates is thus difficult to probe. Previous studies using whole cells have reported reduction rates that do not correlate with particle size. In the present study we isolate the interaction between the decaheme outer-membrane cytochrome OmcA of Shewanella oneidensis and nanoparticulate hematite, examining the reduction rate as a function of particle size and reaction products through detailed characterization of the electron balance and the structure and valence of iron at particle surfaces. By comparison with abiotic reduction via the smaller molecule ascorbic acid, we show that the reduction rate is systematically controlled by the sterically accessible interfacial contact area between OmcA and hematite in particle aggregates; rates increase once pore throat sizes in aggregates become as large as OmcA. Simultaneous measure of OmcA oxidation against Fe(II) release shows a ratio of 1:10, consistent with a cascade OmcA oxidation mechanism heme by heme. X-ray absorption spectroscopies reveal incipient magnetite on the reacted surfaces of the hematite nanoparticles after reaction. The collective findings establish the importance of accessibility of physical contact between the terminal reductases and iron oxide surfaces, and through apparent consistency of observations help reconcile behavior reported at the larger

  8. Mathematical Modeling of Plate−gap Biosensors with an Outer Porous Membrane

    Directory of Open Access Journals (Sweden)

    Valdas Laurinavicius

    2006-07-01

    Full Text Available A plate−gap model of a porous enzyme doped electrode covered by a porousinert membrane has been proposed and analyzed. The two−dimensional−in−spacemathematical model of the plate−gap biosensors is based on the reaction−diffusionequations containing a nonlinear term related to the Michaelis−Menten kinetics. Usingnumerical simulation of the biosensor action, the influence of the geometry of the outermembrane on the biosensor response was investigated at wide range of analyteconcentrations as well as of the reaction rates. The numerical simulation was carried outusing finite−difference technique. The behavior of the plate−gap biosensors was comparedwith that of a flat electrode deposited with a layer of enzyme and covered with the sameouter membrane.

  9. Evolutionarily evolved discriminators in the 3-TPR domain of the Toc64 family involved in protein translocation at the outer membrane of chloroplasts and mitochondria.

    Science.gov (United States)

    Mirus, Oliver; Bionda, Tihana; von Haeseler, Arndt; Schleiff, Enrico

    2009-08-01

    Transport of polypeptides across membranes is a general and essential cellular process utilised by molecular machines. At least one component of these complexes contains a domain composed of three tetratricopeptide repeat (3-TPR) motifs. We have focussed on the receptor Toc64 to elucidate the evolved functional specifications of its 3-TPR domain. Toc64 is a component of the Toc core complex and functionally replaces Tom70 at the outer membrane of mitochondria in plants. Its 3-TPR domain recognises the conserved C-terminus of precursor-bound chaperones. We built homology models of the 3-TPR domain of chloroplastic Toc64 from different species and of the mitochondrial isoform from Arabidopsis. Guided by modelling, we identified residues essential for functional discrimination of the differently located isoforms to be located almost exclusively on the convex surface of the 3-TPR domain. The only exception is at568Ser/ps557Met, which is positioned in the ligand-binding groove. The functional implications of the homology models are discussed.

  10. Development of a cost-effective vaccine candidate with outer membrane vesicles of a tolA-disrupted Shigella boydii strain.

    Science.gov (United States)

    Mitra, Soma; Sinha, Ritam; Mitobe, Jiro; Koley, Hemanta

    2016-04-04

    Our previous studies on outer membrane vesicles based vaccine development against shigellosis, revealed the inability of Shigella to release significant amount of vesicles naturally, during growth. Disruption of tolA, one of the genes of the Tol-Pal system of Gram negative bacterial membrane, has increased the vesicle release rate of a Shigella boydii type 4 strain to approximately 60% higher. We also noticed the vesicles, released from tolA-disrupted strain captured more OmpA protein and lipopolysaccharide, compared to the vesicles released from its wild type prototype. Six to seven weeks old BALB/c mice, immunized with 25 μg of three oral doses of the vesicles, released by tolA mutant, conferred 100% protection against lethal homologous challenge through nasal route, compared to only 60% protection after the same dose of wild type immunogen. Mice, immunized with the vesicles from tolA-mutant, manifested significant secretion of mucosal IgG and IgA. A sharp and significant response of pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) were also observed in the lung lavage of these groups of mice, within 6h post challenge; but at 24h, these inflammatory cytokines showed the sign of subsidence and the system was taken over by the release of anti-inflammatory cytokines (IL-4 and IL-10). Studies with naïve peritoneal macrophages, proved further, the potency of these vesicles to stimulate nitric oxide and TNF-α, IL-12p70, IL-6 and IL-10 productions in-vitro. The ability of these vesicles to trigger polarization of CD4(+) T cells toward Th1 adaptive immune response, had also been observed along with the presence of anti-inflammatory cytokines in the system. Our study demonstrated, the vesicles from tolA-disrupted Shigella were able to suppress Shigella-mediated inflammation in the host and could balance between inflammation and anti-inflammation, promoting better survival and health of the infected mice. Outer membrane vesicles from tolA-mutant, could be a potential

  11. Collagen-based mechanical anisotropy of the tectorial membrane: implications for inter-row coupling of outer hair cell bundles.

    Directory of Open Access Journals (Sweden)

    Núria Gavara

    Full Text Available BACKGROUND: The tectorial membrane (TM in the mammalian cochlea displays anisotropy, where mechanical or structural properties differ along varying directions. The anisotropy arises from the presence of collagen fibrils organized in fibers of approximately 1 microm diameter that run radially across the TM. Mechanical coupling between the TM and the sensory epithelia is required for normal hearing. However, the lack of a suitable technique to measure mechanical anisotropy at the microscale level has hindered understanding of the TM's precise role. METHODOLOGY/PRINCIPAL FINDINGS: Here we report values of the three elastic moduli that characterize the anisotropic mechanical properties of the TM. Our novel technique combined Atomic Force Microscopy (AFM, modeling, and optical tracking of microspheres to determine the elastic moduli. We found that the TM's large mechanical anisotropy results in a marked transmission of deformations along the direction that maximizes sensory cell excitation, whereas in the perpendicular direction the transmission is greatly reduced. CONCLUSIONS/SIGNIFICANCE: Computational results, based on our values of elastic moduli, suggest that the TM facilitates the directional cooperativity of sensory cells in the cochlea, and that mechanical properties of the TM are tuned to guarantee that the magnitude of sound-induced tip-link stretching remains similar along the length of the cochlea. Furthermore, we anticipate our assay to be a starting point for other studies of biological tissues that require directional functionality.

  12. Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria.

    Science.gov (United States)

    Hooda, Yogesh; Lai, Christine Chieh-Lin; Judd, Andrew; Buckwalter, Carolyn M; Shin, Hyejin Esther; Gray-Owen, Scott D; Moraes, Trevor F

    2016-02-29

    Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism.

  13. Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

    Institute of Scientific and Technical Information of China (English)

    罗依惠; 严杰; 毛亚飞; 李淑萍

    2004-01-01

    Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.

  14. Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

    Institute of Scientific and Technical Information of China (English)

    罗依惠; 严杰; 毛亚飞; 李淑萍

    2004-01-01

    Objective: To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups. The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence, and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972). OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results: All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa. A positive protein fragment with approximately 32 KDa confirmed by Western blot, was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira. Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.

  15. Epidemiological observation on effect of Leptospiral outer membrane vaccine%钩端螺旋体外膜菌苗流行病学效果研究

    Institute of Scientific and Technical Information of China (English)

    程均福; 秦进才; 谢广中; 张锦麟; 张流波; 丁建平; 严有望; 张新炳

    2001-01-01

    Objective To study the safety and effect of Leptospiral outer membrane vaccine. Methods Eighty thousands dosages of Leptospiral outer membrane vaccine were vaccinated in Jingzhou and Shishou city Hubei prevince. Temperaure, side-effects such as Local edema with in 48 hours as well as the incidence of Leptospirsis within a year among those were vaccinated and unvaccinated were observed. Results (1)No any severe side-effect and abnormal reaction, was found, only 2 case suffered from slight fever and local edema which receded in 48 hrs. (2) Effects of Leptospiral outer membrane vaccine were as follows: 2 cases were attacked by lcterohaemorrhagiae in vaccination group and 47 cases in control group, so the protection rate 95.57% and confidence interval (CI) was 85.43%-98.20%. Fifteen hebdomadis cases were found in control group. The protection rate of this vaccine reached 100.00%, CI 77.08%-100.00%. Conclusion Safty and protective effect were well showed when type of bacteria was concordant with that in vaccination district.%目的 证实钩端螺旋体外膜菌苗的安全性和免疫效果。方法 在湖北省荆州市和石首市现场接种钩端螺旋体外膜菌苗80 000人份,观察48 h内体温、局部红肿等副反应和一年内接种钩端螺旋体外膜菌苗者发病情况,同时设对照组。结果 所有钩端螺旋体外膜菌苗接种者未见严重副反应和异常反应,仅2例菌苗接种者有低热和局部红肿,48 h后消失,安全性良好。接种组发生黄疸出血群钩端螺旋体病人2例,对照组发生黄疸出血群钩端螺旋体病人47例,黄疸出血群钩端螺旋体外膜菌苗保护效果95.57%,95%可信限为85.43%~98.20%;对照组发生七日热群钩端螺旋体病人15例,七日热群钩端螺旋体外膜菌苗有效率100.00%,95%可信限下限77.08%。结论 钩端螺旋体外膜菌苗安全性良好,接种与疫区流行菌群一致的钩端螺旋体外膜菌苗,可以取

  16. Chlamydial salpingitis in female guinea pigs receiving oral contraceptives.

    Science.gov (United States)

    Barron, A L; Pasley, J N; Rank, R G; White, H J; Mrak, R E

    1988-01-01

    Female guinea pigs were given daily doses of a combination of oral contraceptive (OC) agents, consisting of mestranol and norethynodrel suspended in sesame oil or distilled H2O, and were infected in the genital tract with the chlamydial agent of guinea pig inclusion conjunctivitis (GPIC). Counts of chlamydial inclusions in cells of vaginal smears collected during infection, showed prolongation and enhancement of infection in OC-treated animals as compared with controls. Appearance of IgG and IgA antibodies to GPIC in genital secretions, as determined by enzyme-linked immunosorbent assay (ELISA), was also delayed in OC-treated animals as compared with controls. OC-treated infected animals were killed on days 15 and 43, and gross pathological evidence for ascending infection culminating in salpingitis was found in all of five and four of five animals, respectively. On the other hand, among untreated infected controls on each sacrifice day, only one of five animals had any evidence for ascending infection. Chlamydiae were detected by light and electron microscopy in fallopian tube tissue collected on day 15 following OC-treatment but not in tissue from control animals.

  17. Loss of outer membrane integrity in Gram-negative bacteria by silver nanoparticles loaded with Camellia sinensis leaf phytochemicals: plausible mechanism of bacterial cell disintegration

    Indian Academy of Sciences (India)

    M SINGH; A K MALLICK; M BANERJEE; R KUMAR

    2016-12-01

    New generation antimicrobial and smart drugs are the needs of the present era in fighting microbial infection and various chronic diseases. Nowadays nanoparticles (NPs) are widely applied in biomedical fields by virtue of their surface modification, which enhances both target selectivity and function. This study is a continuation of our earlier study that demonstrated antimicrobial property of NPs against both Gram-positive and Gramnegative organisms (Goswami et al, 2015). Silver NPs were synthesized using tea leaves (Camellia sinensis) decoction and were characterized using UV-vis spectrophotometry, transmission electron microscopy (TEM) and Fouriertransformed infrared spectroscopy (FTIR). The silver NPs were stable at various environmental conditions. The stability of the particles may be due to various phytochemicals of tea that were bound to the surface of reducedsilver ions as a capping agent. The antimicrobial activity of NP was investigated against three Gram-negative pathogenic bacteria (Shigella dysentriae, Salmonella infestis and Vibrio parahaemolyticus). The outer membrane ofGram-negative bacteria is a lipopolysaccharide (LPS) in nature and provides protection from various stress conditions and antibiotics. But a silver NP destroys its membrane integrity and thus helps in cell killing. Spectral changesconfirmed NP interaction with hydrophobic moiety of LPS. Minimum inhibitory concentrations for S. dysentriae, S. infestis and V. parahaemolyticus were 3.75, 5.25 and 5.25 $\\mu$g ml$^{−1}$, respectively. Inhibition of biofilm formationwas significant with the three bacterial strains. Cytoplasmic leakage from each bacterial strain was also demonstrated on account of NP treatment. The particles demonstrated good biocompatibility. No damage of human buccal mucosal cells was recorded even at concentration of 10 mg ml$^{−1}$. Thus, silver NPs would be potential oral therapeutic molecules against Gram-negative bacteria.

  18. Association of the Cytoplasmic Membrane Protein XpsN with the Outer Membrane Protein XpsD in the Type II Protein Secretion Apparatus of Xanthomonas campestris pv. Campestris

    Science.gov (United States)

    Lee, Hsien-Ming; Wang, Kuan-Cheng; Liu, Yi-Ling; Yew, Hsin-Yan; Chen, Ling-Yun; Leu, Wei-Ming; Chen, David Chanhen; Hu, Nien-Tai

    2000-01-01

    An xps gene cluster composed of 11 open reading frames is required for the type II protein secretion in Xanthomonas campestris pv. campestris. Immediately upstream of the xpsD gene, which encodes an outer membrane protein that serves as the secretion channel by forming multimers, there exists an open reading frame (previously designated ORF2) that could encode a protein of 261 amino acid residues. Its N-terminal hydrophobic region is a likely membrane-anchoring sequence. Antibody raised against this protein could detect in the wild-type strain of X. campestris pv. campestris a protein band with an apparent molecular mass of 36 kDa by Western blotting. Its aberrant slow migration in sodium dodecyl sulfate-polyacrylamide gels might be due to its high proline content. We designated this protein XpsN. By constructing a mutant strain with an in-frame deletion of the chromosomal xpsN gene, we demonstrated that it is required for the secretion of extracellular enzyme by X. campestris pv. campestris. Subcellular fractionation studies indicated that the XpsN protein was tightly associated with the membrane. Sucrose gradient sedimentation followed by immunoblot analysis revealed that it primarily appeared in the cytoplasmic membrane fractions. Immune precipitation experiments indicated that the XpsN protein was coprecipitated with the XpsD protein. In addition, the XpsN protein was co-eluted with the (His)6-tagged XpsD protein from the metal affinity chromatography column. All observations suggested that the XpsN protein forms a stable complex with the XpsD protein. In addition, immune precipitation analysis of the XpsN protein with various truncated XpsD proteins revealed that the C-terminal region of the XpsD protein between residues 650 and 759 was likely to be involved in complex formation between the two. PMID:10692359

  19. Partial Characterisation of Salmonella gallinarum Clinical Isolate and Expression of Its Antigenic Outer Membrane Protein C (OmpC Gene In Planta

    Directory of Open Access Journals (Sweden)

    Ee Leen Pang

    2015-05-01

    Full Text Available Fowl typhoid’s epidemiology and disease intervention have been extensively studied since 1950's owing to its high mortality and morbidity rates. Even up-to-date, outbreaks are incessantly haunting poultry industries of major continents. Salmonella gallinarum, the etiologic agent of fowl typhoid, was used to develop a series of vaccination regime. However, treatments are gradually losing effectiveness due to residual virulence from mutated strains and rapid evolution of multi-drug resistance isolates. Hence, in planta subunit vaccine production is proposed to surpass current limitations. The homotrimeric osmoporin (outer membrane protein C is a potent candidate antigen that confers momentous stimulation of humoral and cell-mediated immune responses in broilers. This research signified the potential development of a plant-expressed OmpC immunogen. The project scope embarked on the identification of S. gallinarum clinical isolate, construction of expression cassette and delivery of constructs into Nicotiana benthamiana via agroinfiltration. The OmpC transcripts and proteins were detected successfully at the molecular weights of ~1.002 kbp and ~35 kDa, respectively. These preliminary findings pave the feasibility of biomanufacturing a safe and cost-effective fowl typhoid vaccine that would confer multi-protection against other significant Salmonella infections attributed to the high sequence homology of the OmpC gene. Speed improvement is demonstrated and transient expression appears to outperform conventional platforms in expediting vaccine production for an emerging pandemic strain.

  20. Outer membrane vesicles from flagellin-deficient Salmonella enterica serovar Typhimurium induce cross-reactive immunity and provide cross-protection against heterologous Salmonella challenge

    Science.gov (United States)

    Liu, Qiong; Liu, Qing; Yi, Jie; Liang, Kang; Hu, Bo; Zhang, Xiangmin; Curtiss, Roy; Kong, Qingke

    2016-01-01

    Outer membrane vesicles (OMVs) isolated from Salmonella Typhimurium are potentially useful for developing subunit vaccines because of high immunogenicity and protective efficacy. However, flagella might remain in OMV pellets following OMV purification, resulting in non-essential immune responses and counteraction of bacterial protective immune responses when developing a vaccine against infection of multiple serotypes Salmonella. In this study, a flagellin-deficient S. Typhimurium mutant was constructed. Lipopolysaccharide profiles, protein profiles and cryo-electron microscopy revealed that there were no significant differences between the wild-type and mutant OMVs, with the exception of a large amount of flagellin in the wild-type OMVs. Neither the wild-type OMVs nor the non-flagellin OMVs were toxic to macrophages. Mice immunized with the non-flagellin OMVs produced high concentrations of IgG. The non-flagellin OMVs elicited strong mucosal antibody responses in mice when administered via the intranasal route in addition to provoking higher cross-reactive immune responses against OMPs isolated from S. Choleraesuis and S. Enteritidis. Both intranasal and intraperitoneal immunization with the non-flagellin OMVs provided efficient protection against heterologous S. Choleraesuis and S. Enteritidis challenge. Our results indicate that the flagellin-deficient OMVs may represent a new vaccine platform that could be exploited to facilitate the production of a broadly protective vaccine. PMID:27698383

  1. Induction of immune response in mice with a DNA vaccine encoding outer membrane protein (omp31) of Brucella melitensis 16M.

    Science.gov (United States)

    Gupta, V K; Rout, P K; Vihan, V S

    2007-06-01

    Brucellosis causes serious economic losses to goat farmers by way of reproductive losses in the form of abortions and stillbirths. Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. In this study, we evaluated the ability of DNA vaccine encoding the omp31 protein of Brucella melitensis 16M to induce cellular and humoral immune responses in mice. We constructed eukaryotic expression vectors called pTargeTomp31, encoding outer membrane protein (omp31) of B. melitensis 16M. pTargeTomp31 was injected intramuscularly three times, at 3-week intervals in groups of mice 6 weeks of age. pTargeTomp31 induced good antibody response in ELISA . pTargeTomp31 elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the omp31 antigen or B. melitensis 16M extract. We also demonstrate that animals immunized with this plasmid elicited a strong and long-lived memory immune response. Furthermore, pTargeTomp31 elicited a typical T-helper 1-dominated immune response in mice, as determined by immunoglobulin G isotype analysis. This vaccine also provided the moderate degree of protection to the mice. This study for the first time focuses on DNA immunization of a gene from B. melitensis. These results may lead to the development of a DNA-based vaccine for the control of brucellosis in goats.

  2. Fine Mapping of the Interaction between C4b-Binding Protein and Outer Membrane Proteins LigA and LigB of Pathogenic Leptospira interrogans.

    Science.gov (United States)

    Breda, Leandro C D; Hsieh, Ching-Lin; Castiblanco Valencia, Mónica M; da Silva, Ludmila B; Barbosa, Angela S; Blom, Anna M; Chang, Yung-Fu; Yung-Fu, Chang; Isaac, Lourdes

    2015-01-01

    The complement system consists of more than 40 proteins that participate in the inflammatory response and in pathogen killing. Complement inhibitors are necessary to avoid the excessive consumption and activation of this system on host cells. Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Pathogenic leptospires are able to escape from complement activation by binding to host complement inhibitors Factor H [FH] and C4b-binding protein (C4BP) while non-pathogenic leptospires are rapidly killed in the presence of fresh serum. In this study, we demonstrate that complement control protein domains (CCP) 7 and 8 of C4BP α-chain interact with the outer membrane proteins LcpA, LigA and LigB from the pathogenic leptospire L. interrogans. The interaction between C4BP and LcpA, LigA and LigB is sensitive to ionic strength and inhibited by heparin. We fine mapped the LigA and LigB domains involved in its binding to C4BP and heparin and found that both interactions are mediated through the bacterial immunoglobulin-like (Big) domains 7 and 8 (LigA7-8 and LigB7-8) of both LigA and LigB and also through LigB9-10. Therefore, C4BP and heparin may share the same binding sites on Lig proteins.

  3. Mechanistic insight into the TH1-biased immune response to recombinant subunit vaccines delivered by probiotic bacteria-derived outer membrane vesicles.

    Science.gov (United States)

    Rosenthal, Joseph A; Huang, Chung-Jr; Doody, Anne M; Leung, Tiffany; Mineta, Kaho; Feng, Danielle D; Wayne, Elizabeth C; Nishimura, Nozomi; Leifer, Cynthia; DeLisa, Matthew P; Mendez, Susana; Putnam, David

    2014-01-01

    Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV's ability to induce self-adjuvanted robust and protective humoral and T(H)1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered "pathogen-like particles" derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion.

  4. The outer membrane protein TolC is required for phytoalexin resistance and virulence of the fire blight pathogen Erwinia amylovora.

    Science.gov (United States)

    Al-Karablieh, Nehaya; Weingart, Helge; Ullrich, Matthias S

    2009-07-01

    Erwinia amylovora causes fire blight on several plant species such as apple and pear, which produce diverse phytoalexins as defence mechanisms. An evolutionary successful pathogen thus must develop resistance mechanisms towards these toxic compounds. The E. amylovora outer membrane protein, TolC, might mediate phytoalexin resistance through its interaction with the multidrug efflux pump, AcrAB. To prove this, a tolC mutant and an acrB/tolC double mutant were constructed. The minimal inhibitory concentrations of diverse antimicrobials and phytoalexins were determined for these mutants and compared with that of a previously generated acrB mutant. The tolC and arcB/tolC mutants were considerably more susceptible than the wild type but showed similar levels as the acrB mutant. The results clearly indicated that neither TolC nor AcrAB significantly interacted with other transport systems during the efflux of the tested toxic compounds. Survival and virulence assays on inoculated apple plants showed that pathogenicity and the ability of E. amylovora to colonize plant tissue were equally impaired by mutations of tolC and acrB/tolC. Our results allowed the conclusion that TolC plays an important role as a virulence and fitness factor of E. amylovora by mediating resistance towards phytoalexins through its exclusive interaction with AcrAB.

  5. Protection against Salmonella typhi infection in mice after immunization with outer membrane proteins isolated from Salmonella typhi 9,12,d, Vi.

    Science.gov (United States)

    Isibasi, A; Ortiz, V; Vargas, M; Paniagua, J; González, C; Moreno, J; Kumate, J

    1988-01-01

    The current studies were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce protection against challenge with the bacteria in mucin. OMPs were isolated as described by Schnaitman (J. Bacteriol. 108:553-556, 1971) and were found to be contaminated with approximately 4% lipopolysaccharide (LPS). Immunization with as little as 30 micrograms of OMPs conferred 100% protection to mice challenged with up to 1,000 50% lethal doses (LD50) of two strains of S. typhi (9,12,d, Vi and Ty2). In addition, 30% protection against challenge with up to 500 LD50 of Salmonella typhimurium was achieved. Immunization with LPS at doses equivalent to those found in the OMPs was considerably inferior to the OMPs in the induction of an immune status. Moreover, LPS was effective only when the challenge was performed with S. typhi 9,12,d, Vi (40% protection to 100 LD50). An antiserum raised in rabbits reacted mainly against the bands of the molecular weights corresponding to the so-called porins contained in the OMP preparation as shown by Western blotting (immunoblotting). This rabbit antiserum protected 100% of mice against challenge with 100 LD50 of either strain of S. typhi and 80% of mice against challenge with the same LD50 of S. typhimurium. These results indicate the usefulness of OMPs in the induction of active immunity against S. typhi in mice. Images PMID:2844676

  6. Immunoblot detection of class-specific humoral immune response to outer membrane proteins isolated from Salmonella typhi in humans with typhoid fever.

    Science.gov (United States)

    Ortiz, V; Isibasi, A; García-Ortigoza, E; Kumate, J

    1989-07-01

    The studies reported here were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce a humoral immune response in humans with typhoid fever. OMPs were isolated with the nonionic detergent Triton X-100 and were found to be contaminated with approximately 4% lipopolysaccharide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns showed protein bands with molecular size ranges from 17 to 70 kilodaltons; the major groups of proteins were those that correspond to the porins and OmpA of gram-negative bacteria. Rabbit antiserum to OMPs or to S. typhi recognized OMPs after absorption with lipopolysaccharide. Sera from patients with typhoid fever contained immunoglobulin M antibodies which reacted with a protein of 28 kilodaltons and immunoglobulin G antibodies which reacted mainly with the porins, as determined by immunoblotting. These results indicate that the porins are the major immunogenic OMPs from S. typhi and that the immune response induced in the infection could be related to the protective status.

  7. Polyphosphate kinases modulate Campylobacter jejuni outer membrane constituents and alter its capacity to invade and survive in intestinal epithelial cells in vitro.

    Science.gov (United States)

    Pina-Mimbela, Ruby; Madrid, Jesús Arcos; Kumar, Anand; Torrelles, Jordi B; Rajashekara, Gireesh

    2015-12-30

    Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Polyphosphate kinases 1 and 2 (PPK1 and PPK2) regulate several cellular processes, including the biosynthesis of the bacterial cell wall. Despite their importance, whether PPK1 and PPK2 modulate the composition of C. jejuni outer membrane constituents (OMCs) and consequently impact its interaction with host cells remains unknown. Our comparative analysis between C. jejuni wild type, Δppk1, and Δppk2 strains showed qualitative and quantitative differences in the total OMC composition among these strains. Importantly, these OMC variations observed on the C. jejuni polyphosphate kinase mutants are directly related to their capacity to invade, survive, and alter the immune response of intestinal epithelial cells in vitro. Specifically, sub-fractionation of the C. jejuni OMC indicated that OMC proteins are uniquely associated with bacterial invasion, whereas C. jejuni OMC proteins, lipids, and lipoglycans are all associated with C. jejuni intracellular survival. This study provides new insights regarding the function of polyphosphate kinases and their role in C. jejuni infection.

  8. Evaluation of recombinant outer membrane protein C based indirect enzyme-linked immunoassay for the detection of Salmonella antibodies in poultry

    Directory of Open Access Journals (Sweden)

    Jinu Manoj

    2015-08-01

    Full Text Available Aim: To evaluate the efficacy of recombinant outer membrane proteinC (rOmpC based enzyme-linked immunoassay (ELISA for the diagnosis of salmonellosis in poultry. Materials and Methods: Three antigens were prepared, and the indirect ELISA was standardized using the antigens and the antiserum raised in chicken against Omp and rOmpC. Sera were collected from a total of 255 apparently healthy field chickens and screened for the presence of Salmonella antibodies by this ELISA. Results: The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Omp revealed major polypeptides at 36, 42 and 52 kDa, and the rOmpC was evident by a single protein band of 43 kDa. The Omp and rOmpC antigen revealed an optimum concentration of 78 and 156 ng, respectively, in the assay, while the whole cell antigen gave an optimum reaction at a concentration of 106 organisms/ml. The test was found to be specific as it did not react with any of the antisera of seven other organisms. The developed ELISA detected Salmonella antibodies from 22 (8.62% samples with rOmpC antigen, while 24 (9.41% samples gave a positive reaction with both Omp and whole cell antigens. Conclusion: We suggest rOmpC based indirect ELISA as a suitable screening tool for serological monitoring of poultry flocks.

  9. DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates.

    Directory of Open Access Journals (Sweden)

    Marcus Thein

    Full Text Available Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

  10. Two outer membrane lipoproteins from Histophilus somni are immunogenic in rabbits and sheep and induce protection against bacterial challenge in mice.

    Science.gov (United States)

    Guzmán-Brambila, Carolina; Rojas-Mayorquín, Argelia E; Flores-Samaniego, Beatriz; Ortuño-Sahagún, Daniel

    2012-11-01

    Histophilus somni is an economically important pathogen of cattle and other ruminants and is considered one of the key components of the bovine respiratory disease (BRD) complex, the leading cause of economic loss in the livestock industry. BRD is a multifactorial syndrome, in which a triad of agents, including bacteria, viruses, and predisposing factors or "stressors," combines to induce disease. Although vaccines against H. somni have been used for many decades, traditional bacterins have failed to demonstrate effective protection in vaccinated animals. Hence, the BRD complex continues to produce strong adverse effects on the health and well-being of stock and feeder cattle. The generation of recombinant proteins may facilitate the development of more effective vaccines against H. somni, which could confer better protection against BRD. In the present study, primers were designed to amplify, clone, express, and purify two recombinant lipoproteins from H. somni, p31 (Plp4) and p40 (LppB), which are structural proteins of the outer bacterial membrane. The results presented here demonstrate, to our knowledge for the first time, that when formulated, an experimental vaccine enriched with these two recombinant lipoproteins generates high antibody titers in rabbits and sheep and exerts a protective effect in mice against septicemia induced by H. somni bacterial challenge.

  11. Outer membrane mutants of Salmonella typhimurium LT2 have lipopolysaccharide-dependent resistance to the bactericidal activity of anaerobic human neutrophils.

    Science.gov (United States)

    Okamura, N; Spitznagel, J K

    1982-06-01

    The capacity of neutrophil polymorphonuclear granulocytes (PMNs) to phagocytize bacteria under anaerobic as well as aerobic conditions afforded the opportunity to compare the bactericidal activities of oxygen-independent and oxygen-dependent antimicrobial mechanisms in human PMNs challenged with Salmonella typhimurium LT2 and its lipopolysaccharide mutants (outer membrane mutants). Anaerobic human PMNs challenged with either opsonized LT2 or serum-treated zymosan failed to produce detectable superoxide anion (O2-) or to reduce nitroblue tetrazolium, although aerobic PMNs readily produced O2- in response to such challenge. Anaerobic PMNs killed these bacteria in an ordered fashion that appeared to be dependent on their lipopolysaccharide chemotype. As the carbohydrate content of the mutant lipopolysaccharide decreased, the bacteria became less resistant to the oxygen-independent bactericidal activity. The results resembled the ordered resistance to oxygen-independent killing observed with LT2 and its mutants in PMN-free systems with PMN granule proteins. Studies on the kinetics of killing showed these to be less rapid in anaerobic as compared with aerobic conditions. Opsonization increased the rate of phagocytosis, but such factors as opsonization and the rate of phagocytosis did not appear to affect intraleukocytic bactericidal capacity in that the resultant proportion of bacteria remaining viable after ingestion was similar regardless of which serum was used (normal serum, C6-deficient serum, C8-deficient serum, or no serum at all). The results are consistent with an active and substantial participation by oxygen-independent systems in the antimicrobial effects of neutrophils.

  12. Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA–HasR from Serratia marcescens

    Energy Technology Data Exchange (ETDEWEB)

    Huché, Frédéric, E-mail: huche@pasteur.fr [Fachbereich Biologie, Universität Konstanz, 78457 Konstanz (Germany); Unité des Membranes Bactériennes, CNRS URA 2172, Département de Microbiologie Fondamentale et Médicale, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris CEDEX 15 (France); Delepelaire, Philippe; Wandersman, Cécile [Unité des Membranes Bactériennes, CNRS URA 2172, Département de Microbiologie Fondamentale et Médicale, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris CEDEX 15 (France); Welte, Wolfram [Fachbereich Biologie, Universität Konstanz, 78457 Konstanz (Germany)

    2006-01-01

    The expression, purification, and crystallization in space group P2{sub 1}2{sub 1}2{sub 1} of the complex HasA-HasR from S. marcescens are reported. Diffraction data have been collected and processed to 6.8 Å. Serratia marcescens is able to acquire iron using its haem-acquisition system (‘has’), which contains an outer membrane receptor HasR and a soluble haemophore HasA. After secretion, HasA binds free haem in the extracellular medium or extracts it from haemoproteins and delivers it to the receptor. Here, the crystallization of a HasA–HasR complex is reported. HasA and HasR have been overexpressed in Escherichia coli and the complex formed and crystallized. Small platelets and bunches of needles of dimensions 0.01 × 0.1 × 1 mm were obtained. A native data set has been collected to 6.8 Å.

  13. Biogenesis of outer membrane vesicles in Serratia marcescens is thermoregulated and can be induced by activation of the Rcs phosphorelay system.

    Science.gov (United States)

    McMahon, Kenneth J; Castelli, Maria E; García Vescovi, Eleonora; Feldman, Mario F

    2012-06-01

    Outer membrane vesicles (OMVs) have been identified in a wide range of bacteria, yet little is known of their biogenesis. It has been proposed that OMVs can act as long-range toxin delivery vectors and as a novel stress response. We have found that the formation of OMVs in the gram-negative opportunistic pathogen Serratia marcescens is thermoregulated, with a significant amount of OMVs produced at 22 or 30°C and negligible quantities formed at 37°C under laboratory conditions. Inactivation of the synthesis of the enterobacterial common antigen (ECA) resulted in a hypervesiculation phenotype, supporting the hypothesis that OMVs are produced in response to stress. We demonstrate that the phenotype can be reversed to wild-type (WT) levels upon the loss of the Rcs phosphorelay response regulator RcsB, but not RcsA, suggesting a role for the Rcs phosphorelay in the production of OMVs. MS fingerprinting of the OMVs provided evidence of cargo selection within wild-type cells, suggesting a possible role for Serratia OMVs in toxin delivery. In addition, OMV-associated cargo proved toxic upon injection into the haemocoel of Galleria mellonella larvae. These experiments demonstrate that OMVs are the result of a regulated process in Serratia and suggest that OMVs could play a role in virulence.

  14. Enhancement of protective immunity in European eel(Anguilla anguilla)against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein

    Institute of Scientific and Technical Information of China (English)

    Ruizhang Guan; Jing Xiong; Wenshu Huang; Songlin Guo

    2011-01-01

    To develop a vaccine,which can simultaneously prevent the diseases caused by various pathogenic bacteria in fish,we try to find a conserved outer membrane protein(OMP)antigen from different bacterial pathogens.In this study,an OMP fragment of 747 by(named as Omp-G),whichwas highly conserved in seven Aeromonas OMP sequences from the NCBI database,was amplified by PCR from one Aeromonas sobria strain(1110)and two Aeromonas hydrophila strains(1327 and B33)with the designed specific primers.The sequence was cloned into pGEX-2T(6 × His-tag)vector,expressed in Escherichia coli system,and then the recombinant protein(named as rOmp-G)was purified with nickel chelating affinity chromatography.The purified romp-G showed a good immunogenicity in rabbits and well-conserved characteristics in these three pathogens by enzyme-linked immunosorbed assay.Furthermore,the rOmp-G also showed good immunogenicity in eels(Anguilla anguilla)for eliciting significantly increased specific antibodies(P<0.01),and providing higher protection efficiencies(P<0.05)after the pathogens challenge.The values of the relative percent survival in eels were 70% and 50% for two A.hydrophila strain challenge,and 75% for A.sobria strain challenge.This is the first report of a potential vaccination in eels that simultaneously provide protectiveness against different Aeromonas pathogens with a conserved partial OMP.

  15. Expression and Comparative Analysis of Genes Encoding Outer Membrane Proteins LipL21, LipL32 and OmpL1 in Epidemic Leptospires

    Institute of Scientific and Technical Information of China (English)

    Xiang-Yan ZHANG; Xiao-Kui GUO; Yang YU; Ping HE; Yi-Xuan ZHANG; Bao-Yu HU; Yang YANG; Yi-Xin NIE; Xiu-Gao JIANG; Guo-Ping ZHAO

    2005-01-01

    Leptospiral outer membrane proteins (OMPs) are highly conserved in different species, and play an essential role in the development of new immunoprotection and serodiagnosis strategies. The genes encoding LipL21, LipL32 and OmpL1 were cloned from the complete genome sequence of Leptospira interrogans serovar lai strain Lai and expressed in vitro. Sequence comparison analysis revealed that the three genes were highly conserved among distinct epidemic leptospires, including three major epidemic species Leptospira interrogans, Leptospira borgpetersenii and Leptospira weilii, in China. Immunoblot analysis was further performed to scrutinize 15 epidemic Leptospira reference strains using the antisera of the recombinant OMPs. Both immunoblot assay and reverse transcription-polymerase chain reaction demonstrated that these three OMPs were conservatively expressed in pathogenic L. interrogans strains and other pathogenic leptospires.Additionally, the use of these recombinant OMPs as antigens in enzyme-linked immunosorbent assay (ELISA)for serodiagnosis of leptospirosis was evaluated. The recombinant LipL32 and OmpL1 proteins showed a high degree of ELISA reactivity with sera from patients infected with L. interrogans strain Lai and other pathogenic leptospires. These results may contribute to the identification of candidates for broad-range vaccines and immunodiagnostic antigens in further research.

  16. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain.

    Science.gov (United States)

    de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A; Enghild, Jan J; Thøgersen, Ida B; Gao, Jinlong; Kwan, Ann H; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F Xavier; Nguyen, Ky-Anh; Potempa, Jan

    2016-03-23

    In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.

  17. Immunogenicity and protective efficacy of the recombinant Pasteurella lipoprotein E and outer membrane protein H from Pasteurella multocida A:3 in mice.

    Science.gov (United States)

    Okay, Sezer; Özcengiz, Erkan; Gürsel, Ihsan; Özcengiz, Gülay

    2012-12-01

    Pasteurella multocida serotype A:3 is a Gram-negative bacterial pathogen, one of the causative agents of shipping fever of cattle. In this study, outer membrane protein H (ompH) and Pasteurella lipoprotein E (plpE) genes were cloned and plpEC-ompH fusion was constructed and expressed in Escherichia coli. Recombinant PlpE, OmpH and PlpEC-OmpH fusion proteins were purified and formulated with oil-based and oil-based CpG ODN adjuvants. Antibody responses in mice vaccinated with recombinant PlpE and PlpEC-OmpH proteins formulated with both adjuvants were significantly (pmultocida A:3. The recombinant proteins PlpE and PlpEC-OmpH fusion conferred 100% protection when formulated with oil-based CpG ODN while the protectivity was found to be 80% and 60%, respectively when only oil-based adjuvant was used in respective formulations. These findings indicated that the recombinant PlpE or PlpEC-OmpH fusion proteins formulated with oil-based CpG ODN adjuvant are possible acellular vaccine candidates against shipping fever.

  18. Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia.

    Science.gov (United States)

    Bertot, Gustavo M; Restelli, Marcela A; Galanternik, Laura; Aranibar Urey, Rene C; Valvano, Miguel A; Grinstein, Saúl

    2007-06-01

    Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis.

  19. Nasal Immunization with Burkholderia multivorans Outer Membrane Proteins and the Mucosal Adjuvant Adamantylamide Dipeptide Confers Efficient Protection against Experimental Lung Infections with B. multivorans and B. cenocepacia▿

    Science.gov (United States)

    Bertot, Gustavo M.; Restelli, Marcela A.; Galanternik, Laura; Aranibar Urey, Rene C.; Valvano, Miguel A.; Grinstein, Saúl

    2007-01-01

    Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis. PMID:17296759

  20. Intranasal Vaccination with Recombinant Outer Membrane Protein CD and Adamantylamide Dipeptide as the Mucosal Adjuvant Enhances Pulmonary Clearance of Moraxella catarrhalis in an Experimental Murine Model▿

    Science.gov (United States)

    Becker, Pablo D.; Bertot, Gustavo M.; Souss, David; Ebensen, Thomas; Guzmán, Carlos A.; Grinstein, Saúl

    2007-01-01

    Moraxella catarrhalis causes acute otitis media in children and lower respiratory tract infections in adults and elderly. In children the presence of antibodies against the highly conserved outer membrane protein CD correlates with protection against infection, suggesting that this protein may be useful as a vaccine antigen. However, native CD is difficult to purify, and it is still unclear if recombinant CD (rCD) is a valid alternative. We performed a side-by-side comparison of the immunogenicities and efficacies of vaccine formulations containing native CD and rCD with adamantylamide dipeptide as the mucosal adjuvant. Intranasal vaccination of mice stimulated the production of high CD-specific antibody titers in sera and of secretory immunoglobulin A in mucosal lavages, which cross-recognized both antigens. While vaccination with native CD increased the number of interleukin-2 (IL-2)- and gamma interferon-producing cells, rCD mainly stimulated IL-4-secreting cells. Nevertheless, efficient bacterial clearance was observed in the lungs of challenged mice receiving native CD and in the lungs of challenged mice receiving rCD (96% and 99%, respectively). Thus, rCD is a promising candidate for incorporation in vaccine formulations for use against M. catarrhalis. PMID:17101651

  1. Intranasal vaccination with recombinant outer membrane protein CD and adamantylamide dipeptide as the mucosal adjuvant enhances pulmonary clearance of Moraxella catarrhalis in an experimental murine model.

    Science.gov (United States)

    Becker, Pablo D; Bertot, Gustavo M; Souss, David; Ebensen, Thomas; Guzmán, Carlos A; Grinstein, Saúl

    2007-04-01

    Moraxella catarrhalis causes acute otitis media in children and lower respiratory tract infections in adults and elderly. In children the presence of antibodies against the highly conserved outer membrane protein CD correlates with protection against infection, suggesting that this protein may be useful as a vaccine antigen. However, native CD is difficult to purify, and it is still unclear if recombinant CD (rCD) is a valid alternative. We performed a side-by-side comparison of the immunogenicities and efficacies of vaccine formulations containing native CD and rCD with adamantylamide dipeptide as the mucosal adjuvant. Intranasal vaccination of mice stimulated the production of high CD-specific antibody titers in sera and of secretory immunoglobulin A in mucosal lavages, which cross-recognized both antigens. While vaccination with native CD increased the number of interleukin-2 (IL-2)- and gamma interferon-producing cells, rCD mainly stimulated IL-4-secreting cells. Nevertheless, efficient bacterial clearance was observed in the lungs of challenged mice receiving native CD and in the lungs of challenged mice receiving rCD (96% and 99%, respectively). Thus, rCD is a promising candidate for incorporation in vaccine formulations for use against M. catarrhalis.

  2. Chlamydial infections of fish: diverse pathogens and emerging causes of disease in aquaculture species.

    Science.gov (United States)

    Stride, M C; Polkinghorne, A; Nowak, B F

    2014-05-14

    Chlamydial infections of fish are emerging as an important cause of disease in new and established aquaculture industries. To date, epitheliocystis, a skin and gill disease associated with infection by these obligate intracellular pathogens, has been described in over 90 fish species, including hosts from marine and fresh water environments. Aided by advances in molecular detection and typing, recent years have seen an explosion in the description of these epitheliocystis-related chlamydial pathogens of fish, significantly broadening our knowledge of the genetic diversity of the order Chlamydiales. Remarkably, in most cases, it seems that each new piscine host studied has revealed the presence of a phylogenetically unique and novel chlamydial pathogen, providing researchers with a fascinating opportunity to understand the origin, evolution and adaptation of their traditional terrestrial chlamydial relatives. Despite the advances in this area, much still needs to be learnt about the epidemiology of chlamydial infections in fish if these pathogens are to be controlled in farmed environments. The lack of in vitro methods for culturing of chlamydial pathogens of fish is a major hindrance to this field. This review provides an update on our current knowledge of the taxonomy and diversity of chlamydial pathogens of fish, discusses the impact of these infections on the health, and highlights further areas of research required to understand the biology and epidemiology of this important emerging group of fish pathogens of aquaculture species.

  3. Helicobacter pylori ATCC 43629/NCTC 11639 outer membrane vesicles (OMVs from biofilm and planktonic phase associated with extracellular DNA (eDNA.

    Directory of Open Access Journals (Sweden)

    Rossella eGrande

    2015-12-01

    Full Text Available Helicobacter pylori persistence is associated to its capability of developing biofilms as a response to environmental stress and changes. Extracellular DNA (eDNA is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances and/or Outer Membrane Vesicles (OMVs, which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori biofilm (bOMVs and its planktonic phase (pOMVs and to characterize the physical-chemical properties of bOMVs and pOMVs. The presence of eDNA in bOMVs and pOMVs was carried out using a DNase I-gold complex and Transmission Electron Microscope analysis (TEM. bOMVs and pOMVs were further isolated and physical-chemical characterized using the dynamic light scattering (DLS analysis. The eDNA associated to OMVs was detected and quantified by using PicoGreen assay and spectrophotometer, while its extraction was performed through a DNA Kit. The TEM images showed that the eDNA was mainly isolated and identified on OMVs-membrane surface; while the PicoGreen staining showed a 4-fold increase of dsDNA in bOMVs compared to pOMVs. The eDNA extracted from OMVs was visualized by using gel electrophoresis. The DLS analysis demonstrated that H. pylori generate vesicles, both in its planktonic and biofilm phenotypes, with sizes in the nanometer scales and a broad size distribution. The DLS aggregation study of H. pylori OMVs demonstrated that eDNA may play a role in the OMVs aggregation, particularly for biofilm phenotype. The eDNA associated with vesicle membrane can affect the DNase I activity on H. pylori biofilms. OMVs derived from H. pylori ATCC 43629/NCTC 11639, particularly its biofilm phenotype, may play a structural role by preventing eDNA degradation by nucleases and provide a bridging function between eDNA strands.

  4. The Periplasmic Chaperone Network of Campylobacter jejuni: Evidence that SalC (Cj1289) and PpiD (Cj0694) Are Involved in Maintaining Outer Membrane Integrity

    Science.gov (United States)

    Taylor, Aidan J.; Zakai, Shadi A. I.; Kelly, David J.

    2017-01-01

    The outer membrane (OM) of Gram-negative pathogenic bacteria is a key structure in host–pathogen interactions that contains a plethora of proteins, performing a range of functions including adhesion, nutrient uptake, export of effectors and interaction with innate and adaptive components of the immune system. In addition, the OM can exclude drugs and thus contribute to antimicrobial resistance. The OM of the food-borne pathogen Campylobacter jejuni contains porins, adhesins and other virulence factors that must be specifically localized to this membrane, but the protein sorting mechanisms involved are only partially understood. In particular, chaperones are required to ferry OM proteins across the periplasm after they emerge from the Sec translocation system. The SurA-related chaperone PEB4 (Cj0596) is the only protein with a proven role in OM biogenesis and integrity in C. jejuni. In this work, we have constructed a set of isogenic deletion mutants in genes encoding both known and predicted chaperones (cj0596, cj0694, cj1069, cj1228c, and cj1289) using NCTC 11168H as the parental strain. These mutants were characterized using a range of assays to determine effects on growth, agglutination, biofilm formation, membrane permeability and hydrophobicity. We focused on Cj1289 and Cj0694, which our previous work suggested possessed both chaperone and peptidyl-proyl cis/trans isomerase (PPIase) domains. Mutants in either cj1289 or cj0694 showed growth defects, increased motility, agglutination and biofilm formation and severe OM permeability defects as measured by a lysozyme accessibility assay, that were comparable to those exhibited by the isogenic peb4 mutant. 2D-gel comparisons showed a general decrease in OM proteins in these mutants. We heterologously overproduced and purified Cj0694 and obtained evidence that this protein was an active PPIase, as judged by its acceleration of the refolding rate of reduced and alkylated ribonuclease T1 and that it also possessed

  5. The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H

    Directory of Open Access Journals (Sweden)

    Rauna eRiva

    2015-02-01

    Full Text Available The virulence factor PgtE is an outer membrane protease (omptin of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e. rough LPS, as observed e.g. in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B and H (H, key proteins controlling formation and inactivation of the complement protein C3b and thereby the activity of the complement system. S. enterica serovar Typhimurium or omptin-expressing recombinant E. coli bacteria were incubated with purified human complement proteins or recombinant H fragments. PgtE cleaved both B and H, whereas its close homolog Pla of Yersinia pestis cleaved only H. H was cleaved at both N- and C-termini, while the central region resisted proteolysis. Because of multiple effects of PgtE on complement components (cleavage of C3, C3b, B and H we assessed its effect on the opsonophagocytosis of Salmonella. In human serum, C3 cleavage was dependent on proteolytically active PgtE. Human neutrophils interacted less with serum-opsonized FITC-stained S. enterica 14028R than with the isogenic ΔpgtE strain, as analyzed by flow cytometry. In conclusion, cleavage of B and H by PgtE, together with C3 cleavage, affects the C3-mediated recognition of S. enterica by human neutrophils, thus thwarting the immune protection against Salmonella.

  6. The Bradyrhizobium japonicum fegA gene encodes an iron-regulated outer membrane protein with similarity to hydroxamate-type siderophore receptors.

    Science.gov (United States)

    LeVier, K; Guerinot, M L

    1996-12-01

    Iron is important in the symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, yet little is known about rhizobial iron acquisition strategies. Analysis of outer membrane proteins (OMPs) from B. japonicum 61A152 identified three iron-regulated OMPs in the size range of several known receptors for Fe(III)-scavenging siderophores. One of the iron-regulated proteins, FegA, was purified and microsequenced, and a reverse genetics approach was used to clone a fegA-containing DNA fragment. Sequencing of this fragment revealed a single open reading frame of 750 amino acids. A putative N-terminal signal sequence of 14 amino acids which would result in a mature protein of 736 amino acids with a molecular mass of 80,851 Da was predicted. FegA shares significant amino acid similarity with several Fe(III)-siderophore receptors from gram-negative bacteria and has greater than 50% amino acid similarity and 33% amino acid identity with two [corrected] bacterial receptors for hydroxamate-type Fe(III)-siderophores. A dendrogram describing total inferred sequence similarity among 36 TonB-dependent OMPs was constructed; FegA grouped with Fe(III)-hydroxamate receptors. The transcriptional start site of fegA was mapped by primer extension analysis, and a putative Fur-binding site was found in the promoter. Primer extension and RNA slot blot analysis demonstrated that fegA was expressed only in cells grown under iron-limiting conditions. This is the first report of the cloning of a gene encoding a putative Fe(III)-siderophore receptor from nitrogen-fixing rhizobia.

  7. Using Major Outer Membrane Protein Typing as an Epidemiological Tool To Investigate Outbreaks Caused by Milk-Borne Campylobacter jejuni Isolates in California

    Science.gov (United States)

    Mandrell, Robert E.; Yuan, Jean; Bates, Anna; Manalac, Rosa; Mohle-Boetani, Janet; Kimura, Akiko; Lidgard, Janice; Miller, William G.

    2013-01-01

    We describe using major outer membrane protein (MOMP) typing as a screen to compare the Campylobacter jejuni porA gene sequences of clinical outbreak strains from human stool with the porA sequences of dairy farm strains isolated during two milk-borne campylobacteriosis outbreak investigations in California. The genetic relatedness of clinical and environmental strains with identical or closely related porA sequences was confirmed by multilocus sequence typing and pulsed-field gel electrophoresis analysis. The first outbreak involved 1,644 C. jejuni infections at 11 state correctional facilities and was associated with consumption of pasteurized milk supplied by an on-site dairy (dairy A) at a prison in the central valley. The second outbreak involved eight confirmed and three suspect C. jejuni cases linked to consumption of commercial raw milk and raw chocolate colostrum at another central valley dairy (dairy B). Both dairies bottled fluid milk on the farm and distributed the finished product to off-site locations. Altogether, C. jejuni was isolated from 7 of 15 (46.7%) bovine fecal, 12 of 20 (60%) flush alley water, and 1 of 20 (5%) lagoon samples collected on dairy A. At dairy B, C. jejuni was cultured from 9 of 26 (34.6%) bovine fecal samples. Environmental strains indistinguishable from the clinical outbreak strains were found in five flush alley water samples (dairy A) and four bovine fecal samples (dairy B). The findings demonstrate that MOMP typing is a useful tool to triage environmental isolates prior to conducting more labor-intensive molecular typing methods. PMID:23115263

  8. Characterization of the key antigenic components and pre-clinical immune responses to a meningococcal disease vaccine based on Neisseria lactamica outer membrane vesicles.

    Science.gov (United States)

    Finney, Michelle; Vaughan, Thomas; Taylor, Stephen; Hudson, Michael J; Pratt, Catherine; Wheeler, Jun X; Vipond, Caroline; Feavers, Ian; Jones, Christopher; Findlow, Jamie; Borrow, Ray; Gorringe, Andrew

    2008-01-01

    Serogroup B strains are now responsible for over 80% of meningococcal disease in the UK and no suitable vaccine is available that confers universal protection against all serogroup B strains. Neisseria lactamica shares many antigens with the meningococcus, except capsule and the surface protein PorA. Many of these antigens are thought to be responsible for providing cross-protective immunity to meningococcal disease. We have developed an N. lactamica vaccine using methods developed for meningococcal outer membrane vesicle (OMV) vaccines. The major antigenic components were identified by excision of 11 major protein bands from an SDS-PAGE gel, followed by mass spectrometric identification. These bands contained at least 22 proteins identified from an unassembled N. lactamica genome, 15 of which having orthologues in published pathogenic Neisseria genomes. Western blotting revealed that most of these bands were immunogenic, and antibodies to these proteins generally cross-reacted with N. meningitidis proteins. Sera from mice and rabbits immunized with either N. lactamica or N. meningitidis OMVs produced comparable cross-reactive ELISA titres against OMVs prepared from a panel of diverse meningococcal strains. Mice immunized with either N. meningitidis or N. lactamica OMVs showed no detectable serum bactericidal activity against the panel of target strains except N. meningitidis OMV sera against the homologous strain. Similarly, rabbit antisera to N. lactamica OMVs elicited little or no bactericidal antibodies against the panel of serogroup B meningococcal strains. However, such antisera did mediate opsonophagocytosis, suggestingthat this may did mediate opsonophagocytosis, suggesting that this may be a mechanism by which this vaccine protects in a mouse model of meningococcal bacteraemia.

  9. TprC/D (Tp0117/131), a trimeric, pore-forming rare outer membrane protein of Treponema pallidum, has a bipartite domain structure.

    Science.gov (United States)

    Anand, Arvind; Luthra, Amit; Dunham-Ems, Star; Caimano, Melissa J; Karanian, Carson; LeDoyt, Morgan; Cruz, Adriana R; Salazar, Juan C; Radolf, Justin D

    2012-05-01

    Identification of Treponema pallidum rare outer membrane proteins (OMPs) has been a longstanding objective of syphilis researchers. We recently developed a consensus computational framework that employs a battery of cellular localization and topological prediction tools to generate ranked clusters of candidate rare OMPs (D. L. Cox et al., Infect. Immun. 78:5178-5194, 2010). TP0117/TP0131 (TprC/D), a member of the T. pallidum repeat (Tpr) family, was a highly ranked candidate. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning, and liposome incorporation confirmed that full-length, recombinant TprC (TprC(Fl)) forms a β-barrel capable of integrating into lipid bilayers. Moreover, TprC(Fl) increased efflux of terbium-dipicolinic acid complex from large unilamellar vesicles and migrated as a trimer by blue-native PAGE. We found that in T. pallidum, TprC is heat modifiable, trimeric, expressed in low abundance, and, based on proteinase K accessibility and opsonophagocytosis assays, surface exposed. From these collective data, we conclude that TprC is a bona fide rare OMP as well as a functional ortholog of Escherichia coli OmpF. We also discovered that TprC has a bipartite architecture consisting of a soluble N-terminal portion (TprC(N)), presumably periplasmic and bound directly or indirectly to peptidoglycan, and a C-terminal β-barrel (TprC(C)). Syphilitic rabbits generate antibodies exclusively against TprC(C), while secondary syphilis patients fail to mount a detectable antibody response against either domain. The syphilis spirochete appears to have resolved a fundamental dilemma arising from its extracellular lifestyle, namely, how to enhance OM permeability without increasing its vulnerability to the antibody-mediated defenses of its natural human host.

  10. Expression and purification of the recombinant outer membrane protein Tp0453 of Treponema pallidum and its characterization of immuno-competence

    Institute of Scientific and Technical Information of China (English)

    SHUANGQUANLIU; YIMOUWU; FEIJUNZHAO; TIEBINGZENG; WEIGUOYIN

    2005-01-01

    To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T. pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E. coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the presentstudy, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T. pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2 %. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1:1280 after 3 successive doses of immunization.These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T. pallidum infections.

  11. Activation of immune and defense responses in the intestinal mucosa by outer membrane vesicles of commensal and probiotic Escherichia coli strains

    Directory of Open Access Journals (Sweden)

    Maria José eFábrega

    2016-05-01

    Full Text Available The influence of microbiota in human health is well known. Imbalances in microbiome structure have been linked to several diseases. Modulation of microbiota composition through probiotic therapy is an attempt to harness the beneficial effects of commensal microbiota. Although there is wide knowledge of the responses induced by gut microbiota, the microbial factors that mediate these effects are not well known. Gram-negative bacteria release outer membrane vesicles (OMVs as a secretion mechanism of microbial factors, which have an important role in intercellular communication. Here, we investigated whether OMVs from the probiotic Escherichia coli strain Nissle 1917 or the commensal E. coli strain ECOR12 trigger immune responses in various cellular models: (i peripheral blood mononuclear cells (PBMCs as a model of intestinal barrier disruption, (ii apical stimulation of Caco-2/PMBCs co-culture as a model of intact intestinal mucosa, and (iii colonic mucosa explants as an ex vivo model. Stimulations with bacterial lysates were also performed. Whereas both OMVs and lysates activated expression and secretion of several cytokines and chemokines in PBMCs, only OMVs induced basolateral secretion and mRNA upregulation of these mediators in the co-culture model. We provide evidence that OMVs are internalized in polarized Caco-2 cells. The activated epithelial cells elicit a response in the underlying immunocompetent cells. The OMVs effects were corroborated in the ex vivo model. This experimental study shows that OMVs are an effective strategy used by beneficial gut bacteria to communicate with and modulate host responses, activating signaling events through the intestinal epithelial barrier.

  12. Development and comparative evaluation of a plate enzyme-linked immunosorbent assay based on recombinant outer membrane antigens Omp28 and Omp31 for diagnosis of human brucellosis.

    Science.gov (United States)

    Tiwari, Sapana; Kumar, Ashu; Thavaselvam, Duraipandian; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

    2013-08-01

    Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and

  13. Transcriptional regulation of the outer membrane porin gene ompW reveals its physiological role during the transition from the aerobic to the anaerobic lifestyle of Escherichia coli

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    Minfeng eXiao

    2016-05-01

    Full Text Available Understanding bacterial physiology relies on elucidating the regulatory mechanisms and cellular functions of those differentially expressed genes in response to environmental changes. A widespread Gram-negative bacterial outer membrane protein OmpW has been implicated in the adaptation to stresses in various species. It is recently found to be present in the regulon of the global anaerobic transcription factor FNR and ArcA in E. coli. However, little is known about the physiological implications of this regulatory disposition. In this study, we demonstrate that transcription of ompW is indeed mediated by a series of global regulators involved in the anaerobiosis of E. coli. We show that FNR can both activate and repress the expression of ompW through its direct binding to two distinctive sites, -81.5 and -126.5 bp respectively, on ompW promoter. ArcA also participates in repression of ompW under anaerobic condition, but in an FNR dependent manner. Additionally, ompW is also subject to the regulation by CRP and NarL which senses the availability and types of carbon sources and respiration electron acceptors in the environment respectively, implying a role of OmpW in the carbon and energy metabolism of E. coli during its anaerobic adaptation. Molecular docking reveals that OmpW can bind fumarate, an alternative electron acceptor in anaerobic respiration, with sufficient affinity. Moreover, supplement of fumarate or succinate which belongs to the C4-dicarboxylates family of metabolite, to E. coli culture rescues OmpW-mediated colicin S4 killing. Taken together, we propose that OmpW is involved in anaerobic carbon and energy metabolism to mediate the transition from aerobic to anaerobic lifestyle in E. coli.

  14. Flux control exerted by mitochondrial outer membrane carnitine palmitoyltransferase over beta-oxidation, ketogenesis and tricarboxylic acid cycle activity in hepatocytes isolated from rats in different metabolic states.

    Science.gov (United States)

    Drynan, L; Quant, P A; Zammit, V A

    1996-08-01

    The Flux Control Coefficients of mitochondrial outer membrane carnitine palmitoyltransferase (CPT I) with respect to the overall rates of beta-oxidation, ketogenesis and tricarboxylic acid cycle activity were measured in hepatocytes isolated from rats in different metabolic states (fed, 24 h-starved, starved-refed and starved/insulin-treated). These conditions were chosen because there is controversy as to whether, when significant control ceases to be exerted by CPT I over the rate of fatty oxidation [Moir and Zammit (1994) Trends Biochem. Sci. 19, 313-317], this is transferred to one or more steps proximal to acylcarnitine synthesis (e.g. decreased delivery of fatty acids to the liver) or to the reaction catalysed by mitochondrial 3-hydroxy-3-methyl-glutaryl-CoA synthase [Hegardt (1995) Biochem. Soc. Trans. 23, 486-490]. Therefore isolated hepatocytes were used in the present study to exclude the involvement of changes in the rate of delivery of non-esterified fatty acids (NEFA) to the liver, such as occur in vivo, and to ascertain whether, under conditions of constant supply of NEFA, CPT I retains control over the relevant fluxes of fatty acid oxidation to ketones and carbon dioxide, or whether control is transferred to another (intrahepatocytic) site. The results clearly show that the Flux Control Coefficients of CPT I with respect to overall beta-oxidation and ketogenesis are very high under all conditions investigated, indicating that control is not lost to another intrahepatic site during the metabolic transitions studied. The control of CPT I over tricarboxylic acid cycle activity was always very low. The significance of these findings for the integration of fatty acid and carbohydrate metabolism in the liver is discussed.

  15. Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

    Science.gov (United States)

    Fulcher, Robert A; Cole, Leah E; Janowicz, Diane M; Toffer, Kristen L; Fortney, Kate R; Katz, Barry P; Orndorff, Paul E; Spinola, Stanley M; Kawula, Thomas H

    2006-05-01

    Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

  16. A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8.

    Science.gov (United States)

    Ahlstrand, Tuuli; Tuominen, Heidi; Beklen, Arzu; Torittu, Annamari; Oscarsson, Jan; Sormunen, Raija; Pöllänen, Marja T; Permi, Perttu; Ihalin, Riikka

    2017-02-17

    Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI(-) mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

  17. Roles of the outer membrane protein AsmA of Salmonella enterica in the control of marRAB expression and invasion of epithelial cells.

    Science.gov (United States)

    Prieto, Ana I; Hernández, Sara B; Cota, Ignacio; Pucciarelli, M Graciela; Orlov, Yuri; Ramos-Morales, Francisco; García-del Portillo, Francisco; Casadesús, Josep

    2009-06-01

    A genetic screen for suppressors of bile sensitivity in DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the Escherichia coli asmA gene. Disruption of asmA suppressed bile sensitivity also in phoP and wec mutants of S. enterica and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of marA mRNA were found in asmA, asmA dam, asmA phoP, and asmA wec strains of S. enterica, suggesting that lack of AsmA activates expression of the marRAB operon. Hence, asmA mutations may enhance bile resistance by inducing gene expression changes in the marRAB-controlled Mar regulon. In silico analysis of AsmA structure predicted the existence of one transmembrane domain. Biochemical analysis of subcellular fractions revealed that the asmA gene of S. enterica encodes a protein of approximately 70 kDa located in the outer membrane. Because AsmA is unrelated to known transport and/or efflux systems, we propose that activation of marRAB in asmA mutants may be a consequence of envelope reorganization. Competitive infection of BALB/c mice with asmA(+) and asmA isogenic strains indicated that lack of AsmA attenuates Salmonella virulence by the oral route but not by the intraperitoneal route. Furthermore, asmA mutants showed a reduced ability to invade epithelial cells in vitro.

  18. BIOCHEMICAL AND HEMATOLOGICAL ALTERATIONS IN MICE INOCULATED WITH OUTER MEMBRANE PROTEIN, LIPOPOLYSACCHARIDES AND WHOLE CELLS OF PASTEURELLA MULTOCIDA TYPE B: 2

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    Faez Firdaus Jesse Abdullah

    2013-01-01

    Full Text Available Haemorrhagic septicaemia in cattle and buffaloes is an economically important livestock disease in Asia including Malaysia. Therefore, the aim of this study was to investigate the biochemical and hematological alterations in mice model inoculated with outer membrane protein, lipopolysaccharides and whole cells of Pasteurella multocida type B: 2. Two hundred healthy male mice of eight to ten weeks old were used in this study. The mice were divided into four equal groups of 50 mice each. Mice of group 1 were inoculated intra-peritoneal with 1.0 mL sterile Phosphate Buffered Saline (PBS pH7, group 2 were inoculated with 1.0 mL of 109 colony forming unit (cfu of P. multocida B: 2. Mice of groups 3 and 4 were inoculated intra-peritoneal with 1.0 mL of LPS and 1.0 mL of OMP, respectively. Blood samples were collected from the moribund animals and the biochemical and hematological parameters were assessed using ANOVA and Tukey-Kramer test. In the hematology, significant decreases were observed in the treatment groups compared to the control group. Increases were only observed in band neutrophils, eosinophils and plasma proteins in the treatment groups compared to the control group. In the serum biochemistry, significant increases were observed in the treatment groups compared to the control group. Decreases were only observed in AP and albumin: globulin ratio. In electrolytes, significant increases were observed in chloride and calcium in the treatment groups compared to the control. In conclusion, all the immunogen group of mice showed changes in complete blood count and biochemistry profiles with some differences between the groups.

  19. Sporozoite neutralizing antibodies elicited in mice and rhesus macaques immunized with a Plasmodium falciparum repeat peptide conjugated to meningococcal outer membrane protein complex

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    Craig ePrzysiecki

    2012-11-01

    Full Text Available Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS protein, which in Plasmodium falciparum is comprised primarily of 30-40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP6 peptide conjugated to an outer membrane protein complex (OMPC derived from Neisseria meningitidis, a carrier protein used in a licensed H. influenzae pediatric vaccine. Mice immunized with alum-adsorbed (NANP6-OMPC, with or without Iscomatrix© as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P. berghei sporozoites that express P. falciparum CS repeats (PfPb. Inhibition of sporozoite invasion in vitro correlated with in vivo resistance to challenge by the bites of PfPb infected mosquitoes. Challenged mice had > 90% reduction of hepatic stage parasites as measured by real-time PCR, and either sterile immunity, i.e. no detectable blood stage parasites, or delayed prepatent periods which indicate neutralization of a majority, but not all, sporozoites. Rhesus macaques immunized with two doses of (NANP6-OMPC/MAA formulated with Iscomatrix© developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP6-OMPC/MAA+ Iscomatrix© at that time elicited strong anamnestic antibody responses. Rhesus macaque immune sera obtained post second and third dose of vaccine displayed high levels of sporozoite neutralizing activity in vitro that correlated with presence of high anti-repeat antibody titers. These preclinical studies in mice of different MHC haplotypes and a non-human primate support use of CS peptide-OMPC conjugates as a highly immunogenic platform to evaluate CS protective epitopes. Potential pre-erythrocytic vaccines can be combined with sexual blood stage vaccines as a multi-antigen malaria vaccine to block invasion and transmission of Plasmodium parasites

  20. Regulated delayed expression of rfaH in an attenuated Salmonella enterica serovar typhimurium vaccine enhances immunogenicity of outer membrane proteins and a heterologous antigen.

    Science.gov (United States)

    Kong, Qingke; Liu, Qing; Roland, Kenneth L; Curtiss, Roy

    2009-12-01

    RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A DeltarfaH mutant strain is attenuated in mice (50% lethal dose [LD(50)], >10(8) CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC P(BAD) promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the P(BAD) promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, DeltaP(rfaH178), was introduced into the attenuated vaccine strain chi9241 (DeltapabA DeltapabB DeltaasdA) expressing the pneumococcal surface protein PspA from an Asd(+) balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic DeltarfaH derivative or the isogenic RfaH(+) parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD(50) of virulent Streptococcus pneumoniae WU2. Immunization with DeltaP(rfaH178) mutant strains led to increased levels of protection compared to that of the parent chi9241 and of a DeltarfaH derivative of chi9241.

  1. Effect of estradiol on chlamydial genital infection of female guinea pigs.

    OpenAIRE

    Rank, R G; White, H. J.; Hough, A. J.; Pasley, J N; Barron, A L

    1982-01-01

    Female guinea pigs were treated daily with 1 mg of beta-estradiol-3-benzoate intramuscularly beginning 14 days before intravaginal inoculation with the chlamydial agent of guinea pig inclusion conjunctivitis and continuing during the course of the infection. Treatment with estradiol was found to markedly influence the course of genital infection with the chlamydial agent of guinea pig inclusion conjunctivitis, producing infections of greater intensity and longer duration than those in control...

  2. The conserved Tarp actin binding domain is important for chlamydial invasion.

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    Travis J Jewett

    Full Text Available The translocated actin recruiting phosphoprotein (Tarp is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.

  3. Comparison of Murine Cervicovaginal Infection by Chlamydial Strains: Identification of Extrusions Shed In vivo

    Science.gov (United States)

    Shaw, Jennifer H.; Behar, Amanda R.; Snider, Timothy A.; Allen, Noah A.; Lutter, Erika I.

    2017-01-01

    Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections (STIs) and preventable blindness. Untreated, asymptomatic infection as well as frequent re-infection are common and may drive pelvic inflammatory disease, ectopic pregnancy, and infertility. In vivo models of chlamydial infection continue to be instrumental in progress toward a vaccine and further elucidating the pathogenesis of this intracellular bacterium, however significant gaps in our understanding remain. Chlamydial host cell exit occurs via two mechanisms, lysis and extrusion, although the latter has yet to be reported in vivo and its biological role is unclear. The objective of this study was to investigate whether chlamydial extrusions are shed in vivo following infection with multiple strains of Chlamydia. We utilized an established C3H/HeJ murine cervicovaginal infection model with C. trachomatis serovars D and L2 and the Chlamydia muridarum strain MoPn to monitor the (i) time course of infection and mode of host cell exit, (ii) mucosal and systemic immune response to infection, and (iii) gross and histopathology following clearance of active infection. The key finding herein is the first identification of chlamydial extrusions shed from host cells in an in vivo model. Extrusions, a recently appreciated mode of host cell exit and potential means of dissemination, had been previously observed solely in vitro. The results of this study demonstrate that chlamydial extrusions exist in vivo and thus warrant further investigation to determine their role in chlamydial pathogenesis. PMID:28217555

  4. Gender-based screening for chlamydial infection and divergent infection trends in men and women.

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    Susan M Rogers

    Full Text Available OBJECTIVES: To assess the potential impact of chlamydial screening policy that recommends routine screening of women but not men. METHODS: Population surveys of probability samples of Baltimore adults aged 18 to 35 years in 1997-1998 and 2006-2009 collected biospecimens to estimate trends in undiagnosed chlamydial infection. Survey estimates are compared to surveillance data on diagnosed chlamydial infections reported to the Health Department. RESULTS: Prevalence of undiagnosed chlamydial infection among men increased from 1.6% to 4.0%, but it declined from 4.3% to 3.1% among women (p = 0.028 for test of interaction. The annual (average number of diagnosed infections was substantially higher among women than men in both time periods and increased among both men and women. Undiagnosed infection prevalence was substantially higher among black than non-black adults (4.0% vs 1.2%, p = 0.042 in 1997-98 and 5.5% vs 0.7%, p<0.001 in 2006-09. CONCLUSION: Divergent trends in undiagnosed chlamydial infection by gender parallel divergent screening recommendations that encourage chlamydial testing for women but not for men.

  5. A pitfall in diagnosis of human prion diseases using detection of protease-resistant prion protein in urine. Contamination with bacterial outer membrane proteins.

    Science.gov (United States)

    Furukawa, Hisako; Doh-ura, Katsumi; Okuwaki, Ryo; Shirabe, Susumu; Yamamoto, Kazuo; Udono, Heiichiro; Ito, Takashi; Katamine, Shigeru; Niwa, Masami

    2004-05-28

    Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrPSc), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrPSc in human prion diseases. To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant proteins in patients affected with prion diseases and control subjects by Western blot, a coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant signal migrating around 32 kDa previously reported by Shaked et al. (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482) was not observed in this study. Instead, discrete protein bands with an apparent molecular mass of approximately 37 kDa were detected in the urine of many patients affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by incubation of secondary antibodies alone, i.e. in the absence of the primary anti-PrP antibodies. Mass spectrometry and N-terminal protein sequencing analysis revealed that the majority of the PK-resistant 37-kDa proteins in the urine of patients were outer membrane proteins (OMPs) of the Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from the Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrPSc. These findings caution that bacterial contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial species should be excluded in the immunological tests for PrPSc in clinical samples, in

  6. Parenteral immunization of PLA/PLGA nanoparticle encapsulating outer membrane protein (Omp) from Aeromonas hydrophila: Evaluation of immunostimulatory action in Labeo rohita (rohu).

    Science.gov (United States)

    Rauta, Pradipta Ranjan; Nayak, Bismita

    2015-05-01

    Advanced vaccine research approaches needs to explore on biodegradable nanoparticles (NPs) based vaccine carrier that can serve as antigen delivery systems as well as immuno-stimulatory action to induce both innate and adaptive immune response in fish. Immunogenicity of PLA and PLGA NPs encapsulating outer membrane protein (Omp) antigen of Aeromonas hydrophila were evaluated through intra-peritoneal injection in fish, Labeo rohita. Antigen loaded PLA-Omp (223.5 ± 13.19 nm) and PLGA-Omp (166.4 ± 21.23 nm) NPs were prepared using double emulsion method by efficiently encapsulating the antigen reaching the encapsulation efficiency 44 ± 4.58% and 59.33 ± 5.13% respectively. Our formulated PLA Omp and PLGA-Omp NPs were in nanometer range (PLA-Omp, it showed considerably slower antigen release in vitro than PLGA-Omp NPs. Other physical properties like zetapotential values and poly dispersity index (PDI) confirmed the stability as well as monodisperse nature of the formulated nanoparticles. The spherical and isolated nature of PLA-Omp and PLGA-Omp NPs were revealed by SEM analysis. Upon immunization of all antigenic formulations (PLA-Omp NP, PLGA-Omp NP, FIA-Omp, PLA NP, PLGA NP, PBS as control), significant higher bacterial agglutination titre and haemolytic activity were observed in case of PLA-Omp and PLGA-Omp immunized groups than rest groups at both 21 days and 42 days. The specific antibody response was significantly increased and persisted up to 42 days of post immunization by PLA-Omp, PLGA-Omp, FIA-Omp. PLA-Omp NPs showed better immune response (higher bacterial agglutination titre, haemolytic activity, specific antibody titre, higher percent survival upon A. hydrophila challenge) than PLGA-Omp in L. rohita confirming its better efficacy. Comparable antibody response of PLA-Omp and PLGA-Omp with FIA-Omp treated groups suggested that PLA and PLGA could be replacement for Freund's adjuvant (for stimulating antibody response) to overcome many side effects

  7. Comparisons of flux control exerted by mitochondrial outer-membrane carnitine palmitoyltransferase over ketogenesis in hepatocytes and mitochondria isolated from suckling or adult rats.

    Science.gov (United States)

    New, K J; Krauss, S; Elliott, K R; Quant, P A

    1999-02-01

    The primary aim of this paper was to calculate and report flux control coefficients for mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I) over hepatic ketogenesis because its role in controlling this pathway during the neonatal period is of academic importance and immediate clinical relevance. Using hepatocytes isolated from suckling rats as our model system, we measured CPT I activity and carbon flux from palmitate to ketone bodies and to CO2 in the absence and presence of a range of concentrations of etomoxir. (This is converted in situ to etomoxir-CoA which is a specific inhibitor of the enzyme.) From these data we calculated the individual flux control coefficients for CPT I over ketogenesis, CO2 production and total carbon flux (0.51 +/- 0.03; -1.30 +/- 0.26; 0.55 +/- 0.07, respectively) and compared them with equivalent coefficients calculated by similar analyses [Drynan, L., Quant, P.A. & Zammit, V.A. (1996) Biochem. J. 317, 791-795] in hepatocytes isolated from adult rats (0.85 +/- 0.20; 0.23 +/- 0.06; 1.06 +/- 0.29). CPT I exerts significantly less control over ketogenesis in hepatocytes isolated from suckling rats than those from adult rats. In the suckling systems the flux control coefficients for CPT I over ketogenesis specifically and over total carbon flux (< 0.6) are not consistent with the enzyme being rate-limiting. Broadly similar results were obtained and conclusions drawn by reanalysis of previous data {from experiments in mitochondria isolated from suckling or adult rats [Krauss, S., Lascelles, C.V., Zammit, V.A. & Quant, P.A. (1996) Biochem. J. 319, 427-433]} using a different approach of control analysis, although it is not strictly valid to compare flux control coefficients from different systems. Our overall conclusion is that flux control coefficients for CPT I over oxidative fluxes from palmitate (or palmitoyl-CoA) differ markedly according to (a) the metabolic state, (b) the stage of development, (c) the specific

  8. Protective immunity against Rickettsia heilongjiangensis in a C3H/HeN mouse model mediated by outer membrane protein B-pulsed dendritic cells.

    Science.gov (United States)

    Meng, YanFen; Xiong, XiaoLu; Qi, Yong; Duan, ChangSong; Gong, WenPing; Jiao, Jun; Wen, BoHai

    2015-03-01

    Rickettsia heilongjiangensis is an obligate intracellular bacterium that causes Far-Eastern tick-borne spotted fever. Outer membrane protein B (OmpB) is an important surface protein antigen of rickettsiae. In the present study, the ompB gene of R. heilongjiangensis was divided into four fragments, resulting in four recombinant proteins (OmpB-p1, OmpB-p2, OmpB-p3, and OmpB-p4). Each OmpB was used in vitro to stimulate murine bone marrow-derived dendritic cells (BMDCs) of C3H/HeN mice, and the OmpB-pulsed BMDCs were transferred to naïve C3H/HeN mice. On day 14 post-transfer of BMDCs, the mice were challenged with R. heilongjiangensis and the rickettsial loads in the mice were quantitatively determined on day 7 post-challenge. Mice receiving BMDCs pulsed with OmpB-p2, OmpB-p3, or OmpB-p4 exhibited significantly lower bacterial load compared with mice receiving OmpB-p1-pulsed BMDCs. CD4(+) and CD8(+) T cells isolated from the spleen of C3H/HeN mice receiving BMDCs pulsed with each OmpB were co-cultured with BMDCs pulsed with the respective cognate protein. In flow cytometric analysis, the expression level of CD69 on CD4(+) or CD8(+) T cells from mice receiving BMDCs pulsed with OmpB-p2, OmpB-p3, or OmpB-p4 was higher than that on cells from mice receiving OmpB-p1-pulsed BMDCs, while the expression level of tumor necrosis factor (TNF)-α on CD8(+) T cells and interferon (IFN)-γ on the CD4(+) and CD8(+) T cells from mice receiving OmpB-p2, -p3, or -p4 was significantly higher than on cells from mice receiving OmpB-p1-pulsed BMDCs. Our results suggest that the protective OmpBs could activate CD4(+) and CD8(+) T cells and drive their differentiation toward CD4(+) Th1 and CD8(+) Tcl cells, respectively, which produce greater amounts of TNF-α and, in particular, IFN-γ, to enhance rickettsicidal activity of host cells.

  9. Effect of Aqueous Extract of Aegle marmelos Fruit on Adherence and β-Lactam Resistance of Enteropathogenic Escherichia coli by Down Regulating Outer Membrane Protein C

    Directory of Open Access Journals (Sweden)

    Subramaniya Bharathi Raja

    2009-01-01

    Full Text Available Problem statement: Enteropathogenic Escherichia Coli (EPEC continue to be a major health problem, leading to death due to diarrhea, predominantly in children below the age of five. Due to evolution of multi drug resistance in EPEC and side effects caused to host by antibiotics necessitated a search for alternative medicines from medicinal plants. One such medicinal plant used since ancient times to cure diarrhea is Aegle marmelos. This study was done to investigate the effect of aqueous extract of Aegle marmelos fruit (AEAM on outer membrane protein C (OmpC of EPEC, which plays a key role in adherence and antibiotic resistance. Approach: Fixation of minimum inhibitory concentration. In presence and absence of AEAM antibiotic susceptibility test was performed. Expression analysis of OmpC and OmpF was carried out by RT-PCR of EPEC in presence and absence of AEAM. Morphological changes of EPEC in presence and absence of AEAM were analyzed by TEM. In infant mouse ileal loop model, histological analysis, adherence of bacteria to ileal loops and Western blotting for caspase-3 and Hsp70 were done. Results: OmpC (~42kDa a porin, played an important role in selective transport of nutrients and also acted as an adhesin, whereas OmpF (~38kDa is also a porin which is non selective. Susceptibility of EPEC to β-lactam antibiotics in presence of AEAM can be attributed to down regulation of OmpC and upregulation of OmpF. The changes in Omp expression also triggered morphological changes in EPEC. Histology and western blot of Hsp70 and Caspase-3 in rat ileal loop confirmed the effect of AEAM on attenuating the virulence of EPEC by preventing its infection due to loss of adherence. Loss of adherence was due to morphological changes and down regulation of OmpC in EPEC. Conclusion: From this study, we concluded that the protection offered by AEAM against EPEC was due to down regulation of OmpC, leading to loss of adherence and up regulation of OmpF, which

  10. Regulatory protein OmpR influences the serum resistance of Yersinia enterocolitica O:9 by modifying the structure of the outer membrane.

    Directory of Open Access Journals (Sweden)

    Karolina Skorek

    Full Text Available The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.

  11. The Waddlia genome: a window into chlamydial biology.

    Directory of Open Access Journals (Sweden)

    Claire Bertelli

    Full Text Available Growing evidence suggests that a novel member of the Chlamydiales order, Waddlia chondrophila, is a potential agent of miscarriage in humans and abortion in ruminants. Due to the lack of genetic tools to manipulate chlamydia, genomic analysis is proving to be the most incisive tool in stimulating investigations into the biology of these obligate intracellular bacteria. 454/Roche and Solexa/Illumina technologies were thus used to sequence and assemble de novo the full genome of the first representative of the Waddliaceae family, W. chondrophila. The bacteria possesses a 2'116'312 bp chromosome and a 15'593 bp low-copy number plasmid that might integrate into the bacterial chromosome. The Waddlia genome displays numerous repeated sequences indicating different genome dynamics from classical chlamydia which almost completely lack repetitive elements. Moreover, W. chondrophila exhibits many virulence factors also present in classical chlamydia, including a functional type III secretion system, but also a large complement of specific factors for resistance to host or environmental stresses. Large families of outer membrane proteins were identified indicating that these highly immunogenic proteins are not Chlamydiaceae specific and might have been present in their last common ancestor. Enhanced metabolic capability for the synthesis of nucleotides, amino acids, lipids and other co-factors suggests that the common ancestor of the modern Chlamydiales may have been less dependent on their eukaryotic host. The fine-detailed analysis of biosynthetic pathways brings us closer to possibly developing a synthetic medium to grow W. chondrophila, a critical step in the development of genetic tools. As a whole, the availability of the W. chondrophila genome opens new possibilities in Chlamydiales research, providing new insights into the evolution of members of the order Chlamydiales and the biology of the Waddliaceae.

  12. The role of NOD1 and NOD2 in host defense against chlamydial infection.

    Science.gov (United States)

    Zou, Yan; Lei, Wenbo; He, Zhansheng; Li, Zhongyu

    2016-09-01

    Chlamydial species are common intracellular parasites that cause various diseases, mainly characterized by persistent infection, which lead to inflammatory responses modulated by pattern recognition receptors (PRRs). The best understood PRRs are the extracellular Toll-like receptors, but recent significant advances have focused on two important proteins, NOD1 and NOD2, which are members of the intracellular nucleotide-binding oligomerization domain receptor family and are capable of triggering the host innate immune signaling pathways. This results in the production of pro-inflammatory cytokines, which is vital for an adequate host defense against intracellular chlamydial infection. NOD1/2 ligands are known to derive from peptidoglycan, and the latest research has resolved the paradox of whether chlamydial species possess this bacterial cell wall component; this finding is likely to promote in-depth investigations into the interaction between the NOD proteins and chlamydial pathogens. In this review, we summarize the basic characteristics and signal transduction functions of NOD1 and NOD2 and highlight the new research on the roles of NOD1 and NOD2 in the host defense against chlamydial infection.

  13. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  14. 气单胞菌外膜蛋白基因工程疫苗的研究进展%Progress in Outer Membrane Protein Genetic Engineering Vaccine against Aeromonas

    Institute of Scientific and Technical Information of China (English)

    单晓枫; 曹亮; 沈锦玉; 陈龙; 康元环; 陈亨利; 钱爱东

    2014-01-01

    This article reviewed the development, immunization and disadvantages of Aeromonas outer membrane protein genetic engineering vaccine, DNA vaccine and recombinant live vector vaccine in order to provide reference for the research of Aeromonas vaccine.%就气单胞菌外膜蛋白的基因工程亚单位疫苗、DNA疫苗、重组活载体疫苗等基因工程疫苗的研究现状、免疫方式以及不足之处进行了综述,以期为气单胞菌疫苗研制提供参考。

  15. Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages.

    Science.gov (United States)

    Hua, Chun-Zhen; Hu, Wei-Lin; Shang, Shi-Qiang; Li, Jian-Ping; Hong, Li-Quan; Yan, Jie

    2015-12-16

    Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T- and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children influenzae.

  16. The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

    Science.gov (United States)

    Kudva, Indira T; Krastins, Bryan; Torres, Alfredo G; Griffin, Robert W; Sheng, Haiqing; Sarracino, David A; Hovde, Carolyn J; Calderwood, Stephen B; John, Manohar

    2015-06-01

    Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006). The E. coli O157 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract.

  17. 碳青霉烯类耐药鲍曼不动杆菌外膜蛋白表达的分析∗%Analysis of the expression of the outer membrane protein in carbapenem-resistant Acinetobacter baumannii

    Institute of Scientific and Technical Information of China (English)

    王同慧; 袁清强; 凌保东

    2015-01-01

    目的:筛选碳青霉烯类耐药鲍曼不动杆菌并了解其耐药情况,分析碳青霉烯类耐药菌及碳青霉烯类敏感菌的外膜蛋白表达情况。方法采用琼脂二倍稀释法筛选碳青霉烯类耐药鲍曼不动杆菌,运用聚合酶链反应(PCR)技术检测外膜蛋白基因carO;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析细菌外膜蛋白的表达情况。结果110株临床分离鲍曼不动杆菌中的32株细菌对碳青霉烯类抗菌药物耐药,此32株碳青霉烯类耐药菌除对多黏菌素 B 敏感性为100%,对其他所试药物均呈不同程度耐药;碳青霉烯类鲍曼不动杆菌均携带 carO 基因,SDS-PAGE 分析显示碳青霉烯类耐药菌株与敏感菌株相比,在相对分子质量约22×103和50×103等处的蛋白条带存在差异。结论鲍曼不动杆菌对抗菌药物的耐药情况严重,且碳青霉烯类耐药菌与敏感菌的外膜蛋白在表达上存在明显差异。%Objective To Screen the carbapenem-resistant acinetobacter baumannii so as to understand the drug resistance,and to analyze the outer membrane protein expression between the carbapenem-resistant Acinetobacter baumannii and the sensitive strains.Methods The twofold agar dilution method was used to screen the carbapenem-resistant acinetobacter baumannii.PCR technique was used to detect the outer membrane protein-encoding gene carO.SDS-PAGE was used to analyze the expression of outer membrane protein.Results 32 strains were carbapenem-resistant acinetobacter baumannii separated from 1 10 strains,which were 100% sensitive to polymyxin B and were resistant to other drugs with different degrees O.They all carried carO gene.There were different protein bands between the carbapenem-resistant strains and the sensitive strains,in which two protein bands of 50× 10 3 and 22×10 3 were recognized as outer membrane proteins through the analysis of SDS-PAGE.Conclusion Drug resistance of acinetobacter baumannii is serious

  18. Effect of estradiol on chlamydial genital infection of female guinea pigs.

    Science.gov (United States)

    Rank, R G; White, H J; Hough, A J; Pasley, J N; Barron, A L

    1982-11-01

    Female guinea pigs were treated daily with 1 mg of beta-estradiol-3-benzoate intramuscularly beginning 14 days before intravaginal inoculation with the chlamydial agent of guinea pig inclusion conjunctivitis and continuing during the course of the infection. Treatment with estradiol was found to markedly influence the course of genital infection with the chlamydial agent of guinea pig inclusion conjunctivitis, producing infections of greater intensity and longer duration than those in control animals. Moreover, pathogenesis was altered in that ascending infection was observed, resulting in endometritis, cystic salpingitis, and cystitis. Infection in the controls was limited to the cervix and vagina. Estradiol treatment increased the apparent number of infected cells in the cervix and vagina as detected by histopathology and immunofluorescent staining. Humoral and cell-mediated immune responses to the chlamydial agent of guinea pig inclusion conjunctivitis were comparable in estradiol-treated and untreated animals. These data indicate that hormonal manipulation may have profound effects on the course of chlamydial genital infections.

  19. Characterization of in vitro chlamydial cultures in low-oxygen atmospheres

    DEFF Research Database (Denmark)

    Juul, Nicolai Stefan; Jensen, Helene; Hvid, Malene;

    2007-01-01

    To mimic in vivo conditions during chlamydial infections, Chlamydia trachomatis serovar D and Chlamydia pneumoniae CWL029 were cultured in low-oxygen atmospheres containing 4% O(2), with parallel controls cultured in atmospheric air. Both were enriched with 5% CO(2). The results showed a dramatic...... increase in the growth of C. pneumoniae but not of C. trachomatis....

  20. Epithelial Cell Invasion and Adherence Directed by the Enterotoxigenic Escherichia coli tib Locus Is Associated with a 104-Kilodalton Outer Membrane Protein

    Science.gov (United States)

    1994-08-01

    copy EIEC (2 strains) - AFRIMS of the upstream region in cis or in trans restores invasion and production of the 104-kDa form of the TibA protein...antibodies ’ EIEC , enteroinvasive E. co/i; EHEC. entcrohemorrhagic E. cobi; EAggEC. against membrane localization and cell attachment epitopes of