WorldWideScience

Sample records for sarcosine-insoluble outer membrane

  1. 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....

  2. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

    NARCIS (Netherlands)

    Kieselbach, Thomas; Zijnge, Vincent; Granstrom, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and

  3. Separation of the outer membrane and identification of major outer membrane proteins from Porphyromonas gingivalis.

    Science.gov (United States)

    Murakami, Yukitaka; Imai, Masashi; Nakamura, Hiroshi; Yoshimura, Fuminobu

    2002-04-01

    The outer membrane of Porphyromonas gingivalis, an oral strict anaerobe, was isolated by sucrose density gradient centrifugation. The outer membrane obtained by the differential detergent extraction method, previously reported, showed an essentially similar protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), confirming that the latter method is suitable for the study of outer membrane proteins in this organism. N-terminal amino acid sequence analysis revealed that major outer membrane proteins in this organism included Arg-gingipain, Lys-gingipain, RagA (a TonB-linked receptor), and putative porins that were homologous to Escherichia coli OmpA.

  4. 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....

  5. Bacterial outer membrane vesicles and vaccine applications.

    Science.gov (United States)

    Acevedo, Reinaldo; Fernández, Sonsire; Zayas, Caridad; Acosta, Armando; Sarmiento, Maria Elena; Ferro, Valerie A; Rosenqvist, Einar; Campa, Concepcion; Cardoso, Daniel; Garcia, Luis; Perez, Jose Luis

    2014-01-01

    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 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 has been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice has shown their protective potential. dOMVB has been evaluated with non-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.

  6. BACTERIAL OUTER MEMBRANE VESICLES AND VACCINE APPLICATIONS

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  8. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    Directory of Open Access Journals (Sweden)

    Thomas Kieselbach

    Full Text Available Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT and leukotoxin (LtxA into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs. To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e using liquid chromatography-tandem mass spectrometry (LC-MS/MS. This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.

  9. Outer membrane vesicles as platform vaccine technology.

    Science.gov (United States)

    van der Pol, Leo; Stork, Michiel; van der Ley, Peter

    2015-09-01

    Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram-negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self-adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV-containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV-producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well-defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications. © 2015 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. The copyright line of the article for this article was changed on 23 February 2016 after original online publication.

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

    Science.gov (United States)

    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.

  11. Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    Jong Sook Jin

    Full Text Available Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA packaged in the OMVs. A. baumannii ATCC 19606(T secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606(T induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA(22-170 was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.

  12. Delivery of Foreign Antigens by Engineered Outer Membrane Vesicle Vaccines

    National Research Council Canada - National Science Library

    David J. Chen; Nikolaus Osterrieder; Stephan M. Metzger; Elizabeth Buckled; Anne M. Dood; Matthew P. DeLisa; David Putnam; Robert Langer

    2010-01-01

    .... We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants...

  13. Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorferi.

    Science.gov (United States)

    Brusca, J S; McDowall, A W; Norgard, M V; Radolf, J D

    1991-01-01

    Borrelia burgdorferi B31 with and without outer membranes contained nearly identical amounts of outer surface proteins A and B. The majority of each immunogen also was localized intracellularly by immunocryoultramicrotomy. These results are inconsistent with the widely held belief that outer surface proteins A and B are exclusively outer membrane proteins. Images FIG. 1 FIG. 2 FIG. 3 PMID:1744059

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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

  17. 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).

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

    Science.gov (United States)

    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.

  19. Simulations of outer membrane channels and their permeability.

    Science.gov (United States)

    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.

  20. Production of outer membrane vesicles and outer membrane tubes by Francisella novicida.

    Science.gov (United States)

    McCaig, William D; Koller, Antonius; Thanassi, David G

    2013-03-01

    Francisella spp. are highly infectious and virulent bacteria that cause the zoonotic disease tularemia. Knowledge is lacking for the virulence factors expressed by Francisella and how these factors are secreted and delivered to host cells. Gram-negative bacteria constitutively release outer membrane vesicles (OMV), which may function in the delivery of virulence factors to host cells. We identified growth conditions under which Francisella novicida produces abundant OMV. Purification of the vesicles revealed the presence of tube-shaped vesicles in addition to typical spherical OMV, and examination of whole bacteria revealed the presence of tubes extending out from the bacterial surface. Recently, both prokaryotic and eukaryotic cells have been shown to produce membrane-enclosed projections, termed nanotubes, which appear to function in cell-cell communication and the exchange of molecules. In contrast to these previously characterized structures, the F. novicida tubes are produced in liquid as well as on solid medium and are derived from the OM rather than the cytoplasmic membrane. The production of the OMV and tubes (OMV/T) by F. novicida was coordinately regulated and responsive to both growth medium and growth phase. Proteomic analysis of purified OMV/T identified known Francisella virulence factors among the constituent proteins, suggesting roles for the vesicles in pathogenesis. In support of this, production of OM tubes by F. novicida was stimulated during infection of macrophages and addition of purified OMV/T to macrophages elicited increased release of proinflammatory cytokines. Finally, vaccination with purified OMV/T protected mice from subsequent challenge with highly lethal doses of F. novicida.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Exploring bacterial outer membrane barrier to combat bad bugs

    Directory of Open Access Journals (Sweden)

    Ghai I

    2017-08-01

    Full Text Available Ishan Ghai,1 Shashank Ghai2 1School of Engineering and Life Sciences, Jacobs University, Bremen, 2Leibniz University, Hannover, Germany Abstract: One of the main fundamental mechanisms of antibiotic resistance in Gram-negative bacteria comprises an effective change in the membrane permeability to antibiotics. The Gram-negative bacterial complex cell envelope comprises an outer membrane that delimits the periplasm from the exterior environment. The outer membrane contains numerous protein channels, termed as porins or nanopores, which are mainly involved in the influx of hydrophilic compounds, including antibiotics. Bacterial adaptation to reduce influx through these outer membrane proteins (Omps is one of the crucial mechanisms behind antibiotic resistance. Thus to interpret the molecular basis of the outer membrane permeability is the current challenge. This review attempts to develop a state of knowledge pertinent to Omps and their effective role in antibiotic influx. Further, it aims to study the bacterial response to antibiotic membrane permeability and hopefully provoke a discussion toward understanding and further exploration of prospects to improve our knowledge on physicochemical parameters that direct the translocation of antibiotics through the bacterial membrane protein channels. Keywords: antibiotics, Gram-negative bacteria, cell envelope, protein channels, nanopores, influx, antibiotic resistance

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

    Science.gov (United States)

    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.

  4. 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.

  5. Polymorphic Regulation of Outer Membrane Lipid A Composition

    Directory of Open Access Journals (Sweden)

    Russell E. Bishop

    2016-11-01

    Full Text Available The importance of the polymorphic-phase behavior of lipid A structural variations in determining their endotoxic activities has been recognized previously, but any potential role for lipid A polymorphism in controlling outer membrane structure and function has been largely ignored until now. In a recent article in mBio [7(5:e01532-16, https://doi.org/10.1128/mBio.01532-16], Katherine E. Bonnington and Meta J. Kuehn of Duke University’s Department of Biochemistry make a compelling case for considering how the molecular shapes of the various lipid A structural subtypes found in the outer membrane contribute to the process of outer membrane vesicle (OMV formation.

  6. Bacterial outer membrane vesicles suppress tumor by interferon-?-mediated antitumor response

    OpenAIRE

    Kim, Oh Youn; Park, Hyun Taek; Dinh, Nhung Thi Hong; Choi, Seng Jin; Lee, Jaewook; Kim, Ji Hyun; Lee, Seung-Woo; Gho, Yong Song

    2017-01-01

    Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteolipids enriched with outer membrane proteins, to the surroundings. Outer membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used outer membrane vesicles in treating cancer thus far. Here we investigate the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show rem...

  7. 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.

  8. 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

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

    Directory of Open Access Journals (Sweden)

    Katherine E. Bonnington

    2016-10-01

    Full Text Available 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.

  10. Outer membrane phospholipase A's roles in Helicobacter pylori acid adaptation

    NARCIS (Netherlands)

    Vollan, H.S.; Tannaes, T.; Caugant, D.A.; Vriend, G.; Bukholm, G.

    2017-01-01

    BACKGROUND: The pH of the human gastric mucosa varies around 2.5 so that only bacteria with strong acidic stress tolerance can colonize it. The ulcer causing Helicobacter pylori thrives in the gastric mucosa. We analyse the roles of the key outer membrane protein OMPLA in its roles in acid

  11. 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

  12. Meningococcal outer membrane vesicles, lipopolysaccharide and innate immunity

    NARCIS (Netherlands)

    Zariri, A.

    2016-01-01

    Adjuvants are molecules that increase the immunogenicity of an antigen and play a key role in initiating an immune response. Traditional whole cell or outer membrane vesicle (OMV) vaccines already have components that are recognized by pattern recognition receptors (PRRs) on immune cells. On the

  13. 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

  14. Contribution of bacterial outer membrane vesicles to innate bacterial defense

    Directory of Open Access Journals (Sweden)

    Manning Andrew J

    2011-12-01

    Full Text Available Abstract Background Outer membrane vesicles (OMVs are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate outer membrane vesiculation as a bacterial mechanism for immediate short-term protection against outer membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection. Results We found that a hyper-vesiculating mutant of Escherichia coli survived treatment by antimicrobial peptides (AMPs polymyxin B and colistin better than the wild-type. Supplementation of E. coli cultures with purified outer membrane vesicles provided substantial protection against AMPs, and AMPs significantly induced vesiculation. Vesicle-mediated protection and induction of vesiculation were also observed for a human pathogen, enterotoxigenic E. coli (ETEC, challenged with polymyxin B. When ETEC with was incubated with low concentrations of vesicles concomitant with polymyxin B treatment, bacterial survival increased immediately, and the culture gained resistance to polymyxin B. By contrast, high levels of vesicles also provided immediate protection but prevented acquisition of resistance. Co-incubation of T4 bacteriophage and OMVs showed fast, irreversible binding. The efficiency of T4 infection was significantly reduced by the formation of complexes with the OMVs. Conclusions These data reveal a role for OMVs in contributing to innate bacterial defense by adsorption of antimicrobial peptides and bacteriophage. Given the increase in vesiculation in response to the antimicrobial peptides, and loss in efficiency of infection with the T4-OMV complex, we conclude that OMV production may be an important factor in neutralizing environmental agents that target the outer membrane of Gram-negative bacteria.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Wael Elhenawy

    2016-07-01

    Full Text Available 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.

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

    Science.gov (United States)

    Bonnington, Katherine E; Kuehn, Meta J

    2016-10-18

    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. All Gram-negative bacteria alter the structural composition of LPS present in their OM in response to various environmental stimuli. We developed a system to track the native dynamics of lipid A change in Salmonella enterica serovar Typhimurium

  18. Functional motor microdomains of the outer hair cell lateral membrane.

    Science.gov (United States)

    Santos-Sacchi, Joseph

    2002-12-01

    The outer hair cell (OHC) of the mammalian inner ear is a highly partitioned neuroepithelial cell whose lateral membrane is devoted to electromotility, a fast mechanical length change owing to the motor protein, prestin. Spatially restricted measures of prestin-derived nonlinear capacitance or gating charge, using either electrical amputation or discrete membrane mechanical deformation, were used to determine that functional variation exists within the extensive lateral membrane of the cell. This was evidenced by variation in the motor's operating voltage range and sensitivity among microdomains within the lateral membrane. That is, localized regions of the membrane evidenced Boltzmann distributions of motor charge whose midpoint voltage and slope differed from those obtained for the whole cell. These data highlight the functional independence of microdomains and imply that measured whole cell characteristics may differ from the microscopic characteristics of elementary motors.

  19. 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.

  20. 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.

  1. DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis.

    Directory of Open Access Journals (Sweden)

    Haruyuki Nakayama-Imaohji

    Full Text Available Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343 in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON. ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.

  2. DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis.

    Science.gov (United States)

    Nakayama-Imaohji, Haruyuki; Hirota, Katsuhiko; Yamasaki, Hisashi; Yoneda, Saori; Nariya, Hirofumi; Suzuki, Motoo; Secher, Thomas; Miyake, Yoichiro; Oswald, Eric; Hayashi, Tetsuya; Kuwahara, Tomomi

    2016-01-01

    Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.

  3. Spheres of influence: Porphyromonas gingivalis outer membrane vesicles.

    Science.gov (United States)

    Gui, M J; Dashper, S G; Slakeski, N; Chen, Y-Y; Reynolds, E C

    2016-10-01

    Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram-negative bacteria important for bacterial interaction with the environment. Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P. gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A-LPS) and associated C-terminal domain (CTD) -family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A-LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD-family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non-motile bacteria by motile bacteria. The P. gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro- and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors.

    Science.gov (United States)

    Veith, Paul D; Chen, Yu-Yen; Gorasia, Dhana G; Chen, Dina; Glew, Michelle D; O'Brien-Simpson, Neil M; Cecil, Jessica D; Holden, James A; Reynolds, Eric C

    2014-05-02

    Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC-MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs' volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. Protein selection and export via outer membrane vesicles.

    Science.gov (United States)

    Bonnington, K E; Kuehn, M J

    2014-08-01

    Outer membrane vesicles (OMVs) are constitutively produced by all Gram-negative bacteria. OMVs form when buds from the outer membrane (OM) of cells encapsulate periplasmic material and pinch off from the OM to form spheroid particles approximately 10 to 300nm in diameter. OMVs accomplish a diversity of functional roles yet the OMV's utility is ultimately determined by its unique composition. Inclusion into OMVs may impart a variety of benefits to the protein cargo, including: protection from proteolytic degradation, enhancement of long-distance delivery, specificity in host-cell targeting, modulation of the immune response, coordinated secretion with other bacterial effectors, and/or exposure to a unique function-promoting environment. Many enriched OMV-associated components are virulence factors, aiding in host cell destruction, immune system evasion, host cell invasion, or antibiotic resistance. Although the mechanistic details of how proteins become enriched as OMV cargo remain elusive, recent data on OM biogenesis and relationships between LPS structure and OMV-cargo inclusion rates shed light on potential models for OM organization and consequent OMV budding. In this review, mechanisms based on pre-existing OM microdomains are proposed to explain how cargo may experience differing levels of enrichment in OMVs and degrees of association with OMVs during extracellular export. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. 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.

  16. Bacterial outer membrane vesicles suppress tumor by interferon-γ-mediated antitumor response.

    Science.gov (United States)

    Kim, Oh Youn; Park, Hyun Taek; Dinh, Nhung Thi Hong; Choi, Seng Jin; Lee, Jaewook; Kim, Ji Hyun; Lee, Seung-Woo; Gho, Yong Song

    2017-09-20

    Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteolipids enriched with outer membrane proteins, to the surroundings. Outer membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used outer membrane vesicles in treating cancer thus far. Here we investigate the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show remarkable capability of bacterial outer membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects. Moreover, systematically administered bacterial outer membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such outer membrane vesicle-mediated immune response. Together, our results herein demonstrate the potential of bacterial outer membrane vesicles as effective immunotherapeutic agent that can treat various cancers without apparent adverse effects.Bacterial outer membrane vesicles (OMVs) contain immunogens but no study has yet examined their potential in treating cancer. Here, the authors demonstrate that OMVs can suppress established tumours and prevent tumour metastasis by an interferon-γ mediated antitumor response.

  17. Outer Membrane Vesicle Production by Escherichia coli Is Independent of Membrane Instability

    OpenAIRE

    McBroom, Amanda J.; Johnson, Alexandra P.; Vemulapalli, Sreekanth; Kuehn, Meta J.

    2006-01-01

    It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we co...

  18. Phylogenetic Analysis of Mitochondrial Outer Membrane β-Barrel Channels

    Science.gov (United States)

    Wojtkowska, Małgorzata; Jąkalski, Marcin; Pieńkowska, Joanna R.; Stobienia, Olgierd; Karachitos, Andonis; Przytycka, Teresa M.; Weiner, January; Kmita, Hanna; Makałowski, Wojciech

    2012-01-01

    Transport of molecules across mitochondrial outer membrane is pivotal for a proper function of mitochondria. The transport pathways across the membrane are formed by ion channels that participate in metabolite exchange between mitochondria and cytoplasm (voltage-dependent anion-selective channel, VDAC) as well as in import of proteins encoded by nuclear genes (Tom40 and Sam50/Tob55). VDAC, Tom40, and Sam50/Tob55 are present in all eukaryotic organisms, encoded in the nuclear genome, and have β-barrel topology. We have compiled data sets of these protein sequences and studied their phylogenetic relationships with a special focus on the position of Amoebozoa. Additionally, we identified these protein-coding genes in Acanthamoeba castellanii and Dictyostelium discoideum to complement our data set and verify the phylogenetic position of these model organisms. Our analysis show that mitochondrial β-barrel channels from Archaeplastida (plants) and Opisthokonta (animals and fungi) experienced many duplication events that resulted in multiple paralogous isoforms and form well-defined monophyletic clades that match the current model of eukaryotic evolution. However, in representatives of Amoebozoa, Chromalveolata, and Excavata (former Protista), they do not form clearly distinguishable clades, although they locate basally to the plant and algae branches. In most cases, they do not posses paralogs and their sequences appear to have evolved quickly or degenerated. Consequently, the obtained phylogenies of mitochondrial outer membrane β-channels do not entirely reflect the recent eukaryotic classification system involving the six supergroups: Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta. PMID:22155732

  19. 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.

  20. 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).

  1. 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.

  2. Outer membrane vesicle: a macromolecule with multifunctional activity.

    Science.gov (United States)

    Moshiri, Arfa; Dashtbani-Roozbehani, Abolfazl; Najar Peerayeh, Shahin; Siadat, Seyed Davar

    2012-07-01

    Nowadays adjuvants are extensively used as immuno-stimulatory and immuno-modulatory compounds as components of subunit and combination vaccine formulations. The adjuvants of microbial origin are more frequently used among currently used licensed or experimental adjuvants. The outer membrane vesicle (OMV) of Neisseria meningitidis is among the newly studied components of microbial origin, which could be applied as an adjuvant. Although the potency of OMV as a carrier (conjugated to a hapten) is now proven, the adjuvant properties of OMV have particular significance as a potential target for protective immunity. Since it has immune-stimulatory activity, OMV has been utilized in vaccine development. This commentary reviews the different applications of OMV as potential adjuvant in the field of vaccine development.

  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. The Lethal Cargo of Myxococcus xanthus Outer Membrane Vesicles

    Directory of Open Access Journals (Sweden)

    James E Berleman

    2014-09-01

    Full Text Available Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including MXAN_3564 (mepA, an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is the long-predicted (yet to date unknown primary extracellular protease in M. xanthus.

  6. Outer membrane vesicle production by Escherichia coli is independent of membrane instability.

    Science.gov (United States)

    McBroom, Amanda J; Johnson, Alexandra P; Vemulapalli, Sreekanth; Kuehn, Meta J

    2006-08-01

    It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the sigma(E) cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.

  7. 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.

  8. Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

    Science.gov (United States)

    Harner, Max; Neupert, Walter; Deponte, Marcel

    2011-07-15

    The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.

  9. 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.

  10. 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...

  11. 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

  12. 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.

  13. Vibrio fischeri-derived outer membrane vesicles trigger host development.

    Science.gov (United States)

    Aschtgen, Marie-Stephanie; Wetzel, Keith; Goldman, William; McFall-Ngai, Margaret; Ruby, Edward

    2016-04-01

    Outer membrane vesicles (OMV) are critical elements in many host-cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that within 12 h of colonizing crypts deep within the squid's light organ, the symbionts trigger an irreversible programme of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage-like cells called haemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin released by the symbionts, which can induce haemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that haemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host's detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis. © 2015 John Wiley & Sons Ltd.

  14. Mechanisms of outer membrane vesicle entry into host cells.

    Science.gov (United States)

    O'Donoghue, Eloise J; Krachler, Anne Marie

    2016-11-01

    Bacterial outer membrane vesicles (OMVs) are nano-sized compartments consisting of a lipid bilayer that encapsulates periplasm-derived, luminal content. OMVs, which pinch off of Gram-negative bacteria, are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial cells as well as eukaryotic cells. Compared with other secretion systems, OMVs can transfer a chemically extremely diverse range of cargo, including small molecules, nucleic acids, proteins, and lipids to proximal cells. Although it is well recognized that OMVs can enter and release cargo inside host cells during infection, the mechanisms of host association and uptake are not well understood. This review highlights existing studies focusing on OMV-host cell interactions and entry mechanisms, and how these entry routes affect cargo processing within the host. It further compares the wide range of methods currently used to dissect uptake mechanisms, and discusses potential sources of discrepancy regarding the mechanism of OMV uptake across different studies. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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...

  20. Preferential Packing of Acidic Glycosidases and Proteases into Bacteroides Outer Membrane Vesicles

    OpenAIRE

    Elhenawy, Wael; Debelyy, Mykhaylo O.; Feldman, Mario F.

    2014-01-01

    ABSTRACT Outer membrane vesicles (OMV) are spherical membranous structures released from the outer membrane (OM) of Gram-negative bacteria. OMV have been proposed to play several different roles during both pathogenesis and symbiosis. Despite the fact that OMV were described several decades ago, their biogenesis is a poorly characterized process. Whether OMV are produced by an active mechanism or by passive disintegration of the OM is a still matter of controversy. Bacteroides fragilis and Ba...

  1. 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....

  2. Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa.

    Science.gov (United States)

    Macdonald, Ian A; Kuehn, Meta J

    2013-07-01

    As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.

  3. Outer membrane machinery and alginate synthesis regulators control membrane vesicle production in Pseudomonas aeruginosa.

    Science.gov (United States)

    Tashiro, Yosuke; Sakai, Ryosuke; Toyofuku, Masanori; Sawada, Isao; Nakajima-Kambe, Toshiaki; Uchiyama, Hiroo; Nomura, Nobuhiko

    2009-12-01

    The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways.

  4. 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.

  5. 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.

  6. 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...

  7. 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.

  8. Next-generation outer membrane vesicle vaccines from concept to clinical trials

    NARCIS (Netherlands)

    Waterbeemd, van de B.

    2013-01-01

    Only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. The OMV vaccines, however, provide limited coverage and are difficult to produce. This is caused by an obligatory detergent treatment, which removes lipopolysaccharide

  9. Quantitative Proteomics Reveals Distinct Differences in the Protein Content of Outer Membrane Vesicle Vaccines

    NARCIS (Netherlands)

    Waterbeemd, van de B.; Mommen, G.P.M.; Pennings, J.L.A.; Eppink, M.H.M.; Wijffels, R.H.; Pol, van der L.A.; Jong, de A.P.J.M.

    2013-01-01

    At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in

  10. Meningococcal outer membrane vesicle composition-dependent activation of the innate immune response

    NARCIS (Netherlands)

    Zariri, Afshin; Beskers, Joep; van de Waterbeemd, Bas; Hamstra, Hendrik Jan; Bindels, Tim H E; van Riet, Elly; van Putten, Jos P M|info:eu-repo/dai/nl/069916527; van der Ley, Peter

    2016-01-01

    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

  11. 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.

  12. Genomic analysis indicates the presence of an asymmetric bilayer outer membrane in Planctomycetes and Verrucomicrobia

    Directory of Open Access Journals (Sweden)

    Daan R Speth

    2012-08-01

    Full Text Available Bacteria of the phylum Planctomycetes are of special interest for the study of compartmental cellular organization. Members of this phylum share a very unusual prokaryotic cell plan, featuring several membrane-bound compartments. Recently, it was shown that this cellular organization might extend to certain members of the phylum Verrucomicrobia. The Planctomycete cell plan has been defined as featuring a proteinaceous cell wall, a cytoplasmic membrane surrounding the paryphoplasm and an intracytoplasmic membrane defining the riboplasm. So far it was presumed that Planctomycetes did not have an asymmetric bilayer outer membrane as observed in Gram-negative bacteria. However, recent work on outer membrane biogenesis has provided several marker genes in the outer membrane protein (OMP assembly and the lipopolysaccharide (LPS insertion complexes. Additionally, advances in computational prediction of OMPs provided new tools to perform more accurate genomic screening for such proteins.Here we searched all 22 Planctomycetes and Verrucomicrobia genomes available in Genbank, plus the recently published genome of ‘Candidatus Scalindua profunda’, for markers of outer membrane biogenesis and OMPs. We were able to identify the key components of LPS insertion, OMP assembly and at least eight OMPs in all genomes tested. Additionally, we have analyzed the transcriptome and proteome data of the Planctomycetes ‘Candidatus Kuenenia stuttgartiensis’ and ‘Ca. S. profunda’ and could confirm high expression of several predicted OMPs, including the biomarkers of outer membrane biogenesis.

  13. 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.

  14. 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.

  15. Antigenic Properties of HpaA and Omp18, Two Outer Membrane Proteins of Helicobacter pylori

    OpenAIRE

    Voland, Petra; Hafsi, Nadia; Zeitner, Marco; Laforsch, Stephanie; Wagner, Hermann; Prinz, Christian

    2003-01-01

    Outer membrane proteins (OMPs) are incorporated into the outer plasma membrane of Helicobacter pylori and are important for, e.g., ion transport, adherence, structural and osmotic stability, and bacterial virulence but may also be antigenic due to their surface exposure. Previous proteome-based approaches with H. pylori lysates determined a strong serological reaction towards two H. pylori OMPs, HpaA (TIGR HP0797) and Omp18 (TIGR HP1125). PCR was used to detect DNA encoding the two proteins, ...

  16. Degradation of transplanted rat liver mitochondrial-outer-membrane proteins in hepatoma cells.

    OpenAIRE

    Russell, S.M.; Mayer, R J

    1983-01-01

    Reductively [3H]methylated 3H mitochondrial-outer-membrane vesicles from rat liver and vesicles where monoamine oxidase has been derivatized irreversibly by [3H]-pargyline have been deliberately miscompartmentalized by heterologous transplantation into hepatoma (HTC) cells by poly(ethylene glycol)-mediated vesicle-cell fusion. Fluorescein-conjugated mitochondrial-outer-membrane vesicles have also been used to show that transplanted material is patched, capped and internalized. Reductively met...

  17. Role of Pseudomonas aeruginosa peptidoglycan-associated outer membrane proteins in vesicle formation.

    Science.gov (United States)

    Wessel, Aimee K; Liew, Jean; Kwon, Taejoon; Marcotte, Edward M; Whiteley, Marvin

    2013-01-01

    Gram-negative bacteria produce outer membrane vesicles (OMVs) that package and deliver proteins, small molecules, and DNA to prokaryotic and eukaryotic cells. The molecular details of OMV biogenesis have not been fully elucidated, but peptidoglycan-associated outer membrane proteins that tether the outer membrane to the underlying peptidoglycan have been shown to be critical for OMV formation in multiple Enterobacteriaceae. In this study, we demonstrate that the peptidoglycan-associated outer membrane proteins OprF and OprI, but not OprL, impact production of OMVs by the opportunistic pathogen Pseudomonas aeruginosa. Interestingly, OprF does not appear to be important for tethering the outer membrane to peptidoglycan but instead impacts OMV formation through modulation of the levels of the Pseudomonas quinolone signal (PQS), a quorum signal previously shown by our laboratory to be critical for OMV formation. Thus, the mechanism by which OprF impacts OMV formation is distinct from that for other peptidoglycan-associated outer membrane proteins, including OprI.

  18. A bilayer-couple model of bacterial outer membrane vesicle biogenesis.

    Science.gov (United States)

    Schertzer, Jeffrey W; Whiteley, Marvin

    2012-01-01

    Gram-negative bacteria naturally produce outer membrane vesicles (OMVs) that arise through bulging and pinching off of the outer membrane. OMVs have several biological functions for bacteria, most notably as trafficking vehicles for toxins, antimicrobials, and signaling molecules. While their biological roles are now appreciated, the mechanism of OMV formation has not been fully elucidated. We recently demonstrated that the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) is required for OMV biogenesis in P. aeruginosa. We hypothesized that PQS stimulates OMV formation through direct interaction with the outer leaflet of the outer membrane. To test this hypothesis, we employed a red blood cell (RBC) model that has been used extensively to study small-molecule-membrane interactions. Our results revealed that addition of PQS to RBCs induced membrane curvature, resulting in the formation of membrane spicules (spikes), consistent with small molecules that are inserted stably into the outer leaflet of the membrane. Radiotracer experiments demonstrated that sufficient PQS was inserted into the membrane to account for this curvature and that curvature induction was specific to PQS structure. These data suggest that a low rate of interleaflet flip-flop forces PQS to accumulate in and expand the outer leaflet relative to the inner leaflet, thus inducing membrane curvature. In support of PQS-mediated outer leaflet expansion, the PQS effect was antagonized by chlorpromazine, a molecule known to be preferentially inserted into the inner leaflet. Based on these data, we propose a bilayer-couple model to describe P. aeruginosa OMV biogenesis and suggest that this is a general mechanism for bacterial OMV formation. Despite the ubiquity and importance of outer membrane vesicle (OMV) production in Gram-negative bacteria, the molecular details of OMV biogenesis are not fully understood. Early experiments showed that 2-heptyl-3-hydroxy-4-quinolone (PQS) induces OMV formation

  19. Acinetobacter baumannii outer membrane vesicles elicit a potent innate immune response via membrane proteins.

    Directory of Open Access Journals (Sweden)

    So Hyun Jun

    Full Text Available Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606(T induced expression of pro-inflammatory cytokine genes, interleukin (IL-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host.

  20. 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.

  1. 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

  2. Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis.

    Science.gov (United States)

    Große, Lena; Wurm, Christian A; Brüser, Christian; Neumann, Daniel; Jans, Daniel C; Jakobs, Stefan

    2016-02-15

    The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  3. A β-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in Cupriavidus necator

    Science.gov (United States)

    Belchik, Sara Mae; Schaeffer, Scott M.; Hasenoehrl, Shelley

    2010-01-01

    The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a β-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds. PMID:19937267

  4. A beta-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in Cupriavidus necator.

    Science.gov (United States)

    Belchik, Sara Mae; Schaeffer, Scott M; Hasenoehrl, Shelley; Xun, Luying

    2010-06-01

    The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a beta-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds.

  5. 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

  6. 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.

  7. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

    Science.gov (United States)

    Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

    2016-12-06

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

  8. Outer membrane active transport: structure of the BtuB:TonB complex.

    Science.gov (United States)

    Shultis, David D; Purdy, Michael D; Banchs, Christian N; Wiener, Michael C

    2006-06-02

    In Gram-negative bacteria, the import of essential micronutrients across the outer membrane requires a transporter, an electrochemical gradient of protons across the inner membrane, and an inner membrane protein complex (ExbB, ExbD, TonB) that couples the proton-motive force to the outer membrane transporter. The inner membrane protein TonB binds directly to a conserved region, called the Ton-box, of the transporter. We solved the structure of the cobalamin transporter BtuB in complex with the C-terminal domain of TonB. In contrast to its conformations in the absence of TonB, the Ton-box forms a beta strand that is recruited to the existing beta sheet of TonB, which is consistent with a mechanical pulling model of transport.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Adam J Kulp

    Full Text Available The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection. The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.

  18. Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli.

    Science.gov (United States)

    Kulp, Adam J; Sun, Bo; Ai, Teresa; Manning, Andrew J; Orench-Rivera, Nichole; Schmid, Amy K; Kuehn, Meta J

    2015-01-01

    The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.

  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. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions

    Science.gov (United States)

    Schwechheimer, Carmen; Kuehn, Meta J.

    2017-01-01

    Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles. PMID:26373371

  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. 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.

  8. 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.

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

    Science.gov (United States)

    Yamashita, Akihiro; Fujimoto, Masaru; Katayama, Kenta; Yamaoka, Shohei; Tsutsumi, Nobuhiro; Arimura, Shin-Ichi

    2016-01-01

    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.

  10. Outer membrane lipoprotein VacJ is required for the membrane integrity, serum resistance and biofilm formation of Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Xie, Fang; Li, Gang; Zhang, Wanjiang; Zhang, Yanhe; Zhou, Long; Liu, Shuanghong; Liu, Siguo; Wang, Chunlai

    2016-02-01

    The outer membrane proteins of Actinobacillus pleuropneumoniae are mediators of infection, acting as targets for the host's defense system. The outer membrane lipoprotein VacJ is involved in serum resistance and intercellular spreading in several pathogenic bacteria. To investigate the role of VacJ in the pathogenicity of Actinobacillus pleuropneumoniae, the vacJ gene-deletion mutant MD12 ΔvacJ was constructed. The increased susceptibility to KCl, SDS plus EDTA, and several antibiotics in the MD12ΔvacJ mutant suggested that the stability of the outer membrane was impaired as a result of the mutation in the vacJ gene. The increased NPN fluorescence and significant cellular morphological variation in the MD12ΔvacJ mutant further demonstrated the crucial role of the VacJ lipoprotein in maintaining the outer membrane integrity of A. pleuropneumoniae. In addition, the MD12ΔvacJ mutant exhibited decreased survival from the serum and complement killing compared to the wild-type strain. Interestingly, the MD12ΔvacJ mutant showed reduced biofilm formation compared to the wild-type strain. To our knowledge, this is the first description of the VacJ lipoprotein contributing to bacterial biofilm formation. The data presented in this study illustrate the important role of the VacJ lipoprotein in the maintenance of cellular integrity, serum resistance, and biofilm formation in A. pleuropneumoniae. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Membrane Distribution of the Pseudomonas Quinolone Signal Modulates Outer Membrane Vesicle Production in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Catalina Florez

    2017-08-01

    Full Text Available The Pseudomonas quinolone signal (PQS is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis.

  12. Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains.

    Science.gov (United States)

    Remis, Jonathan P; Wei, Dongguang; Gorur, Amita; Zemla, Marcin; Haraga, Jessica; Allen, Simon; Witkowska, H Ewa; Costerton, J William; Berleman, James E; Auer, Manfred

    2014-02-01

    The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  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. A TonB-dependent outer membrane receptor of Pseudomonas fluorescens: virulence and vaccine potential.

    Science.gov (United States)

    Hu, Yong-hua; Dang, Wei; Sun, Li

    2012-09-01

    Pseudomonas fluorescens is a Gram-negative bacterium and a common aquaculture pathogen. In this study, we identified from a pathogenic P. fluorescens strain a TonB-dependent outer membrane receptor, TdrA, as a secreted protein and examined its function and vaccine potential. TdrA is composed of 746 residues and possesses conserved structural domains of TonB-dependent outer membrane receptors. Quantitative real-time reverse transcriptase-PCR analysis showed that expression of tdrA was upregulated under conditions of iron starvation and during infection of host cells. Consistently, iron depletion induced increased production of TdrA protein in the outer membrane. Compared to the wild type, a tdrA-knock out mutant (1) was unable to grow in the absence of iron, (2) exhibited drastically attenuated overall bacterial virulence, and (3) was impaired in the ability to establish lethal infection in host tissues. Purified recombinant TdrA (rTdrA), when used as a subunit vaccine to immunize flounder, was able to induce strong protective immunity, including production of serum-specific antibodies that resulted in effective protection against lethal-dose P. fluorescens challenge. Together, these results indicate that TdrA is an outer membrane receptor and a protective immunogen that is likely to be involved in iron acquisition and, as a result, required for optimal bacterial virulence.

  15. 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....

  16. Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

    Science.gov (United States)

    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 well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

  17. 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

  18. 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...

  19. 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...

  20. Improved Production Process for Native Outer Membrane Vesicle Vaccine against Neisseria meningitidis

    NARCIS (Netherlands)

    Waterbeemd, van de B.; Wijffels, R.H.; Zomer, G.; Kaaijk, P.; Ruiterkamp, N.; Dobbelsteen, van den G.J.M.; Pol, van der L.A.

    2013-01-01

    An improved detergent-free process has been developed to produce vaccine based on native outer membrane vesicles (NOMV) against Neisseria meningitidis serogroup B. Performance was evaluated with the NonaMen vaccine concept, which provides broad coverage based on nine distinct PorA antigens. Scalable

  1. Porin Loss Impacts the Host Inflammatory Response to Outer Membrane Vesicles of Klebsiella pneumoniae

    Science.gov (United States)

    Turner, Kelli L.; Cahill, Bethaney K.; Dilello, Sarah K.; Gutel, Dedra; Brunson, Debra N.; Albertí, Sebastián

    2015-01-01

    Antibiotic-resistant strains of Klebsiella pneumoniae often exhibit porin loss. In this study, we investigated how porin loss impacted the composition of secreted outer membrane vesicles as well as their ability to trigger proinflammatory cytokine secretion by macrophages. We hypothesize that porin loss associated with antibiotic resistance will directly impact both the composition of outer membrane vesicles and their interactions with phagocytic cells. Using clonally related clinical isolates of extended-spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae with different patterns of porin expression, we demonstrated that altered expression of OmpK35 and OmpK36 results in broad alterations to the protein profile of secreted vesicles. Additionally, the level of OmpA incorporation was elevated in strains lacking a single porin. Porin loss significantly impacted macrophage inflammatory responses to purified vesicles. Outer membrane vesicles lacking both OmpK35 and OmpK36 elicited significantly lower levels of proinflammatory cytokine secretion than vesicles from strains expressing one or both porins. These data demonstrate that antibiotic resistance-associated porin loss has a broad and significant effect on both the composition of outer membrane vesicles and their interactions with phagocytic cells, which may impact bacterial survival and inflammatory reactions in the host. PMID:26666932

  2. [Obtaining monoclonal antibodies against outer membrane glycoproteins of Entamoeba histolytica].

    Science.gov (United States)

    Agundis, C; Isibasi, A; Ortíz, V; Reyes, J L; Paniagua, J; Ramírez, A; Kumate, J

    1990-01-01

    The goal of this paper was the production of monoclonal antibodies capable of detecting relevant antigens from the surface of Entamoeba histolytica trophozoites, with the purpose of using them as a diagnostic test. The cellular fusion for obtaining the monoclonal antibodies (mAb) was done with spleen cells from BALB/c mice, previously immunized with glycoproteins from the membrane, as well as Sp2/0 cells. The hybridoma supernatants were tested with ELISA, using glycoproteins and lipopeptide phosphoglycans (LPPG) as antigens. Seven hybridomas producing mAb against the glycoproteins were found. Among these, three recognize LPPG. The ability of reacting with the mAb against two molecules disappeared for all the LPPG positive ones when were treated with meta-periodate, and only three reacted against the glycoproteins. All of the mAb were of the Ig M isotypes. They were characterized by Dot blot and Western blot assays. From the results, one may deduce that some mAb recognize as epitopes the polysaccharide portion, and thus infer that they are directed of against the surface and therefore, in the future, could be used with a diagnostic purpose.

  3. 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

  4. 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

  5. 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.

  6. NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes.

    Science.gov (United States)

    Mahalakshmi, Radhakrishnan; Franzin, Carla M; Choi, Jungyuen; Marassi, Francesca M

    2007-12-01

    The beta-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D2O for days and is assigned to the transmembrane beta-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D2O are assigned to the extracellular and periplasmic loops. The two-dimensional 1H/15N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5 degrees tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to alpha-helical membrane proteins.

  7. 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.

  8. 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.

  9. 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.

  10. Cross-linking of anaplasma marginale outer membrane proteins enhances immunogenicity, but is not required for protection from challenge

    Science.gov (United States)

    Bacterial outer membrane proteins are the primary targets of a protective immune response. The specific characteristics of outer membrane-based immunogens, in terms of antigen content and context that are required for protective immunity remain unknown for a wide variety of bacterial pathogens. Usin...

  11. Comprehensive in silico prediction and analysis of chlamydial outer membrane proteins reflects evolution and life style of the Chlamydiae

    Directory of Open Access Journals (Sweden)

    Myers Garry

    2009-12-01

    Full Text Available Abstract Background Chlamydiae are obligate intracellular bacteria comprising some of the most important bacterial pathogens of animals and humans. Although chlamydial outer membrane proteins play a key role for attachment to and entry into host cells, only few have been described so far. We developed a comprehensive, multiphasic in silico approach, including the calculation of clusters of orthologues, to predict outer membrane proteins using conservative criteria. We tested this approach using Escherichia coli (positive control and Bacillus subtilis (negative control, and applied it to five chlamydial species; Chlamydia trachomatis, Chlamydia muridarum, Chlamydia (a.k.a. Chlamydophila pneumoniae, Chlamydia (a.k.a. Chlamydophila caviae, and Protochlamydia amoebophila. Results In total, 312 chlamydial outer membrane proteins and lipoproteins in 88 orthologous clusters were identified, including 238 proteins not previously recognized to be located in the outer membrane. Analysis of their taxonomic distribution revealed an evolutionary conservation among Chlamydiae, Verrucomicrobia, Lentisphaerae and Planctomycetes as well as lifestyle-dependent conservation of the chlamydial outer membrane protein composition. Conclusion This analysis suggested a correlation between the outer membrane protein composition and the host range of chlamydiae and revealed a common set of outer membrane proteins shared by these intracellular bacteria. The collection of predicted chlamydial outer membrane proteins is available at the online database pCOMP http://www.microbial-ecology.net/pcomp and might provide future guidance in the quest for anti-chlamydial vaccines.

  12. Expression of human CEACAM1 in transgenic mice limits the Opa-specific immune response against meningococcal outer membrane vesicles.

    NARCIS (Netherlands)

    Zariri, A.; Dijken, H. van; Hamstra, H.J.; Flier, M. van der; Vidarsson, G.; Putten, J.P. van; Boog, C.J.; Dobbelsteen, G. van den; Ley, P. van der

    2013-01-01

    Outer membrane vesicles (OMVs) have been extensively investigated as meningococcal vaccine candidates. Among their major components are the opacity (Opa) proteins, a family of surface-exposed outer membrane proteins important for bacterial adherence and entry into host cells. Many Opa-dependent

  13. Structure of TonB in complex with FhuA, E. coli outer membrane receptor.

    Science.gov (United States)

    Pawelek, Peter D; Croteau, Nathalie; Ng-Thow-Hing, Christopher; Khursigara, Cezar M; Moiseeva, Natalia; Allaire, Marc; Coulton, James W

    2006-06-02

    The cytoplasmic membrane protein TonB spans the periplasm of the Gram-negative bacterial cell envelope, contacts cognate outer membrane receptors, and facilitates siderophore transport. The outer membrane receptor FhuA from Escherichia coli mediates TonB-dependent import of ferrichrome. We report the 3.3 angstrom resolution crystal structure of the TonB carboxyl-terminal domain in complex with FhuA. TonB contacts stabilize FhuA's amino-terminal residues, including those of the consensus Ton box sequence that form an interprotein beta sheet with TonB through strand exchange. The highly conserved TonB residue arginine-166 is oriented to form multiple contacts with the FhuA cork, the globular domain enclosed by the beta barrel.

  14. 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.

  15. Structure of TonB in Complex with FhuA, E. Coli Outer Membrane Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Pawelek,P.; Croteau, N.; Ng-Thow-Hing, C.; Khursigara, C.; Moiseeva, N.; Allaire, M.; Coulton, J.

    2006-01-01

    The cytoplasmic membrane protein TonB spans the periplasm of the Gram-negative bacterial cell envelope, contacts cognate outer membrane receptors, and facilitates siderophore transport. The outer membrane receptor FhuA from Escherichia coli mediates TonB-dependent import of ferrichrome. We report the 3.3 angstrom resolution crystal structure of the TonB carboxyl-terminal domain in complex with FhuA. TonB contacts stabilize FhuA's amino-terminal residues, including those of the consensus Ton box sequence that form an interprotein {beta} sheet with TonB through strand exchange. The highly conserved TonB residue arginine-166 is oriented to form multiple contacts with the FhuA cork, the globular domain enclosed by the {beta} barrel.

  16. Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

    Directory of Open Access Journals (Sweden)

    Bunai Christine L

    2009-02-01

    Full Text Available Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr, an alkaline solution (180 mM Na2CO3, pH 11.3 and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14. Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries; (ii peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries; (iii cytoplasmic or ribosomal membrane-contaminating proteins (80 entries. Thirty-one proteins were experimentally associated with the outer membrane (OM. Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM proteins with two or more predicted transmembrane domains (TMDs were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

  17. Membrane Distribution of the Pseudomonas Quinolone Signal Modulates Outer Membrane Vesicle Production in Pseudomonas aeruginosa.

    Science.gov (United States)

    Florez, Catalina; Raab, Julie E; Cooke, Adam C; Schertzer, Jeffrey W

    2017-08-08

    The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis.IMPORTANCE Bacterial secretion has been recognized as an essential facet of microbial pathogenesis and human disease. Numerous virulence factors have been found to be transported within outer membrane vesicles (OMVs), and delivery using these biological nanoparticles often results in increased potency. OMV biogenesis is an important but poorly

  18. Outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria.

    Directory of Open Access Journals (Sweden)

    Carla Pérez-Cruz

    Full Text Available Outer-inner membrane vesicles (O-IMVs were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM and Cryo-transmission electron microscopy (Cryo-TEM was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles.

  19. Outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria.

    Science.gov (United States)

    Pérez-Cruz, Carla; Delgado, Lidia; López-Iglesias, Carmen; Mercade, Elena

    2015-01-01

    Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles.

  20. Immunoproteomic analysis of outer membrane proteins and extracellular proteins of Actinobacillus pleuropneumoniae JL03 serotype 3

    Directory of Open Access Journals (Sweden)

    Hu Yong

    2009-08-01

    Full Text Available Abstract Background Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia, a highly contagious respiratory infection in pigs, and all the 15 serotypes are able to cause disease. Current vaccines including subunit vaccines could not provide satisfactory protection against A. pleuropneumoniae. In this study, the immunoproteomic approach was applied to the analysis of extracellular and outer membrane proteins of A. pleuropneumoniae JL03 serotype 3 for the identification of novel immunogenic proteins for A. pleuropneumoniae. Results A total of 30 immunogenic proteins were identified from outer membrane and extracellular proteins of JL03 serotype 3, of which 6 were known antigens and 24 were novel immunogenic proteins for A. pleuropneumoniae. Conclusion These data provide information about novel immunogenic proteins for A. pleuropneumoniae serotype 3, and are expected to aid in development of novel vaccines against A. pleuropneumoniae.

  1. Effect of the morphogene bolA on the permeability of the Escherichia coli outer membrane.

    Science.gov (United States)

    Freire, Patrick; Vieira, Helena L A; Furtado, Ana R; de Pedro, Miguel A; Arraiano, Cecília M

    2006-07-01

    Escherichia coli bolA is a morphogene involved in stress response and cell division. Overexpression of bolA induces biofilm formation and affects the levels of carboxypeptidases PBP5, PBP6 and beta-lactamase AmpC. In this study, we have shown that changes in the expression of bolA result in alterations in the properties of the outer membrane. The sensitivity to detergents and vancomycin was reduced when bolA was overexpressed and fluorescent probes indicated that different levels of bolA had an effect on outer membrane protein accessibility. Moreover, bolA was shown to be involved in the modulation of the OmpF/OmpC balance.

  2. Studying Factors Involved in Biogenesis of Lysobacter sp. XL1 Outer Membrane Vesicles.

    Science.gov (United States)

    Kudryakova, I V; Suzina, N E; Vinokurova, N G; Shishkova, N A; Vasilyeva, N V

    2017-04-01

    The Gram-negative bacterium Lysobacter sp. XL1 produces outer membrane vesicles that are heterogeneous in size, density, and protein composition. One of the subpopulations is secretory vesicles for lytic protease L5 of Lysobacter sp. XL1 (Kudryakova et al. (2015) FEMS Microbiol. Lett., 362, fnv137). Protein L5 was assumed to influence biogenesis of these secretory vesicles that contain it. Using a Pseudomonas fluorescens Q2-87/B expression system, it was shown that the recombinant L5 protein may act as a factor of vesicle biogenesis. This points to a possible involvement of L5 protein in Lysobacter sp. XL1 vesicle biogenesis. Furthermore, it was established that the main phospholipid of Lysobacter sp. XL1 vesicles is cardiolipin, and vesicles are formed predominantly of outer membrane regions enriched with this phospholipid. This indicates that cardiolipin participates in biogenesis of all vesicle subpopulations in Lysobacter sp. XL1.

  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. Discrimination of outer membrane proteins using a K-nearest neighbor method.

    Science.gov (United States)

    Yan, C; Hu, J; Wang, Y

    2008-06-01

    Identification of outer membrane proteins (OMPs) from genome is an important task. This paper presents a k-nearest neighbor (K-NN) method for discriminating outer membrane proteins (OMPs). The method makes predictions based on a weighted Euclidean distance that is computed from residue composition. The method achieves 89.1% accuracy with 0.668 MCC (Matthews correlation coefficient) in discriminating OMPs and non-OMPs. The performance of the method is improved by including homologous information into the calculation of residue composition. The final method achieves an accuracy of 96.1%, with 0.873 MCC, 87.5% sensitivity, and 98.2% specificity. Comparisons with multiple recently published methods show that the method proposed in this study outperforms the others.

  5. 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.

  6. Envelope control of outer membrane vesicle production in Gram-negative bacteria.

    Science.gov (United States)

    Schwechheimer, Carmen; Sullivan, Claretta J; Kuehn, Meta J

    2013-05-07

    All Gram-negative bacteria studied to date have been shown to produce outer membrane vesicles (OMVs), which are budded, released spheres of outer membrane with periplasmic content. OMVs have been implicated in the delivery of virulence factors in pathogenesis. However, OMVs also benefit nonpathogenic species by delivering degradative enzymes to defend an ecological niche against competing bacterial species, and they can serve as an envelope stress response. Despite these important roles, very little is known about the mechanism of production of OMVs. Here we review the advantage of vesiculation, particularly in a nonpathogenic context, as well as the hurdles that have to be overcome in Gram-negative envelope architecture before a vesicle can form and bud. Lastly, we address the question of whether OMV production is a stochastic or regulated process.

  7. Voltage-dependent changes in specific membrane capacitance caused by prestin, the outer hair cell lateral membrane motor.

    Science.gov (United States)

    Santos-Sacchi, Joseph; Navarrete, Enrique

    2002-05-01

    In the outer hair cell (OHC), membrane capacitance principally derives from two components - that associated with lateral membrane sensor/motor charge movement, and that proportional to the membrane surface area (C(sa)). We used measures of membrane capacitance to test a model hypothesis that OHC lateral membrane molecular motors, recently identified as the protein prestin, fluctuate between two area states. By measuring membrane capacitance in native OHCs or prestin-transfected HEK cells at extreme voltages (+/-200 mV) where motor-derived charge movement is small or absent, we observed that C(sa) depends on the state of the motors, or correspondingly on membrane voltage. Deiters cells or control HEK cells, which lack motors, do not show this dependence. We modeled the voltage-dependent change in C(sa) as a Boltzmann process with the same parameters that describe the charge movement of the motors' voltage sensors. C(sa) is 3.28+/-0.75 pF (mean +/-SD; n=23) larger during extreme hyperpolarization, and the number of motors in OHCs and prestin-transfected HEK cells correlates with the magnitude of Delta C(sa)( r=0.78). Although these data are consistent with the area motor model, the corresponding area change, assuming 0.5 microF/cm(2) under constant membrane thickness, is unphysiologically large, and indicates that the capacitance change must result from changes not only in lateral membrane area but also specific capacitance. Thus, we conclude that a conformational change in the lateral membrane motor, prestin, additionally alters the dielectric constant and/or thickness of the lateral plasma membrane.

  8. 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.

  9. 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.

  10. 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...

  11. 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 ...

  12. 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...

  13. 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...

  14. 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...

  15. Haemophilus influenzae outer membrane vesicle-induced blood-brain barrier permeability during experimental meningitis.

    OpenAIRE

    Wispelwey, B; Hansen, E J; Scheld, W M

    1989-01-01

    Haemophilus influenzae type b (Hib) lipopolysaccharide (LPS) may be present in the cerebrospinal fluid largely as part of outer membrane vesicles (OMV), which could possibly alter its activity. Similar to inoculation of purified Hib LPS, intracisternal inoculation of Hib OMV into adult rats resulted in dose- and time-dependent increases in blood-brain barrier permeability. Polymyxin B, but not an oligosaccharide-specific monoclonal antibody, significantly inhibited the activity of Hib OMV. No...

  16. Next-generation outer membrane vesicle vaccines from concept to clinical trials

    OpenAIRE

    Waterbeemd, van de, B.

    2013-01-01

    Only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. The OMV vaccines, however, provide limited coverage and are difficult to produce. This is caused by an obligatory detergent treatment, which removes lipopolysaccharide (LPS), a toxic OMV component. This thesis explored an alternative approach, based on OMV with attenuated lpxL1-LPS and a detergent-free process. The alternative approach is referred to as ‘next-ge...

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Production of outer membrane vesicles by the plague pathogen Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Justin L Eddy

    Full Text Available Many Gram-negative bacteria produce outer membrane vesicles (OMVs during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37 °C compared to 26 °C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp. In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand.

  4. Enterohemorrhagic Escherichia coli OmpT regulates outer membrane vesicle biogenesis.

    Science.gov (United States)

    Premjani, Veena; Tilley, Derek; Gruenheid, Samantha; Le Moual, Hervé; Samis, John A

    2014-06-01

    Enterohemorrhagic Escherichia coli (EHEC) infection from food or water often results in severe diarrheal disease and is a leading cause of death globally. Outer membrane vesicles (OMVs) secreted from E. coli induce lethality in mice. The omptin outer membrane protease OmpT from E. coli inactivates antimicrobial peptides and may enhance colonization of the uroepithelium, but its precise function remains unclear. Given OmpT is an outer membrane protease, we hypothesized it may have a role in OMV biogenesis. To further characterize the effect of OmpT on OMV production, a genetic approach using wild type, an ompT deletion mutant and an ompT overexpressing construct in EHEC were employed. ompT gene deletion markedly decreased OMV production and stainable lipid but increased vesicle diameter. Conversely, ompT overexpression profoundly increased OMV biogenesis but decreased stainable lipid, protein content, and vesicle diameter. Alterations in EHEC ompT gene expression have an impact on the biogenesis, composition, and size of OMVs. Changes in ompT gene expression may dynamically alter OMV formation, composition, and diameter in response to different host environments and contribute to cell-free intercellular communication to enhance bacterial growth and survival. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  5. 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.

  6. 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...

  7. Fractionation of human liver mitochondria: enzymic and morphological characterization of the inner and outer membranes as compared to rat liver mitochondria.

    Science.gov (United States)

    Benga, G; Hodarnau, A; Tilinca, R; Porutiu, D; Dancea, S; Pop, V; Wrigglesworth, J

    1979-02-01

    The fractionation of human liver mitochondria into inner membrane, outer membrane and matrix material is reported. Compared with rat, human liver mitochondria are more fragile. Fractionation can be achieved in only 2 steps, a digitonin treatment for removal of the outer membrane and centrifugation of the inner membrane plus matrix particles through a linear sucrose gradient resulting in purified inner membranes and matrix.

  8. Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.

    Science.gov (United States)

    Aschtgen, Marie-Stephanie; Lynch, Jonathan B; Koch, Eric; Schwartzman, Julia; McFall-Ngai, Margaret; Ruby, Edward

    2016-08-15

    Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. Determining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

    that allows extraction of the motion of the protein from measurements of the mobility of the bead-molecule complex; these results are equally applicable to analyze bead-protein complexes in other membrane systems. Within a domain of radius approximately 25 nm, the receptor diffuses with a diffusion constant...... of (1.5 +/- 1.0) x 10(-9) cm(2)/s and sits in a harmonic potential as if it were tethered by an elastic spring of spring constant of ~1.0 x 10(-2) pN/nm to the bacterial membrane. The purpose of the protein motion might be to facilitate transport of maltodextrins through the outer bacterial membrane....

  16. Efficient quantification and characterization of bacterial outer membrane derived nano-particles with flow cytometric analysis.

    Science.gov (United States)

    Wieser, Andreas; Storz, Enno; Liegl, Gabriele; Peter, Annabell; Pritsch, Michael; Shock, Jonathan; Wai, Sun Nyunt; Schubert, Sören

    2014-11-01

    There currently exists no efficient and easy method for size profiling and counting of membranous nano-scale particles, such as bacterial outer membrane vesicles (OMVs). We present here a cost-effective and fast method capable of profiling and counting small sample volumes of nano-scale membranous vesicles with standard laboratory equipment without the need for any washing steps. OMV populations of different bacterial species are compared and even subpopulations of OMVs can be identified after a simple labelling procedure. Counting is possible over three orders of magnitude without any changes to the protocol. Protein contaminations do not alter the described measurements. Copyright © 2014 Elsevier GmbH. All rights reserved.

  17. 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.

  18. 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.

  19. Synthetic effect between envelope stress and lack of outer membrane vesicle production in Escherichia coli.

    Science.gov (United States)

    Schwechheimer, Carmen; Kuehn, Meta J

    2013-09-01

    Outer membrane vesicles (OMVs) are composed of outer membrane and periplasmic components and are ubiquitously secreted by Gram-negative bacteria. OMVs can disseminate virulence factors for pathogenic bacteria as well as serve as an envelope stress response. From a transposon mutant screen for OMV phenotypes, it was discovered that an nlpA mutant of Escherichia coli produces fewer OMVs than the wild type, whereas a degP mutant produces higher levels of OMVs. NlpA is an inner-membrane-anchored lipoprotein that has a minor role in methionine import. DegP is a periplasmic chaperone/protease for misfolded envelope proteins that is critical when cells are heat shocked. To reveal how these proteins contribute to OMV production, the mutations were combined and the double mutant analyzed. The ΔnlpA ΔdegP strain displayed a high-temperature growth defect that corresponded to the production of fewer OMVs than produced by the ΔdegP strain. This phenotype also pertained to other undervesiculation mutations in a ΔdegP background. The hypovesiculation phenotype of ΔnlpA in the wild-type strain as well as in the degP deletion strain was found to be a stationary-phase phenomenon. The periplasm of the ΔnlpA ΔdegP strain was determined to contain significantly more protein in stationary phase than the wild type. Additionally, misfolded DegP substrate outer membrane porins were detected in ΔdegP mutant-derived OMVs. These data suggest that an accumulation of envelope proteins resulting from decreased vesiculation was toxic and contributed to the growth defect. We conclude that OMV production contributes to relieve the envelope of accumulated toxic proteins and that NlpA plays an important role in the production of vesicles in stationary phase.

  20. 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.

  1. Linkage between anaplasma marginale outer membrane proteins enhances immunogenicity, but is not required for protection from challenge

    Science.gov (United States)

    Prevention of bacterial infections via immunization presents particular challenges. While outer membrane extracts are often protective; they are difficult and expensive to isolate and standardize, and thus often impractical for development and implementation in vaccination programs. In contrast, ind...

  2. 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

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

    Directory of Open Access Journals (Sweden)

    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

  4. 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...

  5. 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...

  6. 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.

  7. 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.

  8. Predatory activity of Myxococcus xanthus outer-membrane vesicles and properties of their hydrolase cargo.

    Science.gov (United States)

    Evans, Alun G L; Davey, Hazel M; Cookson, Alan; Currinn, Heather; Cooke-Fox, Gillian; Stanczyk, Paulina J; Whitworth, David E

    2012-11-01

    The deltaproteobacterium Myxococcus xanthus predates upon members of the soil microbial community by secreting digestive factors and lysing prey cells. Like other Gram-negative bacteria, M. xanthus produces outer membrane vesicles (OMVs), and we show here that M. xanthus OMVs are able to kill Escherichia coli cells. The OMVs of M. xanthus were found to contain active proteases, phosphatases, other hydrolases and secondary metabolites. Alkaline phosphatase activity was found to be almost exclusively associated with OMVs, implying that there is active targeting of phosphatases into OMVs, while other OMV components appear to be packaged passively. The kinetic properties of OMV alkaline phosphatase suggest that there may have been evolutionary adaptation of OMV enzymes to a relatively indiscriminate mode of action, consistent with a role in predation. In addition, the observed regulation of production, and fragility of OMV activity, may protect OMV-producing cells from exploitation by M. xanthus cheating genotypes and/or other competitors. Killing of E. coli by M. xanthus OMVs was enhanced by the addition of a fusogenic enzyme (glyceraldehyde-3-phosphate dehydrogenase; GAPDH), which triggers fusion of vesicles with target membranes within eukaryotic cells. This suggests that the mechanism of prey killing involves OMV fusion with the E. coli outer membrane. M. xanthus secretes GAPDH, which could potentially modulate the fusion of co-secreted OMVs with prey organisms in nature, enhancing their predatory activity.

  9. 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.

  10. Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium Novosphingobium pentaromativorans US6-1.

    Science.gov (United States)

    Yun, Sung Ho; Lee, Sang-Yeop; Choi, Chi-Won; Lee, Hayoung; Ro, Hyun-Joo; Jun, Sangmi; Kwon, Yong Min; Kwon, Kae Kyoung; Kim, Sang-Jin; Kim, Gun-Hwa; Kim, Seung Il

    2017-01-01

    Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMVNovo) are spherical in shape, and the average diameter of OMVNovo is 25-70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMVNovo. Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMVNovo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.

  11. Proteomics of photoreceptor outer segments identifies a subset of SNARE and Rab proteins implicated in membrane vesicle trafficking and fusion.

    Science.gov (United States)

    Kwok, Michael C M; Holopainen, Juha M; Molday, Laurie L; Foster, Leonard J; Molday, Robert S

    2008-06-01

    The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.

  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. 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.

  14. Bacterial outer membrane vesicle biogenesis: a new mechanism and its implications

    Directory of Open Access Journals (Sweden)

    Sandro Roier

    2016-05-01

    Full Text Available Outer membrane vesicle (OMV release by Gram-negative bacteria has been observed and studied for decades. First considered as a by-product of cell lysis, it soon became evident that OMVs are actively secreted from the outer membrane (OM of Gram-negative bacteria. Accordingly, these small particles (~ 10-300 nm in diameter consist mainly of OM components like phospholipids (PLs, OM proteins, and lipopolysaccharides or lipooligosaccharides. However, OMVs may also comprise periplasmic, inner membrane, or cytoplasmic components. Since the shedding of substantial amounts of OM material represents a significant energy cost to the bacterial cell, OMV production must have some vital biological functions for Gram-negative bacteria. Indeed, intense research on that topic revealed that OMVs play important roles in bacterial physiology and pathogenesis, ranging from secretion and delivery of biomolecules (for example, toxins, DNA, or quorum sensing molecules over stress response and biofilm formation to immunomodulation and adherence to host cells. Only recently researchers have begun to elucidate the mechanistic aspects of OMV formation, but a general mechanism for the biogenesis of these vesicles is still lacking. Here we review the findings and implications of our recent study published in Nature Communications (Roier S, et al. (2016 Nat. Commun. 7:10515, where we propose a novel and highly conserved bacterial OMV biogenesis mechanism based on PL accumulation in the outer leaflet of the OM. This mechanism might not only have important pathophysiological roles in vivo, but also represents the first general mechanism of OMV formation applicable to all Gram-negative bacteria.

  15. NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.

    Science.gov (United States)

    Schwechheimer, Carmen; Rodriguez, Daniel L; Kuehn, Meta J

    2015-06-01

    Outer membrane vesicles (OMVs) are ubiquitously secreted from the outer membrane (OM) of Gram-negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild-type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan (PG) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr, consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin-binding protein 4 (PBP4) during exponential growth. The data support a model in which NlpI negatively regulates PBP4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane (OM-PG) envelope stabilizing cross-links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild-type Gram-negative bacteria can use to modulate vesiculation is by altering PG-OM cross-linking via localized modulation of PG degradation and synthesis. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  16. Bacterial outer membrane vesicle biogenesis: a new mechanism and its implications.

    Science.gov (United States)

    Roier, Sandro; Zingl, Franz G; Cakar, Fatih; Schild, Stefan

    2016-05-10

    Outer membrane vesicle (OMV) release by Gram-negative bacteria has been observed and studied for decades. First considered as a by-product of cell lysis, it soon became evident that OMVs are actively secreted from the outer membrane (OM) of Gram-negative bacteria. Accordingly, these small particles (~ 10-300 nm in diameter) consist mainly of OM components like phospholipids (PLs), OM proteins, and lipopolysaccharides or lipooligosaccharides. However, OMVs may also comprise periplasmic, inner membrane, or cytoplasmic components. Since the shedding of substantial amounts of OM material represents a significant energy cost to the bacterial cell, OMV production must have some vital biological functions for Gram-negative bacteria. Indeed, intense research on that topic revealed that OMVs play important roles in bacterial physiology and pathogenesis, ranging from secretion and delivery of biomolecules (for example, toxins, DNA, or quorum sensing molecules) over stress response and biofilm formation to immunomodulation and adherence to host cells. Only recently researchers have begun to elucidate the mechanistic aspects of OMV formation, but a general mechanism for the biogenesis of these vesicles is still lacking. Here we review the findings and implications of our recent study published in Nature Communications (Roier S, et al. (2016) Nat. Commun. 7:10515), where we propose a novel and highly conserved bacterial OMV biogenesis mechanism based on PL accumulation in the outer leaflet of the OM. This mechanism might not only have important pathophysiological roles in vivo, but also represents the first general mechanism of OMV formation applicable to all Gram-negative bacteria.

  17. 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.

  18. 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...

  19. 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...

  20. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles

    OpenAIRE

    Donato, Gina M.; Goldsmith, Cynthia S.; Paddock, Christopher D.; Eby, Joshua C.; Gray, Mary C.; Hewlett, Erik L.

    2012-01-01

    B.pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B.pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B.pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin’s receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b an...

  1. Bacterial Outer Membrane Vesicles as a Delivery System for Virulence Regulation.

    Science.gov (United States)

    Yoon, Hyunjin

    2016-08-28

    Outer membrane vesicles (OMVs) are spherical nanostructures that are ubiquitously shed from gram-negative bacteria both in vitro and in vivo. Recent findings revealed that OMVs, which contain diverse components derived from the parent bacterium, play an important role in communication with neighboring bacteria and the environment. Furthermore, nanoscale proteoliposomes decorated with pathogen-associated molecules attract considerable attention as a non-replicative carrier for vaccines and drug materials. This review introduces recent advances in OMV biogenesis and discusses the roles of OMVs in the context of bacterial communication and virulence regulation. It also describes the remarkable accomplishments in OMV engineering for diverse therapeutic applications.

  2. 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.

  3. 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.

  4. 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.

  5. Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation.

    Science.gov (United States)

    Gamalier, Juliana P; Silva, Thiago P; Zarantonello, Victor; Dias, Felipe F; Melo, Rossana C N

    2017-01-01

    Secretion of membrane vesicles is an important biological process of both eukaryotic and prokaryotic cells. This process has been characterized in pathogenic bacteria, but is less clear in non-pathogenic bacteria from aquatic ecosystems. Here, we investigated, for the first time, the process of formation of outer membranes vesicles (OMVs), nanoscale vesicles extruded from the outer membrane (OM) of gram-negative bacteria, in cultures of freshwater bacteria after exposure or not to ultraviolet radiation (UVR) as an environmental stressor. Non-axenic cultures of freshwater bacteria isolated from a Brazilian aquatic ecosystem (Funil reservoir) were exposed or not to UVR (UVA+UVB) over a 3h period, during which cell density, viability and ultrastructure were analyzed. First, we showed that UVR induce bacterial death. UVR triggered significant negative effect on cell density after 3h of UVR treatment. This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe that enables the distinction of live/dead bacteria. Transmission electron microscopy (TEM) revealed changes indicative of cell death after 3h of UVR exposure, with significant increase of damaged cells compared to the control group. Second, we demonstrated that gram-negative bacteria release OMVs during normal growth and after UVR exposure. OMVs were clearly identified as round, membrane-bound vesicles budding off from the bacterial OM as isolated or clustered vesicles or free in the extracellular medium. Remarkably, quantitative TEM analyses showed that bacteria respond to UVR with increased formation of OMVs. Moreover, while OMVs numbers per intact or damaged cell did not differ in the untreated group, UVR led to a higher vesiculation by bacteria in process of death. This means that degenerating bacteria release OMVs before lysis and that this secretion might be an adaptive/protective response to rapid changes in environmental conditions such as UV radiation. Copyright

  6. Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant.

    Science.gov (United States)

    Turner, Lorinda; Praszkier, Judyta; Hutton, Melanie L; Steer, David; Ramm, Georg; Kaparakis-Liaskos, Maria; Ferrero, Richard L

    2015-08-01

    Multiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. H. pylori ΔtolB, Δpal and ΔtolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. H. pylori ΔtolB and Δpal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori. Moreover, it shows how H. pylori tolB-pal genes may be manipulated to develop "hypervesiculating" strains for vaccine purposes. © 2015 John Wiley & Sons Ltd.

  7. 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.

  8. Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina.

    Directory of Open Access Journals (Sweden)

    Michael W Stuck

    Full Text Available The neural retinal leucine zipper (Nrl knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl(-/- retina, rods are converted into functional cone-like cells. The Nrl(-/- retina is characterized by large undulations of the outer nuclear layer (ONL commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl(-/- retina. We report that rosettes first appear at postnatal day (P8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM are not uniformly formed in the Nrl(-/- retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl(-/- retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM.

  9. Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina.

    Science.gov (United States)

    Stuck, Michael W; Conley, Shannon M; Naash, Muna I

    2012-01-01

    The neural retinal leucine zipper (Nrl) knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl(-/-) retina, rods are converted into functional cone-like cells. The Nrl(-/-) retina is characterized by large undulations of the outer nuclear layer (ONL) commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl(-/-) retina. We report that rosettes first appear at postnatal day (P)8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM) are not uniformly formed in the Nrl(-/-) retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl(-/-) retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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.

  1. 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.

  2. Association of Acinetobacter baumannii EF-Tu with Cell Surface, Outer Membrane Vesicles, and Fibronectin

    Directory of Open Access Journals (Sweden)

    Shatha F. Dallo

    2012-01-01

    Full Text Available A conundrum has long lingered over association of cytosol elongation factor Tu (EF-Tu with bacterial surface. Here we investigated it with Acinetobacter baumannii, an emerging opportunistic pathogen associated with a wide spectrum of infectious diseases. The gene for A. baumannii EF-Tu was sequenced, and recombinant EF-Tu was purified for antibody development. EF-Tu on the bacterial surface and the outer membrane vesicles (OMVs was revealed by immune electron microscopy, and its presence in the outer membrane (OM and the OMV subproteomes was verified by Western blotting with the EF-Tu antibodies and confirmed by proteomic analyses. EF-Tu in the OM and the OMV subproteomes bound to fibronectin as detected by Western blot and confirmed by a label-free real-time optical sensor. The sensor that originates from photonic crystal structure in a total-Internal-reflection (PC-TIR configuration was functionalized with fibronectin for characterizing EF-Tu binding. Altogether, with a novel combination of immunological, proteomical, and biophysical assays, these results suggest association of A. baumannii EF-Tu with the bacterial cell surface, OMVs, and fibronectin.

  3. Outer membrane vesicles of Porphyromonas gingivalis elicit a mucosal immune response.

    Directory of Open Access Journals (Sweden)

    Ryoma Nakao

    Full Text Available We previously reported that mutation of galE in Porphyromonas gingivalis has pleiotropic effects, including a truncated lipopolysaccharide (LPS O-antigen and deglycosylation of the outer membrane protein OMP85 homolog. In the present study, further analysis of the galE mutant revealed that it produced little or no outer membrane vesicles (OMVs. Using three mouse antisera raised against whole cells of the P. gingivalis wild type strain, we performed ELISAs to examine the reactivity of these antisera with whole cells of the wild type or the galE mutant. All three antisera had significantly lower reactivity against the galE mutant compared to wild type. OMVs, but not LPS, retained the immunodominant determinant of P. gingivalis, as determined by ELISAs (with wild type LPS or OMVs as antigen and absorption assays. In addition, we assessed the capacity of OMVs as a vaccine antigen by intranasal immunization to BALB/c mice. Synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [Poly (I∶C], an agonist of Toll-like receptor 3 (TLR3, was used as the mucosal adjuvant. Vaccination with OMV elicited dramatically high levels of P. gingivalis-specific IgA in nasal washes and saliva, as well as serum IgG and IgA. In conclusion, the OMVs of P. gingivalis have an important role in mucosal immunogenicity as well as in antigenicity. We propose that P. gingivalis OMV is an intriguing immunogen for development of a periodontal disease vaccine.

  4. Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification.

    Science.gov (United States)

    Alves, Nathan J; Turner, Kendrick B; Walper, Scott A

    2016-11-16

    An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

  5. Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells.

    Science.gov (United States)

    Ojima, Yoshihiro; Yamaguchi, Kyota; Taya, Masahito

    2017-08-08

    Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N-terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW-GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW-GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW-GFP into OMVs through the increased OmpW-GFP expression on the ΔnlpI cells. Finally, the amount of OmpW-GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW-GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  6. Outer Membrane Vesicle Biosynthesis in Salmonella: Is There More to Gram-Negative Bacteria?

    Directory of Open Access Journals (Sweden)

    Joachim Reidl

    2016-08-01

    Full Text Available Recent research has focused on the biological role of outer membrane vesicles (OMVs, which are derived from the outer membranes (OMs of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16 provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S. Typhimurium.

  7. Outer Membrane Vesicle Biosynthesis in Salmonella: Is There More to Gram-Negative Bacteria?

    Science.gov (United States)

    Reidl, Joachim

    2016-08-16

    Recent research has focused on the biological role of outer membrane vesicles (OMVs), which are derived from the outer membranes (OMs) of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16) provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS) influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S Typhimurium. Copyright © 2016 Reidl.

  8. Modulation of bacterial outer membrane vesicle production by envelope structure and content.

    Science.gov (United States)

    Schwechheimer, Carmen; Kulp, Adam; Kuehn, Meta J

    2014-12-21

    Vesiculation is a ubiquitous secretion process of Gram-negative bacteria, where outer membrane vesicles (OMVs) are small spherical particles on the order of 50 to 250 nm composed of outer membrane (OM) and lumenal periplasmic content. Vesicle functions have been elucidated in some detail, showing their importance in virulence factor secretion, bacterial survival, and biofilm formation in pathogenesis. Furthermore, OMVs serve as an envelope stress response, protecting the secreting bacteria from internal protein misfolding stress, as well as external envelope stressors. Despite their important functional roles very little is known about the regulation and mechanism of vesicle production. Based on the envelope architecture and prior characterization of the hypervesiculation phenotypes for mutants lacking the lipoprotein, Lpp, which is involved in the covalent OM-peptidoglycan (PG) crosslinks, it is expected that an inverse relationship exists between OMV production and PG-crosslinked Lpp. In this study, we found that subtle modifications of PG remodeling and crosslinking modulate OMV production, inversely correlating with bound Lpp levels. However, this inverse relationship was not found in strains in which OMV production is driven by an increase in "periplasmic pressure" resulting from the accumulation of protein, PG fragments, or lipopolysaccharide. In addition, the characterization of an nlpA deletion in backgrounds lacking either Lpp- or OmpA-mediated envelope crosslinks demonstrated a novel role for NlpA in envelope architecture. From this work, we conclude that OMV production can be driven by distinct Lpp concentration-dependent and Lpp concentration-independent pathways.

  9. Proteomic characterization of the outer membrane vesicle of Pseudomonas putida KT2440.

    Science.gov (United States)

    Choi, Chi-Won; Park, Edmond Changkyun; Yun, Sung Ho; Lee, Sang-Yeop; Lee, Yeol Gyun; Hong, Yeonhee; Park, Kyeong Ryang; Kim, Sang-Hyun; Kim, Gun-Hwa; Kim, Seung Il

    2014-10-03

    Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440.

  10. 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.

  11. Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa.

    Science.gov (United States)

    Adams, Hendrik; Zeder-Lutz, Gabrielle; Schalk, Isabelle; Pattus, Franc; Celia, Hervé

    2006-08-01

    Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.

  12. Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli.

    Science.gov (United States)

    Cama, Jehangir; Bajaj, Harsha; Pagliara, Stefano; Maier, Theresa; Braun, Yvonne; Winterhalter, Mathias; Keyser, Ulrich F

    2015-11-04

    Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.

  13. 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.

  14. 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.

  15. 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

  16. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles.

    Science.gov (United States)

    Elhenawy, Wael; Debelyy, Mykhaylo O; Feldman, Mario F

    2014-03-11

    Outer membrane vesicles (OMV) are spherical membranous structures released from the outer membrane (OM) of Gram-negative bacteria. OMV have been proposed to play several different roles during both pathogenesis and symbiosis. Despite the fact that OMV were described several decades ago, their biogenesis is a poorly characterized process. Whether OMV are produced by an active mechanism or by passive disintegration of the OM is a still matter of controversy. Bacteroides fragilis and Bacteroides thetaiotaomicron are important members of the human microbiota. In this work, we determined and compared the protein compositions of OM and OMV from B. fragilis and B. thetaiotaomicron. SDS-PAGE analysis of both fractions revealed dramatically different protein profiles. Proteomic analysis of OM and OMV in B. fragilis identified more than 40 proteins found exclusively in OMV and more than 30 proteins detectable only in the OM. The OMV-specific proteome showed a high prevalence of glycosidases and proteases, some of which were shown to be active in vitro. Similar results were obtained for B. thetaiotaomicron. Most of the OMV-exclusive proteins were acidic. Based on these results, we propose that these species possess machinery devoted to selectively pack acidic proteins into the OMV. These OMV equipped with hydrolytic enzymes could help in securing nutrients for the benefit of the whole bacterial community present in the microbiota, uncovering a novel function for bacterial OMV. IMPORTANCE The members of genus Bacteroides are key players in the symbiosis between the human host and the gut microbiota. It is known for its ability to degrade a wide variety of glycans that are not substrates for human glycosidases. The cleaved glycans can be utilized by Bacteroides and other microbiota members, resulting in the production of short-chain fatty acids that are beneficial for the host. Although members of the genus Bacteroides are known to secrete different hydrolases, their secretion

  17. 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.

  18. 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.

  19. Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation.

    Science.gov (United States)

    Mashburn-Warren, Lauren; Howe, Jörg; Garidel, Patrick; Richter, Walter; Steiniger, Frank; Roessle, Manfred; Brandenburg, Klaus; Whiteley, Marvin

    2008-07-01

    Bacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4'-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.

  20. 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.

  1. 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.

  2. Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris

    Directory of Open Access Journals (Sweden)

    Niehaus Karsten

    2008-06-01

    Full Text Available Abstract Background Outer membrane vesicles (OMVs are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli. Results We have demonstrated that Xanthomonas campestris pv. campestris (Xcc releases OMVs into the culture supernatant during growth. A proteome study identified 31 different proteins that associate with the OMV fraction of which half are virulence-associated. A comparison with the most abundant outer membrane (OM proteins revealed that some proteins are enriched in the OMV fraction. This may be connected to differences in the LPS composition between the OMVs and the OM. Furthermore, a comparison of the OMV proteomes from two different culture media indicated that the culture conditions have an impact on the protein composition. Interestingly, the proteins that are common to both culture conditions are mainly involved in virulence. Conclusion Outer membrane vesicles released from the OM of Xcc contain membrane- and virulence-associated proteins. Future experiments will prove whether these structures can serve as "vehicles" for the transport of virulence factors into the host membrane.

  3. Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris.

    Science.gov (United States)

    Sidhu, Vishaldeep K; Vorhölter, Frank-Jörg; Niehaus, Karsten; Watt, Steven A

    2008-06-02

    Outer membrane vesicles (OMVs) are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli. We have demonstrated that Xanthomonas campestris pv. campestris (Xcc) releases OMVs into the culture supernatant during growth. A proteome study identified 31 different proteins that associate with the OMV fraction of which half are virulence-associated. A comparison with the most abundant outer membrane (OM) proteins revealed that some proteins are enriched in the OMV fraction. This may be connected to differences in the LPS composition between the OMVs and the OM. Furthermore, a comparison of the OMV proteomes from two different culture media indicated that the culture conditions have an impact on the protein composition. Interestingly, the proteins that are common to both culture conditions are mainly involved in virulence. Outer membrane vesicles released from the OM of Xcc contain membrane- and virulence-associated proteins. Future experiments will prove whether these structures can serve as "vehicles" for the transport of virulence factors into the host membrane.

  4. Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

    Directory of Open Access Journals (Sweden)

    Frederico Guilherme Coutinho Abath

    1990-03-01

    Full Text Available Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76 were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page. Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted.

  5. 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.

  6. 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.

  7. Crystal structure of outer membrane protein NMB0315 from Neisseria meningitidis.

    Directory of Open Access Journals (Sweden)

    Xiangyu Wang

    Full Text Available NMB0315 is an outer membrane protein of Neisseria meningitidis serogroup B (NMB and a potential candidate for a broad-spectrum vaccine against meningococcal disease. The crystal structure of NMB0315 was solved by single-wavelength anomalous dispersion (SAD at a resolution of 2.4 Å and revealed to be a lysostaphin-type peptidase of the M23 metallopeptidase family. The overall structure consists of three well-separated domains and has no similarity to any previously published structure. However, only the topology of the carboxyl-terminal domain is highly conserved among members of this family, and this domain is a zinc-dependent catalytic unit. The amino-terminal domain of the structure blocks the substrate binding pocket in the carboxyl-terminal domain, indicating that the wild-type full-length protein is in an inactive conformational state. Our studies improve the understanding of the catalytic mechanism of M23 metallopeptidases.

  8. Genetic Manipulation of Outer Membrane Permeability: Generating Porous Heterogeneous Catalyst Analogs in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Patel, TN; Park, AHA; Bantat, S

    2014-12-01

    The limited permeability of the E. coli outer membrane can significantly hinder whole-cell biocatalyst performance. In this study, the SARS coronavirus small envelope protein (SCVE) was expressed in E. coli cells previously engineered for periplasmic expression of carbonic anhydrase (CA) activity. This maneuver increased small molecule uptake by the cells, resulting in increased apparent CA activity of the biocatalysts. The enhancements in activity were quantified using methods developed for traditional heterogeneous catalysis. The expression of the SCVE protein was found to significantly reduce the Thiele moduli (phi), as well as increase the effectiveness factors (eta), effective diffusivities (D-e), and permeabilities (P) of the biocatalysts. These catalytic improvements translated into superior performance of the biocatalysts for the precipitation of calcium carbonate from solution which is an attractive strategy for long-term sequestration of captured carbon dioxide. Overall, these results demonstrate that synthetic biology approaches can be used to enhance heterogeneous catalysts incorporated into microbial whole-cell scaffolds.

  9. 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.

  10. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles.

    Science.gov (United States)

    Donato, Gina M; Goldsmith, Cynthia S; Paddock, Christopher D; Eby, Joshua C; Gray, Mary C; Hewlett, Erik L

    2012-02-17

    Bordetella pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B. pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B. pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin's receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

  11. Physiopathological roles of spontaneously released outer membrane vesicles of Bordetella pertussis.

    Science.gov (United States)

    Gasperini, Gianmarco; Arato, Vanessa; Pizza, Mariagrazia; Aricò, Beatrice; Leuzzi, Rosanna

    2017-10-05

    Bordetella pertussis has been shown to release outer membrane vesicles (OMV) both in vitro and in vivo but little is known about their biological role during the initial phases of B. pertussis infection of the airways. We have demonstrated that OMV are released by B. pertussis in a human ciliated-airway cell model and purified vesicles can interact with host cells. Binding and uptake are strictly Bvg-regulated and OMV-associated. Pertussis toxin contributes to host-cell intoxication. Furthermore, we have shown that OMV act as iron-delivery systems complementing the B. pertussis growth defect in iron-limiting conditions. We have proved that OMV play different roles in B. pertussis physiopathology and we opened new perspectives to be further investigated.

  12. Evaluation of a Burkholderia pseudomallei Outer Membrane Vesicle Vaccine in Nonhuman Primates.

    Science.gov (United States)

    Petersen, Hailey; Nieves, Wildaliz; Russell-Lodrigue, Kasi; Roy, Chad J; Morici, Lisa A

    2014-01-01

    Burkholderia pseudomallei (Bps)is the causative agent of melioidosis and is endemic in regions of northern Australia and Southeast Asia. Bps is inherently resistant to multiple antibiotics and is considered a potential biological warfare agent by the U.S. DHHS. Therefore, effective vaccines are necessary to prevent natural infection and to safeguard against biological attack with this organism. In our previous work we have shown that immunization with naturally derived outer membrane vesicles (OMVs) from Bps provides significant protection against lethal aerosol and systemic infection in BALB/c mice. In this work, we evaluated the safety and immunogenicity of escalating doses of OMV vaccine in rhesus macaques. We show that immunization of rhesus macaques with Bps OMVs generates humoral immuneresponses to protective protein and polysaccharide antigens without any associated toxicity or reactogenicity. These results lay the groundwork for evaluation of protective efficacy of the OMV vaccine in the nonhuman primate model of melioidosis.

  13. 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.

  14. 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.

  15. 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.

  16. Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori.

    Science.gov (United States)

    Voland, Petra; Hafsi, Nadia; Zeitner, Marco; Laforsch, Stephanie; Wagner, Hermann; Prinz, Christian

    2003-07-01

    Outer membrane proteins (OMPs) are incorporated into the outer plasma membrane of Helicobacter pylori and are important for, e.g., ion transport, adherence, structural and osmotic stability, and bacterial virulence but may also be antigenic due to their surface exposure. Previous proteome-based approaches with H. pylori lysates determined a strong serological reaction towards two H. pylori OMPs, HpaA (TIGR HP0797) and Omp18 (TIGR HP1125). PCR was used to detect DNA encoding the two proteins, and a positive signal was found in all H. pylori strains tested. Proteins were cloned and expressed in the human kidney cell line HK293 with the QiaExpressionist system with a C-terminal His tag. Only sera from infected persons showed a positive reaction with the recombinant proteins. Recombinant HpaA (rHpaA) and rOmp18 were incubated with human peripheral blood mononuclear cells and induced secretion of interleukin-12 (IL-12) and IL-10 from these cells. To determine the effect on antigen-presenting cells, human blood monocytic and dendritic cells (DCs) were isolated by magnetic cell separation. rOmp18 and rHpaA strongly stimulated major histocompatibility class II and CD83 expression 7- to 10-fold on isolated DCs. rHpaA and rOmp18 failed to stimulate IL-8 secretion from monocytes but increased secretion of IL-12 and IL-10 from DCs significantly. In summary, HpaA and Omp18 are recognized by human dendritic cells and induce their maturation as well as antigen presentation. HpaA and Omp18 of H. pylori thereby appear to have a specific antigenic potential in humans.

  17. A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles.

    Science.gov (United States)

    Roier, Sandro; Blume, Thomas; Klug, Lisa; Wagner, Gabriel E; Elhenawy, Wael; Zangger, Klaus; Prassl, Ruth; Reidl, Joachim; Daum, Günther; Feldman, Mario F; Schild, Stefan

    2015-05-01

    Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. Copyright © 2015. Published by Elsevier GmbH.

  18. [Bacterial outer membrane vesicles as nano carriers to study immunological activities].

    Science.gov (United States)

    Qi, Chen; Min, W U; Hongzhen, Bai; Zeling, Guo; Jun, Zhou; Qingqing, Wang; Guping, Tang

    2017-03-25

    Objective: To prepare a nano-carrier based on combining bacterial outer membrane vesicles (OMV) with three block polymer pluronic F127 (PEO100-PPO65-PEO100) (OMV-F127) and to investigate its immunological activity. Methods: Attenuated salmonella (sal) was cultivated. OMV were separated by centrifugal ultrafiltration or ultrasonication, and OMV-F127 was prepared by mechanical extrudation method. The protein contents and compositions were tested with BCA and SDS-PAGE; the morphology of OMV, F127 and OMV-F127 were observed with FM and TEM; the particle sizes and their zeta potential were determined with DLS. Mouse macrophage RAW246.7 cells were treated with OMV-F127 (50 μg/mL, 100 μg/mL) in vitro, and the concentrations of IL-12, TNF-α and IFN-γ in culture supernatant were measured with ELISA kits. Results: The contents of protein in separated OMV by centrifugal ultrafiltration and ultrasonication were 2.8 mg/mL and 2.7 mg/mL, respectively. SDS-PAGE showed the marker protein OmpF/C in OMV. Under the FM and TEM, ball-like structure of F127 and OMV-F127 was observed. Size analysis revealed that the diameters of OMV, F127 and OMV-F127 were 72±2 nm, 90±3 nm and 92±2 nm, respectively. ELISA tests revealed that OMV-F127 significantly stimulated the secretion of IL-12, TNF-α and IFN-γ in RAW246.7 cells. Conclusion: A nano-carrier based on bacterial outer membrane vesicles has been prepared, which can stimulate the secretion of cytokines and may have immunomodulatory effects.

  19. Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells.

    Science.gov (United States)

    Ojima, Yoshihiro; Mohanadas, Thivagaran; Kitamura, Kosei; Nunogami, Shota; Yajima, Reiki; Taya, Masahito

    2017-04-01

    Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37 kDa. Nanoliquid chromatography-tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.

  20. 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.

  1. 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.

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

    OpenAIRE

    Elza FT Belo; Farhat,Calil K; De Gaspari,Elizabeth N.

    2010-01-01

    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...

  3. 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.

  4. 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.

  5. Focus on the Outer Membrane Factor OprM, the Forgotten Player from Efflux Pumps Assemblies

    Directory of Open Access Journals (Sweden)

    Gilles Phan

    2015-11-01

    Full Text Available Antibiotics have been used extensively during several decades and we are now facing the emergence of multidrug resistant strains. It has become a major public concern, urging the need to discover new strategies to combat them. Among the different ways used by bacteria to resist antibiotics, the active efflux is one of the main mechanisms. In Gram-negative bacteria the efflux pumps are comprised of three components forming a long edifice crossing the complete cell wall from the inside to the outside of the cell. Blocking these pumps would permit the restoration of the effectiveness of the current antibiotherapy which is why it is important to increase our knowledge on the different proteins involved in these complexes. A tremendous number of experiments have been performed on the inner membrane protein AcrB from Escherichia coli and, to a lesser extent, the protein partners forming the AcrAB-TolC pump, but less information is available concerning the efflux pumps from other virulent Gram-negative bacteria. The present review will focus on the OprM outer membrane protein from the MexAB-OprM pump of Pseudomonas aeruginosa, highlighting similarities and differences compare to the archetypal AcrAB-TolC in terms of structure, function, and assembly properties.

  6. 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.

  7. Characterization and Vaccine Potential of Outer Membrane Vesicles Produced by Haemophilus parasuis.

    Directory of Open Access Journals (Sweden)

    William D McCaig

    Full Text Available Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria.

  8. Quantitative proteomics reveals distinct differences in the protein content of outer membrane vesicle vaccines.

    Science.gov (United States)

    van de Waterbeemd, Bas; Mommen, Geert P M; Pennings, Jeroen L A; Eppink, Michel H; Wijffels, René H; van der Pol, Leo A; de Jong, Ad P J M

    2013-04-05

    At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in more detail, the protein content of detergent-extracted OMV is compared with two detergent-free alternatives. A novel proteomics strategy has been developed that allows quantitative analysis of many biological replicates despite inherent multiplex restrictions of dimethyl labeling. This enables robust statistical analysis of relative protein abundance. The comparison with detergent-extracted OMV reveales that detergent-free OMV are enriched with membrane (lipo)proteins and contain less cytoplasmic proteins due to a milder purification process. These distinct protein profiles are substantiated with serum blot proteomics, confirming enrichment with immunogenic proteins in both detergent-free alternatives. Therefore, the immunogenic protein content of OMV vaccines depends at least partially on the purification process. This study demonstrates that detergent-free OMV have a preferred composition.

  9. Outer Membrane Vesicles (OMVs of Gram-negative Bacteria: A Perspective Update

    Directory of Open Access Journals (Sweden)

    Arif Tasleem Jan

    2017-06-01

    Full Text Available Outer Membrane Vesicles (OMVs of Gram-negative bacteria are spherical membrane-enclosed entities of endocytic origin. Reported in the consortia of different bacterial species, production of OMVs into extracellular milieu seems essential for their survival. Enriched with bioactive proteins, toxins, and virulence factors, OMVs play a critical role in the bacteria-bacteria and bacteria-host interactions. Emergence of OMVs as distinct cellular entities helps bacteria in adaptating to diverse niches, in competing with other bacteria to protect members of producer species and more importantly play a crucial role in host-pathogen interaction. Composition of OMV, their ability to modulate host immune response, along with coordinated secretion of bacterial effector proteins, endows them with the armory, which can withstand hostile environments. Study of the OMV production under natural and diverse stress conditions has broadened the horizons, and also opened new frontiers in delineating the molecular machinery involved in disease pathogenesis. Playing diverse biological and pathophysiological functions, OMVs hold a great promise in enabling resurgence of bacterial diseases, in concomitance with the steep decline in the efficiency of antibiotics. Having multifaceted role, their emergence as a causative agent for a series of infectious diseases increases the probability for their exploitation in the development of effective diagnostic tools and as vaccines against diverse pathogenic species of Gram-negative origin.

  10. High throughput nanoparticle tracking analysis for monitoring outer membrane vesicle production.

    Science.gov (United States)

    Gerritzen, Matthias J H; Martens, Dirk E; Wijffels, René H; Stork, Michiel

    2017-01-01

    Outer membrane vesicles (OMVs) are spherical membrane nanoparticles released by Gram-negative bacteria. OMVs can be quantified in complex matrices by nanoparticle tracking analysis (NTA). NTA can be performed in static mode or with continuous sample flow that results in analysis of more particles in a smaller time-frame. Flow measurements must be performed manually despite the availability of a sample changer on the NanoSight system. Here we present a method for automated measurements in flow mode. OMV quantification in flow mode results in lower variance in particle quantification (coefficient of variation (CV) of 6%, CV static measurements of 14%). Sizing of OMVs was expected to be less favorable in flow mode due to the increased movement of the particles. However, we observed a CV of 3% in flow mode and a CV of 8% in static measurements. Flow rates of up to 5 µL/min displayed correct size and particle measurements, however, particle concentration was slightly lower than in static measurements. The automated method was used to assess OMV release of batch cultures of Neisseria meningitidis. The bacteria released more OMVs in stationary growth phase, while the size of the vesicles remained constant throughout the culture. Taken together, this study shows that automated measurements in flow mode can be established with advanced scripting to reduce the workload for the user.

  11. 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.

  12. 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.

  13. Diversity of TonB-dependent outer-membrane proteins in plant-associated strains of Pseudomonas fluorescens

    Science.gov (United States)

    Genomic sequences of ten strains of plant-associated Pseudomonas spp. were surveyed for the presence of TonB-dependent outer-membrane proteins (TBDPs), which function in the uptake of substrates from the environment by many Gram-negative bacteria. The ten strains, representing P. fluorescens, P. ch...

  14. Variation in the TonB-dependent Outer-Membrane Proteins in Plant-Associated Strains of Pseudomonas fluorescens

    Science.gov (United States)

    Nutrient acquisition is key to the ecological fitness of environmental bacteria such as Pseudomonas fluorescens and TonB-dependent outer-membrane proteins are important components of the cellular machinery for the uptake of substrates from the environment. Genomic sequences of ten strains of plant-a...

  15. 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

  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. 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

  18. 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

  19. The use of outer membrane proteins as an exposure surface for foreign antigens in AIDS vaccine methodology and AIDS diagnostics

    NARCIS (Netherlands)

    Soede WWD; Hegger I

    1992-01-01

    A live recombinant bacteria or virus with HIV determinants exposed at the outermembrane is one strategy for AIDS vaccine development. Two HIV determinants that showed neutralization capacity in in- vitro experiments were tested for their expression in PhoE outer membrane protein of E coli K12.

  20. 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

  1. 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

  2. The ultrastructure of Ignicoccus: Evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon

    Directory of Open Access Journals (Sweden)

    Reinhard Rachel

    2002-01-01

    Full Text Available A novel genus of hyperthermophilic, strictly chemolithotrophic archaea, Ignicoccus, has been described recently, with (so far three isolates in pure culture. Cells were prepared for ultrastructural investigation by cultivation in cellulose capillaries and processing by high-pressure freezing, freeze-substitution and embedding in Epon. Cells prepared in accordance with this protocol consistently showed a novel cell envelope structure previously unknown among the Archaea: a cytoplasmic membrane; a periplasmic space with a variable width of 20 to 400 nm, containing membrane-bound vesicles; and an outer sheath, approximately 10 nm wide, resembling the outer membrane of gram-negative bacteria. This sheath contained three types of particles: numerous tightly, irregularly packed single particles, about 8 nm in diameter; pores with a diameter of 24 nm, surrounded by tiny particles, arranged in a ring with a diameter of 130 nm; and clusters of up to eight particles, each particle 12 nm in diameter. Freeze-etched cells exhibited a smooth surface, without a regular pattern, with frequent fracture planes through the outer sheath, indicating the presence of an outer membrane and the absence of an S-layer. The study illustrates the novel complex architecture of the cell envelope of Ignicoccus as well as the importance of elaborate preparation procedures for ultrastructural investigations.

  3. 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...

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. Uptake of Helicobacter pylori outer membrane vesicles by gastric epithelial cells.

    Science.gov (United States)

    Parker, Heather; Chitcholtan, Kenny; Hampton, Mark B; Keenan, Jacqueline I

    2010-12-01

    Helicobacter pylori bacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pylori is considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA(+) strain and OMV from a VacA(-) isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA(+) strain was inhibited by H. pylori LPS (58% inhibition with 50 μg/ml LPS), while uptake of OMV from the VacA(-) mutant strain was less affected (25% inhibition with 50 μg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA(-) mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 μg/ml chlorpromazine), while uptake of OMV from the VacA(+) strain was less affected (25% inhibition with 15 μg/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization.

  11. Uptake of Helicobacter pylori Outer Membrane Vesicles by Gastric Epithelial Cells▿

    Science.gov (United States)

    Parker, Heather; Chitcholtan, Kenny; Hampton, Mark B.; Keenan, Jacqueline I.

    2010-01-01

    Helicobacter pylori bacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pylori is considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA+ strain and OMV from a VacA− isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA+ strain was inhibited by H. pylori LPS (58% inhibition with 50 μg/ml LPS), while uptake of OMV from the VacA− mutant strain was less affected (25% inhibition with 50 μg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA− mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 μg/ml chlorpromazine), while uptake of OMV from the VacA+ strain was less affected (25% inhibition with 15 μg/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization. PMID:20876296

  12. Outer Membrane Permeability of Cyanobacterium Synechocystis sp. Strain PCC 6803: Studies of Passive Diffusion of Small Organic Nutrients Reveal the Absence of Classical Porins and Intrinsically Low Permeability.

    Science.gov (United States)

    Kowata, Hikaru; Tochigi, Saeko; Takahashi, Hideyuki; Kojima, Seiji

    2017-10-01

    The outer membrane of heterotrophic Gram-negative bacteria plays the role of a selective permeability barrier that prevents the influx of toxic compounds while allowing the nonspecific passage of small hydrophilic nutrients through porin channels. Compared with heterotrophic Gram-negative bacteria, the outer membrane properties of cyanobacteria, which are Gram-negative photoautotrophs, are not clearly understood. In this study, using small carbohydrates, amino acids, and inorganic ions as permeation probes, we determined the outer membrane permeability of Synechocystis sp. strain PCC 6803 in intact cells and in proteoliposomes reconstituted with outer membrane proteins. The permeability of this cyanobacterium was >20-fold lower than that of Escherichia coli The predominant outer membrane proteins Slr1841, Slr1908, and Slr0042 were not permeable to organic nutrients and allowed only the passage of inorganic ions. Only the less abundant outer membrane protein Slr1270, a homolog of the E. coli export channel TolC, was permeable to organic solutes. The activity of Slr1270 as a channel was verified in a recombinant Slr1270-producing E. coli outer membrane. The lack of putative porins and the low outer membrane permeability appear to suit the cyanobacterial autotrophic lifestyle; the highly impermeable outer membrane would be advantageous to cellular survival by protecting the cell from toxic compounds, especially when the cellular physiology is not dependent on the uptake of organic nutrients.IMPORTANCE Because the outer membrane of Gram-negative bacteria affects the flux rates for various substances into and out of the cell, its permeability is closely associated with cellular physiology. The outer membrane properties of cyanobacteria, which are photoautotrophic Gram-negative bacteria, are not clearly understood. Here, we examined the outer membrane of Synechocystis sp. strain PCC 6803. We revealed that it is relatively permeable to inorganic ions but is markedly less

  13. A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans.

    Directory of Open Access Journals (Sweden)

    Marija Pinne

    Full Text Available Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via fresh water and colonization of the renal tubules of their reservoir hosts or infection of accidental hosts, including humans. Bacterial outer membrane proteins (OMPs, particularly those with surface-exposed regions, play crucial roles in virulence mechanisms of pathogens and the adaptation to various environmental conditions, including those of the mammalian host. Little is known about the surface-exposed OMPs in Leptospira, particularly those with outer membrane-spanning domains. Herein, we describe a comprehensive strategy for identification and characterization of leptospiral transmembrane OMPs. The genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 allowed us to employ the beta-barrel prediction programs, PRED-TMBB and TMBETA-NET, to identify potential transmembrane OMPs. Several complementary methods were used to characterize four novel OMPs, designated OmpL36, OmpL37, OmpL47 and OmpL54. In addition to surface immunofluorescence and surface biotinylation, we describe surface proteolysis of intact leptospires as an improved method for determining the surface exposure of leptospiral proteins. Membrane integration was confirmed using techniques for removal of peripheral membrane proteins. We also demonstrate deficiencies in the Triton X-114 fractionation method for assessing the outer membrane localization of transmembrane OMPs. Our results establish a broadly applicable strategy for the elucidation of novel surface-exposed outer membrane-spanning proteins of Leptospira, an essential step in the discovery of potential virulence factors, diagnostic antigens and vaccine candidates.

  14. The outer membrane cytochromes of Shewanella oneidensis MR-1 are lipoproteins.

    Science.gov (United States)

    Myers, C R; Myers, J M

    2004-01-01

    To determine if the outer membrane (OM) cytochromes OmcA and OmcB of the metal-reducing bacterium Shewanella oneidensis MR-1 are lipoproteins, and to assess cell surface exposure of the cytochromes by radioiodination. In anaerobic MR-1 cells grown with (3)H-palmitoleic acid, both OmcA and OmcB were radiolabelled. The identities of these bands were confirmed by the absence of each radiolabelled band in the respective mutants lacking individual OM cytochromes. Radioiodination of cell surface proteins in anaerobic cells resulted in (125)I-labelled OmcA. The identity of this band was confirmed by its absence in an OmcA-minus mutant. A ubiquitous radioiodinated band that migrates similarly to OmcB precluded the ability to determine the potential cell surface exposure of OmcB by this method. Both OmcA and OmcB are lipoproteins, and OmcA is cell surface exposed. The lipoprotein modification of these OM cytochromes could be important for their localization or incorporation into the OM. The cell surface exposure of OmcA could allow it to directly transfer electrons to extracellular electron acceptors (e.g. manganese oxides) and is consistent with its in vivo role.

  15. Colistin resistance associated with outer membrane protein change in Klebsiella pneumoniae and Enterobacter asburiae.

    Science.gov (United States)

    Kádár, Béla; Kocsis, Béla; Tóth, Ákos; Kristóf, Katalin; Felső, Péter; Kocsis, Béla; Böddi, Katalin; Szabó, Dóra

    2017-06-01

    In this study, outer membrane proteins (OMPs) of colistin-resistant Klebsiella pneumoniae and Enterobacter asburiae were analyzed. One colistin-susceptible and three colistin-resistant K. pneumoniae sequence type 258 strains as well as one colistin-susceptible E. asburiae and its colistin-heteroresistant counterpart strain were involved in the study. OMP analysis of each strain was performed by microchip method. Matrix-assisted laser desorption ionization time of flight/mass spectrometry (MALDI-TOF/MS) investigation was carried out after separation of OMPs by two-dimensional gel electrophoresis and in-gel digestion. The MALDI-TOF/MS analysis of OMPs in the colistin-susceptible K. pneumoniae found 16 kDa proteins belonging to the LysM domain/BON superfamily, as well as DNA starvation proteins, whereas OmpX and OmpW were detected in the colistin-resistant counterpart strains. OmpC and OmpW were detected in the colistin-susceptible E. asburiae, whereas OmpA and OmpX were identified in the colistin-resistant counterpart. This study demonstrated that OMP differences were between colistin-susceptible and -resistant counterpart strains. The altered Gram-negative cell wall may contribute to acquired colistin resistance in Enterobacteriaceae.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5.

    Science.gov (United States)

    Hartney, Sierra L; Mazurier, Sylvie; Kidarsa, Teresa A; Quecine, Maria Carolina; Lemanceau, Philippe; Loper, Joyce E

    2011-04-01

    The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved β-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.

  1. 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

  2. Outer membrane cytochromes/flavin interactions in Shewanella spp.-A molecular perspective.

    Science.gov (United States)

    Babanova, Sofia; Matanovic, Ivana; Cornejo, Jose; Bretschger, Orianna; Nealson, Kenneth; Atanassov, Plamen

    2017-05-31

    Extracellular electron transfer (EET) is intrinsically associated with the core phenomena of energy harvesting/energy conversion in natural ecosystems and biotechnology applications. However, the mechanisms associated with EET are complex and involve molecular interactions that take place at the "bionano interface" where biotic/abiotic interactions are usually explored. This work provides molecular perspective on the electron transfer mechanism(s) employed by Shewanella oneidensis MR-1. Molecular docking simulations were used to explain the interfacial relationships between two outer-membrane cytochromes (OMC) OmcA and MtrC and riboflavin (RF) and flavin mononucleotide (FMN), respectively. OMC-flavin interactions were analyzed by studying the electrostatic potential, the hydrophilic/hydrophobic surface properties, and the van der Waals surface of the OMC proteins. As a result, it was proposed that the interactions between flavins and OMCs are based on geometrical recognition event. The possible docking positions of RF and FMN to OmcA and MtrC were also shown.

  3. Projection maps of three members of the KdgM outer membrane protein family.

    Science.gov (United States)

    Signorell, Gian A; Chami, Mohamed; Condemine, Guy; Schenk, Andreas D; Philippsen, Ansgar; Engel, Andreas; Remigy, Herve W

    2007-12-01

    We present the projection structures of the three outer membrane porins KdgM and KdgN from Erwinia chrysanthemi and NanC from Escherichia coli, based on 2D electron crystallography. A wide screening of 2D crystallization conditions yielded tubular crystals of a suitable size and quality to perform high-resolution electron microscopy. Data processing of untilted samples allowed us to separate the information of the two crystalline layers and resulted in projection maps to a resolution of up to 7A. All three proteins exhibit a similar putative beta-barrel structure and the three crystal forms have the same symmetry. However, there are differences in the packing arrangements of the monomers as well as the densities of the projections. To interpret these projections, secondary structure prediction was performed using beta-barrel specific prediction algorithms. The predicted transmembrane beta-barrels have a high similarity in the arrangement of the putative beta-strands and the loops, but do not match those of OmpG, a related protein porin whose structure was solved.

  4. 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.

  5. Molecular recognition in myxobacterial outer membrane exchange: Functional, social and evolutionary implications

    Science.gov (United States)

    Wall, Daniel

    2014-01-01

    Summary Through cooperative interactions, bacteria can build multicellular communities. To ensure that productive interactions occur, bacteria must recognize their neighbors and respond accordingly. Molecular recognition between cells is thus a fundamental behavior, and in bacteria important discoveries have been made. This MicroReview focuses on a recently described recognition system in myxobacteria that is governed by a polymorphic cell surface receptor called TraA. TraA regulates outer membrane exchange (OME), whereby myxobacterial cells transiently fuse their OMs to efficiently transfer proteins and lipids between cells. Unlike other transport systems, OME is rather indiscriminate in what OM goods are transferred. In contrast, the recognition of partnering cells is discriminatory and only occurs between cells that bear identical or closely related TraA proteins. Therefore TraA functions in kin recognition and, in turn, OME helps regulate social interactions between myxobacteria. Here, I discuss and speculate on the social and evolutionary implications of OME and suggest it helps to guide their transition from free-living cells into coherent and functional populations. PMID:24261719

  6. 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.

  7. 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.

  8. 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.

  9. Ultrastructural characteristics of ostrich eggshell: outer shell membrane and the calcified layers

    Directory of Open Access Journals (Sweden)

    P.D.G. Richards

    2000-07-01

    Full Text Available The ultrastructure of the eggshell of the domestic hen has been well researched and structural studies of other avian species, such as the ostrich, often base their interpretation of egg shell structure on that of the chicken. In the ostrich, lowered hatchability and hatching trauma may be due to shell ultrastructural abnormalities. In the present study the ultrastructure of the calcified portion, and the outer shell membrane (OSM, of domesticated ostrich eggshells was investigated using standard electron microscopic techniques. Transmission and scanning electron microscopy studies demonstrated intimate contact between cup-shaped structures present on the OSMand the mammillary layer of the calcified portion of the shell. The initial calcium carbonate growth of the calcified shell was of a dendritic nature with nucleation sites on the surface of the cup's contents. The dendritic growth gave way to a more randomly-orientated, smaller crystallite growth structure, which changed in formas it neared the vertical crystal layer (VCL. The VCL is described as being both amorphous and 'crumbly' depending on the plane of fracture. These observations suggest that firstly, initial calcification is contained within the cups and is then directed outwards to formthe shell and that secondly, the VCL may contain an evolutionary, calcified cuticular layer. These observations serve as a baseline for studies investigating the effect of shell structure and strength on hatchling trauma and the influence of maternal diet.

  10. Fast electromechanical amplification in the lateral membrane of the outer hair cell.

    Science.gov (United States)

    Santos-Sacchi, Joseph; Navarrete, Enrique; Song, Lei

    2009-01-01

    Outer hair cells provide amplification within the mammalian cochlea to enhance audition. The mechanism is believed to reside within the lateral membrane of the cell that houses an expansive array of molecular motors, identified as prestin, which drives somatic electromotility. By measuring nonlinear capacitance, the electrical signature of electromotility, at kilohertz rates we have uncovered new details of the early molecular events that arise from voltage perturbations of prestin. We show that dynamic changes in motor state probability occur within the kilohertz range, and signify an amplificatory event. Additionally, we show a lack of effect of Cl driving force, an absence of cell length effect (indicating that the kinetics does not vary across auditory frequency), and the first demonstration of the time dependence of tension induced amplificatory shifts. The process we have identified, where the stimulus-response function shifts in time along the stimulus axis in a multi-exponential manner, bears similarities to those components of adaptation found in the OHC stereociliar transducer identified recently. As with the forward transducer, the speed of the reverse transducer amplificatory event consequently impacts on high frequency peripheral auditory processing.

  11. 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.

  12. 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.

  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. Outer membrane vesicles (OMV) production of Neisseria meningitidis serogroup B in batch process.

    Science.gov (United States)

    Santos, Sílvia; Arauz, Luciana Juncioni de; Baruque-Ramos, Júlia; Lebrun, Ivo; Carneiro, Sylvia Mendes; Barreto, Sandra Alves; Schenkman, Rocilda Perazzini Furtado

    2012-09-14

    Serogroup B outer membrane vesicles (OMV) with iron regulated proteins (IRP) from Neisseria meningitidis constitute the antigen for the vaccine against the disease caused by this bacterium. Aiming to enhance final OMV concentration, seven batch experiments were carried out under four different conditions: (i) with original Catlin medium; (ii) with original Catlin medium and lactate and amino acids pulse at the 6th cultivation hour; (iii) with Catlin medium with double initial concentrations of lactate and amino acids and (iv) Catlin medium without glycerol and with double initial concentrations of lactate and amino acids. The cultivation experiments were carried out in a 7-L bioreactor under the following conditions: 36°C, 0.5atm, overlay air 1L/min, agitation: 250-850 rpm, and O(2) control at 10%, 20 h. After lactate and amino acids exhaustion, cell growth reached stationary phase and a significant release increase of OMV was observed. According to the Luedeking & Piret model, OMV liberation is non-growth associated. Glycerol was not consumed during cultivation. The maximum OMV concentration value attained was 162 mg/L with correspondent productivity of 8.1mg/(Lh) employing Catlin medium with double initial concentrations of lactate and amino acids. The obtained OMV satisfied constitution and protein pattern criteria and were suitable for vaccine production. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Impact of MenBvac, an outer membrane vesicle (OMV) vaccine, on the meningococcal carriage.

    Science.gov (United States)

    Delbos, Valérie; Lemée, Ludovic; Bénichou, Jacques; Berthelot, Gilles; Deghmane, Ala-Eddine; Leroy, Jean-Philippe; Houivet, Estelle; Hong, Eva; Taha, Muhamed-Kheir; Caron, François

    2013-09-13

    The aim of the study was to analyze the impact of MenBvac, an outer membrane vesicle (OMV) vaccine against P1.7,16 strains, on meningococcal carriage. During a B:14:P1.7,16/ST-32 outbreak in Normandy (France), children aged 1-7 years were randomly selected to participate in the study. Among the 1082 volunteers, there were 17 Neisseria meningitidis carriers (carriage rate of 1.57%). MenBvac vaccination appeared associated with lower carriage rate, i.e., 0.31% among the vaccinated children versus 2.10% among the non-vaccinated (p=0.03). The beneficial effect on carriage was observed regardless of the strain serogroup. OMV-vaccinated mice also showed reduction of bacterial acquisition of OMV-homolog and hererolog strains in respiratory pathways after intranasal challenge. These results suggest that meningococcal OMV-based vaccines reduce meningococcal carriage and may hence confer herd immunity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Outer Membrane Vesicles of Helicobacter pylori TK1402 are Involved in Biofilm Formation

    Directory of Open Access Journals (Sweden)

    Ochiai Kuniyasu

    2009-09-01

    Full Text Available Abstract Background Helicobacter pylori forms biofilms on glass surfaces at the air-liquid interface in in vitro batch cultures; however, biofilms of H. pylori have not been well characterized. In the present study, we analyzed the ability of H. pylori strains to form biofilms and characterized the underlying mechanisms of H. pylori biofilm formation. Results Strain TK1402 showed strong biofilm forming ability relative to the other strains in Brucella broth supplemented with 7% FCS. The strong biofilm forming ability of TK1402 is reflected the relative thickness of the biofilms. In addition, outer membrane vesicles (OMV were detected within the matrix of only the TK1402 biofilms. Biofilm formation was strongly correlated with the production of OMV in this strain. We further observed that strain TK1402 did not form thick biofilms in Brucella broth supplemented with 0.2% β-cyclodextrin. However, the addition of the OMV-fraction collected from TK1402 could enhance biofilm formation. Conclusion The results suggested that OMV produced from TK1402 play an important role in biofilm formation in strain TK1402.

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

    Science.gov (United States)

    Chen, Yong; Liu, Liguo; Fu, Hua; Wei, Candong; Jin, Qi

    2014-10-31

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    Pramod K Rompikuntal

    Full Text Available Outer membrane vesicles (OMVs are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV.In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8 cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37.Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

  19. Designer outer membrane vesicles as immunomodulatory systems - Reprogramming bacteria for vaccine delivery.

    Science.gov (United States)

    Gnopo, Yehou M D; Watkins, Hannah C; Stevenson, Taylor C; DeLisa, Matthew P; Putnam, David

    2017-05-15

    Vaccines often require adjuvants to be effective. Traditional adjuvants, like alum, activate the immune response but in an uncontrolled way. Newer adjuvants help to direct the immune response in a more coordinated fashion. Here, we review the opportunity to use the outer membrane vesicles (OMVs) of bacteria as a way to modulate the immune response toward making more effective vaccines. This review outlines the different types of OMVs that have been investigated for vaccine delivery and how they are produced. Because OMVs are derived from bacteria, they have compositions that may not be compatible with parenteral delivery in humans; therefore, we also review the strategies brought to bear to detoxify OMVs while maintaining an adjuvant profile. OMV-based vaccines can be derived from the pathogens themselves, or can be used as surrogate constructs to mimic a pathogen through the heterologous expression of specific antigens in a desired host source strain, and approaches to doing so are reviewed. Additionally, the emerging area of engineered pathogen-specific carbohydrate sequences, or glycosylated OMVs is reviewed and contrasted with protein antigen delivery. Existing OMV-based vaccines as well as their routes of administration round out the text. Overall, this is an exciting time in the OMV field as it matures and leads to more effective and targeted ways to induce desired pathogen-specific immune responses. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Outer membrane vesicles of Lysobacter sp. XL1: biogenesis, functions, and applied prospects.

    Science.gov (United States)

    Kudryakova, Irina V; Shishkova, Nina A; Vasilyeva, Natalia V

    2016-06-01

    Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been intensively investigated in recent times. Vesicle formation models have been proposed, some factors affecting the process were established, and important roles vesicles play in vital activities of their producing cells were determined. Studies of pathogenic bacterial vesicles contribute to understanding the causes of acute infection and developing drugs on their basis. Despite intensive research, issues associated with the understanding of vesicle biogenesis, the mechanisms of bacterium-bacterium and pathogen-host interactions with participation of vesicles, still remain unresolved. This review discusses some results obtained in the research into OMVs of Lysobacter sp. XL1 VKM B-1576. This bacterium secretes into the environment a spectrum of bacteriolytic enzymes that hydrolyze peptidoglycan of competing bacteria, thus leading to their lysis. One of these enzymes, lytic endopeptidase L5, has been shown not only to be secreted by means of vesicles but also to be involved in their formation. As part of vesicles, the antimicrobial potential of L5 enzyme has been found to be considerably expanded. Vesicles have been shown to have a therapeutic effect in respect of anthrax infection and staphylococcal sepsis modelled in mice. The scientific basis for constructing liposomal antimicrobial preparations from vesicle phospholipids and recombinant bacteriolytic enzyme L5 has been formed.

  1. Analysis of bacteria-derived outer membrane vesicles using tunable resistive pulse sensing

    Science.gov (United States)

    Bogomolny, Evgeny; Hong, Jiwon; Blenkiron, Cherie; Simonov, Denis; Dauros, Priscila; Swift, Simon; Phillips, Anthony; Willmott, Geoff R.

    2015-03-01

    Accurate characterization of submicron particles within biological fluids presents a major challenge for a wide range of biomedical research. Detection, characterization and classification are difficult due to the presence of particles and debris ranging from single molecules up to particles slightly smaller than cells. Especial interest arises from extracellular vesicles (EVs) which are known to play a pivotal role in cell-signaling in multicellular organisms. Tunable resistive pulse sensing (TRPS) is increasingly proving to be a useful tool for high throughput particle-by-particle analysis of EVs and other submicron particles. This study examines the capability of TRPS for characterization of EVs derived from bacteria, also called outer membrane vesicles (OMVs). Measurement of a size distribution (124 +/- 3 nm modal diameter) and concentration (lower bound 7.4 x 109 mL-1) are demonstrated using OMVs derived from uropathogenic Escherichia coli. Important aspects of measurement are discussed, including sample preparation and size selection. Application of TRPS to study EVs could assist the development of these particles in clinical diagnostics and therapeutics.

  2. Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori.

    Science.gov (United States)

    Yonezawa, Hideo; Osaki, Takako; Woo, Timothy; Kurata, Satoshi; Zaman, Cynthia; Hojo, Fuhito; Hanawa, Tomoko; Kato, Shuichi; Kamiya, Shigeru

    2011-12-01

    Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air-liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2-3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Improved production process for native outer membrane vesicle vaccine against Neisseria meningitidis.

    Science.gov (United States)

    van de Waterbeemd, Bas; Zomer, Gijsbert; Kaaijk, Patricia; Ruiterkamp, Nicole; Wijffels, René H; van den Dobbelsteen, Germie P J M; van der Pol, Leo A

    2013-01-01

    An improved detergent-free process has been developed to produce vaccine based on native outer membrane vesicles (NOMV) against Neisseria meningitidis serogroup B. Performance was evaluated with the NonaMen vaccine concept, which provides broad coverage based on nine distinct PorA antigens. Scalable aseptic equipment was implemented, replacing undesirable steps like ultracentrifugation, inactivation with phenol, and the use of preservatives. The resulting process is more consistent and gives a higher yield than published reference processes, enabling NOMV production at commercial scale. Product quality met preliminary specifications for 9 consecutive batches, and an ongoing study confirmed real-time stability up to 12 months after production. As the NOMV had low endotoxic activity and induced high bactericidal titres in mice, they are expected to be safe and effective in humans. The production process is not limited to NonaMen and may be applicable for other N. meningitidis serogroups and other gram-negative pathogens. The current results therefore facilitate the late-stage development and clinical evaluation of NOMV vaccines.

  4. Identification and characterization of outer membrane vesicle-associated proteins in Salmonella enterica serovar Typhimurium.

    Science.gov (United States)

    Bai, Jaewoo; Kim, Seul I; Ryu, Sangryeol; Yoon, Hyunjin

    2014-10-01

    Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Next-generation outer membrane vesicle vaccines against Neisseria meningitidis based on nontoxic LPS mutants.

    Science.gov (United States)

    van der Ley, Peter; van den Dobbelsteen, Germie

    2011-08-01

    Outer membrane vesicles (OMVs) have been used extensively as experimental vaccines against Neisseria meningitidis. Classical meningococcal OMV vaccines contain wildtype lipopolysaccharide (LPS) with a hexa-acylated lipid A moiety, which is a very potent activator of the TLR4 receptor. While this may make the LPS an effective "internal" adjuvant, it also contributes to vaccine reactogenicity. Reduction of endotoxic activity has therefore been essential for the application of meningococcal OMV vaccines in humans. Classical OMV vaccines have a reduced LPS content as a result of detergent extraction, mostly with deoxycholate. An alternative method is the use of meningococcal strains with genetically detoxified LPS, in particular where mutation in the lpxL1 gene has resulted in penta-acylated lipid A with strongly attenuated endotoxic activity. This allows the use of native OMVs without any need for LPS removal by detergent extraction, making it a much easier to produce and more versatile vaccine platform. Several groups have now started the development of native OMV vaccines based on non-toxic LPS mutants, and this Commentary provides an overview of the various approaches and results thus far.

  6. 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

    2015-07-02

    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.

  7. Improved production process for native outer membrane vesicle vaccine against Neisseria meningitidis.

    Directory of Open Access Journals (Sweden)

    Bas van de Waterbeemd

    Full Text Available An improved detergent-free process has been developed to produce vaccine based on native outer membrane vesicles (NOMV against Neisseria meningitidis serogroup B. Performance was evaluated with the NonaMen vaccine concept, which provides broad coverage based on nine distinct PorA antigens. Scalable aseptic equipment was implemented, replacing undesirable steps like ultracentrifugation, inactivation with phenol, and the use of preservatives. The resulting process is more consistent and gives a higher yield than published reference processes, enabling NOMV production at commercial scale. Product quality met preliminary specifications for 9 consecutive batches, and an ongoing study confirmed real-time stability up to 12 months after production. As the NOMV had low endotoxic activity and induced high bactericidal titres in mice, they are expected to be safe and effective in humans. The production process is not limited to NonaMen and may be applicable for other N. meningitidis serogroups and other gram-negative pathogens. The current results therefore facilitate the late-stage development and clinical evaluation of NOMV vaccines.

  8. Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae.

    Science.gov (United States)

    Rompikuntal, Pramod K; Vdovikova, Svitlana; Duperthuy, Marylise; Johnson, Tanya L; Åhlund, Monika; Lundmark, Richard; Oscarsson, Jan; Sandkvist, Maria; Uhlin, Bernt Eric; Wai, Sun Nyunt

    2015-01-01

    Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

  9. 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.

  10. Bacterial Nanobioreactors--Directing Enzyme Packaging into Bacterial Outer Membrane Vesicles.

    Science.gov (United States)

    Alves, Nathan J; Turner, Kendrick B; Daniele, Michael A; Oh, Eunkeu; Medintz, Igor L; Walper, Scott A

    2015-11-11

    All bacteria shed outer membrane vesicles (OMVs) loaded with a diverse array of small molecules, proteins, and genetic cargo. In this study we sought to hijack the bacterial cell export pathway to simultaneously produce, package, and release an active enzyme, phosphotriesterase (PTE). To accomplish this goal the SpyCatcher/SpyTag (SC/ST) bioconjugation system was utilized to produce a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin protein (OmpA-ST), known to be abundant in OMVs. Under a range of physiological conditions the SpyTag and SpyCatcher domains interact with one another and form a covalent isopeptide bond driving packaging of PTE into forming OMVs. The PTE-SC loaded OMVs are characterized for size distribution, number of vesicles produced, cell viability, packaged PTE enzyme kinetics, OMV loading efficiency, and enzyme stability following iterative cycles of freezing and thawing. The PTE-loaded OMVs exhibit native-like enzyme kinetics when assayed with paraoxon as a substrate. PTE is often toxic to expression cultures and has a tendency to lose activity with improper handling. The coexpression of OmpA-ST with PTE-SC, however, greatly improved the overall PTE production levels by mitigating toxicity through exporting of the PTE-SC and greatly enhanced packaged enzyme stability against iterative cycles of freezing and thawing.

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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

  16. 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

  17. 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.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  1. 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.

  2. 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.

  3. Analysis of surface protein expression reveals the growth pattern of the gram-negative outer membrane.

    Directory of Open Access Journals (Sweden)

    Tristan S Ursell

    Full Text Available The outer membrane (OM of Gram-negative bacteria is a complex bilayer composed of proteins, phospholipids, lipoproteins, and lipopolysaccharides. Despite recent advances revealing the molecular pathways underlying protein and lipopolysaccharide incorporation into the OM, the spatial distribution and dynamic regulation of these processes remain poorly understood. Here, we used sequence-specific fluorescent labeling to map the incorporation patterns of an OM-porin protein, LamB, by labeling proteins only after epitope exposure on the cell surface. Newly synthesized LamB appeared in discrete puncta, rather than evenly distributed over the cell surface. Further growth of bacteria after labeling resulted in divergence of labeled LamB puncta, consistent with a spatial pattern of OM growth in which new, unlabeled material was also inserted in patches. At the poles, puncta remained relatively stationary through several rounds of division, a salient characteristic of the OM protein population as a whole. We propose a biophysical model of growth in which patches of new OM material are added in discrete bursts that evolve in time according to Stokes flow and are randomly distributed over the cell surface. Simulations based on this model demonstrate that our experimental observations are consistent with a bursty insertion pattern without spatial bias across the cylindrical cell surface, with approximately one burst of ≈ 10(-2 µm(2 of OM material per two minutes per µm(2. Growth by insertion of discrete patches suggests that stochasticity plays a major role in patterning and material organization in the OM.

  4. [Cloning and prokaryotic expression of the outer membrane protein gene PorB of Neisseria gonorrhoeae].

    Science.gov (United States)

    Wang, Yan; Zhang, Lei; Zhang, Li; Wang, Han

    2011-07-01

    To construct a fused expression vector of the outer membrane protein gene PorB of Neisseria gonorrhoeae, express the fusion protein in the prokaryotic system, and obtain a gene recombination protein, for the purpose of preparing the ground for further research on the pathopoiesis and immune protective response of PorB. A pair of primers were designed according to the known sequence of the PorB gene, and the PorB gene was amplified by PCR from the genome of Neisseria gonorrhoeae 29403 and cloned into the prokaryotic expression plasmid pGEX-4T-1 to generate pGEX-4T-PorB recombinants. The recombinant plasmid pGEX4T-PorB was transferred into competent cells E. coli BL21. After confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis, the recombinant protein was induced to express by isopropyl-beta-D-thiogalactoside (IPTG), and examined by SDS-PAGE and Western blotting. Restriction endonuclease digestion, PCR amplification and DNA sequencing analysis showed that the PorB gene of 1 047 bp was amplified from Neisseria gonorrhoeae DNA, and the recombinant plasmid pGEX-4T-PorB was successfully constructed and highly expressed in E. coli. The prokaryotic expression vector of pGEX-4T-PorB was successfully constructed and efficiently expressed in the prokaryotic system, which has provided a basis for further study on the biological activity of the PorB protein, as well as animal immune experiment and detection of Neisseria gonorrhoeae, and its application as a mucosal immune vaccine.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

  7. Cross-reactivity of outer membrane proteins of Campylobacter species with cholera toxin.

    Science.gov (United States)

    Albert, M John

    2011-02-01

    Campylobacter jejuni is a foodborne pathogen and a leading cause of diarrhoea worldwide. It is believed that a cholera toxin-like toxin (CTLT) produced by C. jejuni may mediate watery diarrhoea. However, the production of a CTLT by C. jejuni is controversial. A cholera toxin gene (ctx) homologue has not been identified in Campylobacter species. We investigated the identity of the CT cross-reactive antigen from Campylobacter species previously and the results are reviewed here. Filtrates of C. jejuni grown in four different liquid media, reported to promote CTLT production, were tested by Chinese hamster ovary (CHO) cell elongation assay for functional toxin and for reactivity with CT antibody using GM1 ganglioside ELISA (GM1 ELISA) and immunoblotting. Protein sequence of the CT antibody-reactive band was determined by matrix-assisted laser desorption ionization-time of flight (MALDI TOF-TOF). Non-jejuni species (C. coli, C. lari, C. foetus, C. hyointestinalis and C. upsaliensis) were investigated by CHO cell assay and immunoblotting. Filtrates from seven C. jejuni reference strains reported to produce CTLT and from 80 clinical strains were negative in the CHO cell assay. However, filtrates from three reference strains and 16 clinical strains were positive by GM1 ELISA. All strains irrespective of GM1 ELISA reactivity, possessed a 53-kDa protein which reacted with CT antibody by immunoblotting. This band was identified as the major outer membrane protein (PorA) of C. jejuni. CT antibody reacted with a C. jejuni recombinant PorA on immunoblotting. All non-C. jejuni strains were negative by CHO cell assay, but the common 53-kDa proteins reacted with CT antibody on immunoblots. The cross-reactivity of PorAs of Campylobacter species with CT may lead to the erroneous conclusion that Campylobacter species produce a functional CTLT.

  8. Monoclonal antibody binding to the major outer membrane protein of Campylobacter coli.

    Science.gov (United States)

    Qian, Hongliang; Pang, Ervinna; Chang, Jason; Toh, Say Ling; Ng, Fook Kheong; Tan, Ai Ling; Kwang, Jimmy

    2008-11-30

    Campylobacter species are major enteric pathogens causing diarrhea illness in humans and animals. Immunological tests are needed for accurate and rapid identification of C. coli, in conjunction with the use of standard biochemical tests. We initiated the creation of monoclonal antibodies (MAbs) using whole C. coli cells as antigen. Four positive clones were identified, namely MAb2G6, MAb3B9, MAb4A10 and MAb5B9. Dot-blot assay and ELISA revealed that only MAb2G6 did not cross react with C. jejuni and other Campylobacter isolates. As demonstrated by dot-blot assay, MAb2G6 reacted with all 23 C. coli isolates tested but did not react with 29 isolates of C. jejuni, 3 other Campylobacter spp. isolates and 19 non-Campylobacter isolates, with the lowest detection limit was in the range of 10(3) to 10(4) bacteria. Western blots and dot blots showed that the antigen of MAb2G6 was a native protein, with immunoprecipitation assay showed that MAb2G6 bound to a protein band of approximately 43 kDa in size, corresponding to major outer membrane protein (MOMP) of C. coli revealed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Immunofluorescence assay (IFA) showed that MOMP of C. coli was indeed the antigen of MAb2G6, with immunogold-electron microscopy demonstrated that MAb2G6 conjugated with immunogold particles bound to all over the surface of C. coli cells. MAb2G6 also showed potential usage in direct detection of C. coli in faecal samples.

  9. 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.

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

    Science.gov (United States)

    Oleastro, Mónica; Ménard, Armelle

    2013-01-01

    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. PMID:24833057

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Mucosal immunization with Shigella flexneri outer membrane vesicles induced protection in mice.

    Science.gov (United States)

    Camacho, A I; de Souza, J; Sánchez-Gómez, S; Pardo-Ros, M; Irache, J M; Gamazo, C

    2011-10-26

    Vaccination appears to be the only rational prophylactic approach to control shigellosis. Unfortunately, there is still no safe and efficacious vaccine available. We investigated the protection conferred by a new vaccine containing outer membrane vesicles (OMVs) from Shigella flexneri with an adjuvant based on nanoparticles in an experimental model of shigellosis in mice. OMVs were encapsulated in poly(anhydride) nanoparticles prepared by a solvent displacement method with the copolymer PMV/MA. OMVs loaded into NPs (NP-OMVs) were homogeneous and spherical in shape, with a size of 197nm (PdI=0.06). BALB/c mice (females, 9-week-old, 20±1g) were immunized by intradermal, nasal, ocular (20μg) or oral route (100μg) with free or encapsulated OMV. Thirty-five days after administration, mice were infected intranasally with a lethal dose of S. flexneri (1×10(7)CFU). The new vaccine was able to protect fully against infection when it was administered via mucosa. By intradermal route the NP-OMVs formulation increased the protection from 20%, obtained with free extract, to 100%. Interestingly, both OMVs and OMV-NP induced full protection when administered by the nasal and conjuntival route. A strong association between the ratio of IL-12p40/IL-10 and protection was found. Moreover, low levels of IFN-γ correlate with protection. Under the experimental conditions used, the adjuvant did not induce any adverse effects. These results place OMVs among promising candidates to be used for vaccination against Shigellosis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. 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.

  14. 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.

  15. Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

    Directory of Open Access Journals (Sweden)

    Anna Lena Jung

    2016-04-01

    Full Text Available The formation and release of outer membrane vesicles (OMVs is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila, a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.

  16. Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Jessica D. Cecil

    2017-08-01

    Full Text Available Outer membrane vesicles (OMVs are proteoliposomes blebbed from the surface of Gram-negative bacteria. Chronic periodontitis is associated with an increase in subgingival plaque of Gram-negative bacteria, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. In this study, we investigated the immune-modulatory effects of P. gingivalis, T. denticola, and T. forsythia OMVs on monocytes and differentiated macrophages. All of the bacterial OMVs were phagocytosed by monocytes, M(naïve and M(IFNγ macrophages in a dose-dependent manner. They also induced NF-κB activation and increased TNFα, IL-8, and IL-1β cytokine secretion. P. gingivalis OMVs were also found to induce anti-inflammatory IL-10 secretion. Although unprimed monocytes and macrophages were resistant to OMV-induced cell death, lipopolysaccharide or OMV priming resulted in a significantly reduced cell viability. P. gingivalis, T. denticola, and T. forsythia OMVs all activated inflammasome complexes, as monitored by IL-1β secretion and ASC speck formation. ASC was critical for OMV-induced inflammasome formation, while AIM2−/− and Caspase-1−/− cells had significantly reduced inflammasome formation and NLRP3−/− cells exhibited a slight reduction. OMVs were also found to provide both priming and activation of the inflammasome complex. High-resolution microscopy and flow cytometry showed that P. gingivalis OMVs primed and activated macrophage inflammasomes in vivo with 80% of macrophages exhibiting inflammasome complex formation. In conclusion, periodontal pathogen OMVs were found to have significant immunomodulatory effects upon monocytes and macrophages and should therefore influence pro-inflammatory host responses associated with disease.

  17. Impact of Reducing Complement Inhibitor Binding on the Immunogenicity of Native Neisseria meningitidis Outer Membrane Vesicles.

    Directory of Open Access Journals (Sweden)

    Helene Daniels-Treffandier

    Full Text Available Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs, and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMVlpxl1. nOMVs unable to bind human complement factor H (hfH were generated by additional deletions of the genes encoding factor H binding protein (fHbp and neisserial surface protein A (NspA (nOMVdis. Antibody responses elicited in mice with nOMVdis were compared to those elicited with nOMVlpxl1 in the presence of hfH. Results demonstrate that the administration of human fH to mice immunized with fHbp containing OMVlpxl1 decreased immunogenicity against fHbp (but not against the OMV as a whole. The majority of the OMV-induced bactericidal immune response (OMVlpxl1 or OMVdis was versus PorA. Despite a considerable reduction of hfH binding to nOMVdis, and the absence of the vaccine antigen fHbp, immunogenicity in mice was not different from nOMVlpxl1, in the absence or presence of hfH (serum bactericidal titers of 1:64 vs 1:128 after one dose in the nOMVdis and nOMVlpxl1-immunized groups respectively. Therefore, partial inhibition of fH binding did not enhance immunity in this model.

  18. B cell activation by outer membrane vesicles--a novel virulence mechanism.

    Science.gov (United States)

    Vidakovics, Maria Laura A Perez; Jendholm, Johan; Mörgelin, Matthias; Månsson, Anne; Larsson, Christer; Cardell, Lars-Olaf; Riesbeck, Kristian

    2010-01-15

    Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca(2+) mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19(+) IgD(+) lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.

  19. Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo

    Science.gov (United States)

    Cecil, Jessica D.; O’Brien-Simpson, Neil M.; Lenzo, Jason C.; Holden, James A.; Singleton, William; Perez-Gonzalez, Alexis; Mansell, Ashley; Reynolds, Eric C.

    2017-01-01

    Outer membrane vesicles (OMVs) are proteoliposomes blebbed from the surface of Gram-negative bacteria. Chronic periodontitis is associated with an increase in subgingival plaque of Gram-negative bacteria, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. In this study, we investigated the immune-modulatory effects of P. gingivalis, T. denticola, and T. forsythia OMVs on monocytes and differentiated macrophages. All of the bacterial OMVs were phagocytosed by monocytes, M(naïve) and M(IFNγ) macrophages in a dose-dependent manner. They also induced NF-κB activation and increased TNFα, IL-8, and IL-1β cytokine secretion. P. gingivalis OMVs were also found to induce anti-inflammatory IL-10 secretion. Although unprimed monocytes and macrophages were resistant to OMV-induced cell death, lipopolysaccharide or OMV priming resulted in a significantly reduced cell viability. P. gingivalis, T. denticola, and T. forsythia OMVs all activated inflammasome complexes, as monitored by IL-1β secretion and ASC speck formation. ASC was critical for OMV-induced inflammasome formation, while AIM2−/− and Caspase-1−/− cells had significantly reduced inflammasome formation and NLRP3−/− cells exhibited a slight reduction. OMVs were also found to provide both priming and activation of the inflammasome complex. High-resolution microscopy and flow cytometry showed that P. gingivalis OMVs primed and activated macrophage inflammasomes in vivo with 80% of macrophages exhibiting inflammasome complex formation. In conclusion, periodontal pathogen OMVs were found to have significant immunomodulatory effects upon monocytes and macrophages and should therefore influence pro-inflammatory host responses associated with disease. PMID:28890719

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

    Directory of Open Access Journals (Sweden)

    Deborah Raphaela Leitner

    2015-08-01

    Full Text Available 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.

  1. B Cell Activation by Outer Membrane Vesicles—A Novel Virulence Mechanism

    Science.gov (United States)

    Perez Vidakovics, Maria Laura A.; Jendholm, Johan; Mörgelin, Matthias; Månsson, Anne; Larsson, Christer; Cardell, Lars-Olaf; Riesbeck, Kristian

    2010-01-01

    Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca2+ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19+ IgD+ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys. PMID:20090836

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

    Directory of Open Access Journals (Sweden)

    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

  3. B cell activation by outer membrane vesicles--a novel virulence mechanism.

    Directory of Open Access Journals (Sweden)

    Maria Laura A Perez Vidakovics

    2010-01-01

    Full Text Available Secretion of outer membrane vesicles (OMV is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR clustering and Ca(2+ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19(+ IgD(+ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86, whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.

  4. Nanopods: a new bacterial structure and mechanism for deployment of outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    Ameesha Shetty

    Full Text Available BACKGROUND: Bacterial outer membrane vesicles (OMV are packets of periplasmic material that, via the proteins and other molecules they contain, project metabolic function into the environment. While OMV production is widespread in proteobacteria, they have been extensively studied only in pathogens, which inhabit fully hydrated environments. However, many (arguably most bacterial habitats, such as soil, are only partially hydrated. In the latter, water is characteristically distributed as films on soil particles that are, on average thinner, than are typical OMV (ca. ≤10 nm water film vs. 20 to >200 nm OMV;. METHODOLOGY/PRINCIPAL FINDINGS: We have identified a new bacterial surface structure, termed a "nanopod", that is a conduit for projecting OMV significant distances (e.g., ≥6 µm from the cell. Electron cryotomography was used to determine nanopod three-dimensional structure, which revealed chains of vesicles within an undulating, tubular element. By using immunoelectron microscopy, proteomics, heterologous expression and mutagenesis, the tubes were determined to be an assembly of a surface layer protein (NpdA, and the interior structures identified as OMV. Specific metabolic function(s for nanopods produced by Delftia sp. Cs1-4 are not yet known. However, a connection with phenanthrene degradation is a possibility since nanopod formation was induced by growth on phenanthrene. Orthologs of NpdA were identified in three other genera of the Comamonadaceae family, and all were experimentally verified to form nanopods. CONCLUSIONS/SIGNIFICANCE: Nanopods are new bacterial organelles, and establish a new paradigm in the mechanisms by which bacteria effect long-distance interactions with their environment. Specifically, they create a pathway through which cells can effectively deploy OMV, and the biological activity these transmit, in a diffusion-independent manner. Nanopods would thus allow environmental bacteria to expand their metabolic

  5. 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.

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

    Science.gov (United States)

    Pors, Susanne E; Pedersen, Ida J; Skjerning, Ragnhild Bager; Thøfner, Ida C N; Persson, Gry; Bojesen, Anders M

    2016-11-15

    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 ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re-isolation remained unchanged. Furthermore, a high OMV-specific IgY response was induced by immunization and subsequent challenge of the hens. The results strongly indicate that immunization with G. anatis OMVs provides significant protection against G. anatis challenge and induces specific antibody responses with high titers of OMV-specific IgY in serum. The results therefore show great promise for OMV based vaccines aiming at providing protecting against G. anatis in egg-laying hens. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Nanopods: a new bacterial structure and mechanism for deployment of outer membrane vesicles.

    Science.gov (United States)

    Shetty, Ameesha; Chen, Shicheng; Tocheva, Elitza I; Jensen, Grant J; Hickey, William J

    2011-01-01

    Bacterial outer membrane vesicles (OMV) are packets of periplasmic material that, via the proteins and other molecules they contain, project metabolic function into the environment. While OMV production is widespread in proteobacteria, they have been extensively studied only in pathogens, which inhabit fully hydrated environments. However, many (arguably most) bacterial habitats, such as soil, are only partially hydrated. In the latter, water is characteristically distributed as films on soil particles that are, on average thinner, than are typical OMV (ca. ≤10 nm water film vs. 20 to >200 nm OMV;). We have identified a new bacterial surface structure, termed a "nanopod", that is a conduit for projecting OMV significant distances (e.g., ≥6 µm) from the cell. Electron cryotomography was used to determine nanopod three-dimensional structure, which revealed chains of vesicles within an undulating, tubular element. By using immunoelectron microscopy, proteomics, heterologous expression and mutagenesis, the tubes were determined to be an assembly of a surface layer protein (NpdA), and the interior structures identified as OMV. Specific metabolic function(s) for nanopods produced by Delftia sp. Cs1-4 are not yet known. However, a connection with phenanthrene degradation is a possibility since nanopod formation was induced by growth on phenanthrene. Orthologs of NpdA were identified in three other genera of the Comamonadaceae family, and all were experimentally verified to form nanopods. Nanopods are new bacterial organelles, and establish a new paradigm in the mechanisms by which bacteria effect long-distance interactions with their environment. Specifically, they create a pathway through which cells can effectively deploy OMV, and the biological activity these transmit, in a diffusion-independent manner. Nanopods would thus allow environmental bacteria to expand their metabolic sphere of influence in a manner previously unknown for these organisms.

  8. 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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. 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.

  10. Fimbriae-mediated outer membrane vesicle production and invasion of Porphyromonas gingivalis.

    Science.gov (United States)

    Mantri, Chinmay K; Chen, Chin-Ho; Dong, Xinhong; Goodwin, Jeffery Shawn; Pratap, Siddharth; Paromov, Victor; Xie, Hua

    2015-02-01

    Porphyromonas gingivalis is a keystone periopathogen that plays an essential role in the progress of periodontitis. Like other gram-negative bacteria, the ability of P. gingivalis to produce outer membrane vesicles is a strategy used to interact with, and survive within its biological niches. Here we compared the protein components associated with vesicles derived from a fimbriated strain (33277) and an afimbriated strain (W83) of P. gingivalis using proteomic analyses. Some well-known virulence factors were identified in vesicles from both strains, such as gingipains and hemagglutinin. In contrast, FimC, FimD, and FimE, minor components of long fimbriae were found exclusively in 33277 vesicles, while proteins with a tetratricopeptide repeat (TPR) domain were unique to W83 vesicles. We found that significantly more 33277 than W83 vesicles were internalized into human oral keratinocytes and gingival fibroblasts. Interestingly, FimA, a well-known adhesin responsible for the attachment and invasion of P. gingivalis into host cells, was not essential for the invasive capabilities of P. gingivalis vesicles. Rather minor components of long fimbriae were required for an efficient invasive activity of vesicles. The most striking finding was that P. gingivalis strains lacking or having a reduced FimA expression showed a significant reduction in vesiculation. These results suggest that production and pathogenicity of P. gingivalis vesicles may largely depend on expression of the fim locus, and that the integration of vesicle production and pathogenicity with fimbrial expression may allow P. gingivalis to confer upon itself certain functional advantages. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. A Meningococcal Outer Membrane Vesicle Vaccine Incorporating Genetically Attenuated Endotoxin Dissociates Inflammation from Immunogenicity.

    Science.gov (United States)

    Dowling, David J; Sanders, Holly; Cheng, Wing Ki; Joshi, Sweta; Brightman, Spencer; Bergelson, Ilana; Pietrasanta, Carlo; van Haren, Simon D; van Amsterdam, Sandra; Fernandez, Jeffrey; van den Dobbelsteen, Germie P J M; Levy, Ofer

    2016-01-01

    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 human in vitro culture systems. 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 (DC) arrays. OMVs were benchmarked against licensed vaccines, including Bexsero and whole cell pertussis formulations, with respect to Th-polarizing cytokine and prostaglandin E2 production, as well as cell surface activation markers (HLA-DR, CD86, and CCR7). OMV immunogenicity was assessed in mice. ΔlpxLI native OMVs (nOMVs) demonstrated significantly less cytokine induction in human blood and DCs than Bexsero and most of the other pediatric vaccines (e.g., PedvaxHib, EasyFive, and bacillus Calmette-Guérin) tested. Despite a much lower inflammatory profile in vitro than Bexsero, ΔlpxLI nOMVs still had moderate DC maturing ability and induced robust anti-N. meningitidis antibody responses after murine immunization. A meningococcal vaccine comprised of attenuated LPS-based OMVs with a limited inflammatory profile in vitro induces robust antigen-specific immunogenicity in vivo.

  12. A Burkholderia pseudomallei outer membrane vesicle vaccine provides protection against lethal sepsis.

    Science.gov (United States)

    Nieves, Wildaliz; Petersen, Hailey; Judy, Barbara M; Blumentritt, Carla A; Russell-Lodrigue, Kasi; Roy, Chad J; Torres, Alfredo G; Morici, Lisa A

    2014-05-01

    The environmental Gram-negative encapsulated bacillus Burkholderia pseudomallei is the causative agent of melioidosis, a disease associated with high morbidity and mortality rates in areas of Southeast Asia and northern Australia in which the disease is endemic. B. pseudomallei is also classified as a tier I select agent due to the high level of lethality of the bacterium and its innate resistance to antibiotics, as well as the lack of an effective vaccine. Gram-negative bacteria, including B. pseudomallei, secrete outer membrane vesicles (OMVs) which are enriched with multiple protein, lipid, and polysaccharide antigens. Previously, we demonstrated that immunization with multivalent B. pseudomallei-derived OMVs protects highly susceptible BALB/c mice against an otherwise lethal aerosol challenge. In this work, we evaluated the protective efficacy of OMV immunization against intraperitoneal challenge with a heterologous strain because systemic infection with phenotypically diverse environmental B. pseudomallei strains poses another hazard and a challenge to vaccine development. We demonstrated that B. pseudomallei OMVs derived from strain 1026b afforded significant protection against septicemic infection with B. pseudomallei strain K96243. OMV immunization induced robust OMV-, lipopolysaccharide-, and capsular polysaccharide-specific serum IgG (IgG1, IgG2a, and IgG3) and IgM antibody responses. OMV-immune serum promoted bacterial killing in vitro, and passive transfer of B. pseudomallei OMV immune sera protected naive mice against a subsequent challenge. These results indicate that OMV immunization provides antibody-mediated protection against acute, rapidly lethal sepsis in mice. B. pseudomallei-derived OMVs may represent an efficacious multivalent vaccine strategy against melioidosis.

  13. 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.

  14. 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.

  15. Excitation of fluorescent dyes inactivates the outer hair cell integral membrane motor protein prestin and betrays its lateral mobility.

    Science.gov (United States)

    Santos-Sacchi, Joseph; Zhao, Hong-Bo

    2003-08-01

    The outer hair cell motor protein, prestin, which resides exclusively in the cell's lateral membrane, underlies the mammal's exquisite sense of hearing. Here we show that photoexposure of the commonly used dyes Lucifer yellow, 6-carboxy-fluorescein, and 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)-1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS), that are in contact with the cell's lateral membrane can photo-inactivate the motor irreversibly, as evidenced by reduction in prestin's gating charge displacement or non-linear capacitance. Furthermore, utilizing restricted fiber optic illumination of the lateral membrane, we show that whole-cell, non-linear capacitance is depleted beyond that expected for an immobile population in the exposed area. These data indicate that lateral diffusion of prestin occurs within the cell's lateral plasma membrane.

  16. Quantitative measurement of the outer membrane permeability in Escherichia coli lpp and tol-pal mutants defines the significance of Tol-Pal function for maintaining drug resistance.

    Science.gov (United States)

    Kowata, Hikaru; Tochigi, Saeko; Kusano, Tomonobu; Kojima, Seiji

    2016-12-01

    Ensuring the stability of the outer membrane permeability barrier is crucial for maintaining drug resistance in Gram-negative bacteria. Lpp protein and Tol-Pal complex are responsible for this function and are widely distributed among Gram-negative bacteria. Thus, these proteins are potential targets to permeabilize the outer membrane barrier. Although deleting these proteins is known to impair the outer membrane stability, the effect of the deletion on the outer membrane barrier property and on the drug resistance has not been fully characterized and evaluated in a quantitative manner. Here, we determined the outer membrane permeability of Escherichia coli Δlpp and Δtol-pal mutants by the assay using intact cells and liposomes reconstituted with the outer membrane proteins. We determined that there was 3- to 5-fold increase of the permeability in Δtol-pal mutants, but not in Δlpp mutant, compared with that in the parental strain. The permeability increase in Δtol-pal mutants occurred without affecting the function of outer membrane diffusion channels, and was most pronounced in the cells at exponential growth phase. The impact of tol-pal deletion on the drug resistance was revealed to be almost comparable with that of deletion of acrAB, a major multidrug efflux transporter of E. coli that makes a predominant contribution to drug resistance. Our observations highlight the importance of Tol-Pal as a possible target to combat multidrug-resistant Gram-negative bacteria.

  17. 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...

  18. 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 ...

  19. 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...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against ?-lactam antibiotics

    OpenAIRE

    Stentz, Régis; Horn, Nikki; Cross, Kathryn; Salt, Louise; Brearley, Charles; Livermore, David M.; Carding, Simon R

    2014-01-01

    Objectives: To identify β-lactamase genes in gut commensal Bacteroides species and to assess the impact of these enzymes, when carried by outer membrane vesicles (OMVs), in protecting enteric pathogens and commensals. Methods: A deletion mutant of the putative class A β-lactamase gene (locus tag BT_4507) found in the genome of the human commensal Bacteroides thetaiotaomicron was constructed and a phenotypic analysis performed. A phylogenetic tree was built from an alignment of nine Bacteroide...

  3. 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.

  4. 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.

  5. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification.

    OpenAIRE

    Grodberg, J; Dunn, J J

    1988-01-01

    Bacteriophage T7 RNA polymerase is stable in Escherichia coli but very susceptible to cleavage by at least one endoprotease after cell lysis. The major source of this endoprotease activity was found to be localized to the outer membrane of the cell. A rapid whole-cell assay was developed to screen different strains for the presence of this proteolytic activity. Using this assay, we identified some common laboratory strains that totally lack the protease. Genetic and Southern analyses of these...

  6. 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.

  7. 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

  8. Identification of Neisseria meningitidis outer membrane vesicle complexes using 2-D high resolution clear native/SDS-PAGE.

    Science.gov (United States)

    Marzoa, Juan; Sánchez, Sandra; Ferreirós, Carlos M; Criado, María Teresa

    2010-01-01

    The identification and characterization of meningococcal outer membrane vesicle complexes can be important for gaining an in-depth understaining of their structure and functionality. Analysis of the vesicle complexome by 'traditional' 2-D analysis, in which isoelectrofocusing is used for separation in the first dimension, is hampered by the high hydrophobicity and extreme isoelectric points of many relevant proteins. Analysis of the meningococcal outer membrane vesicle complexome using Blue Native (nondenaturing) electrophoresis instead of isoelectrofocusing in the first dimension showed several porin complexes, but their composition could not be clearly resolved after separation by SDS-PAGE in the second dimension. In this work, using a recently described native separation technique -high resolution Clear Native Electrophoresis-and different bidimensional approaches, we were able to demonstrate the presence of relevant outer membrane complexes which could be resolved with a higher resolution than in previous analysis. The most relevant were nine porin complexes formed by different combinations of the meningococcal PorA, PorB and RmpM proteins, and comparison with the complexes formed in specific knockout mutants allowed us to infer the relevance of each porin in the formation of each complex.

  9. Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

    Science.gov (United States)

    Kushnareva, Yulia; Andreyev, Alexander Y.; Kuwana, Tomomi; Newmeyer, Donald D.

    2012-01-01

    Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event. PMID:23049480

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Allelic Variation in Outer Membrane Protein A and Its Influence on Attachment of Escherichia coli to Corn Stover

    Directory of Open Access Journals (Sweden)

    Chunyu Liao

    2017-05-01

    Full Text Available Understanding the genetic factors that govern microbe-sediment interactions in aquatic environments is important for water quality management and reduction of waterborne disease outbreaks. Although chemical properties of bacteria have been identified that contribute to initiation of attachment, the outer membrane proteins that contribute to these chemical properties still remain unclear. In this study we explored the attachment of 78 Escherichia coli environmental isolates to corn stover, a representative agricultural residue. Outer membrane proteome analysis led to the observation of amino acid variations, some of which had not been previously described, in outer membrane protein A (OmpA at 10 distinct locations, including each of the four extracellular loops, three of the eight transmembrane segments, the proline-rich linker and the dimerization domain. Some of the polymorphisms within loops 1, 2, and 3 were found to significantly co-occur. Grouping of sequences according to the outer loop polymorphisms revealed five distinct patterns that each occur in at least 5% of our isolates. The two most common patterns, I and II, are encoded by 33.3 and 20.5% of these isolates and differ at each of the four loops. Statistically significant differences in attachment to corn stover were observed among isolates expressing different versions of OmpA and when different versions of OmpA were expressed in the same genetic background. Most notable was the increased corn stover attachment associated with a loop 3 sequence of SNFDGKN relative to the standard SNVYGKN sequence. These results provide further insight into the allelic variation of OmpA and implicate OmpA in contributing to attachment to corn stover.

  12. 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.

  13. Cytosolic BNIP3 Dimer Interacts with Mitochondrial BAX Forming Heterodimers in the Mitochondrial Outer Membrane under Basal Conditions.

    Science.gov (United States)

    Hendgen-Cotta, Ulrike B; Esfeld, Sonja; Rudi, Katharina; Miinalainen, Ilkka; Klare, Johann P; Rassaf, Tienush

    2017-03-23

    The primary function of mitochondria is energy production, a task of particular importance especially for cells with a high energy demand like cardiomyocytes. The B-cell lymphoma (BCL-2) family member BCL-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is linked to mitochondrial targeting after homodimerization, where it functions in inner membrane depolarization and permeabilization of the mitochondrial outer membrane (MOM) mediating cell death. We investigated the basal distribution of cardiac BNIP3 in vivo and its physical interaction with the pro-death protein BCL2 associated X, apoptosis regulator (BAX) and with mitochondria using immunoblot analysis, co-immunoprecipitation, and continuous wave and pulsed electron paramagnetic resonance spectroscopy techniques. We found that BNIP3 is present as a dimer in the cytosol and in the outer membrane of cardiac mitochondria under basal conditions. It forms disulfide-bridged, but mainly non-covalent dimers in the cytosol. Heterodimers with BAX are formed exclusively in the MOM. Furthermore, our results suggest that BNIP3 interacts with the MOM directly via mitochondrial BAX. However, the physical interactions with BAX and the MOM did not affect the membrane potential and cell viability. These findings suggest that another stimulus other than the mere existence of the BNIP3/BAX dimer in the MOM is required to promote BNIP3 cell-death activity; this could be a potential disturbance of the BNIP3 distribution homeostasis, namely in the direction of the mitochondria.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  20. Outer Membrane Proteome of Veillonella parvula: A Diderm Firmicute of the Human Microbiome

    Directory of Open Access Journals (Sweden)

    Daniel I. Poppleton

    2017-06-01

    Full Text Available Veillonella parvula is a biofilm-forming commensal found in the lungs, vagina, mouth, and gastro-intestinal tract of humans, yet it may develop into an opportunistic pathogen. Furthermore, the presence of Veillonella has been associated with the development of a healthy immune system in infants. Veillonella belongs to the Negativicutes, a diverse clade of bacteria that represent an evolutionary enigma: they phylogenetically belong to Gram-positive (monoderm Firmicutes yet maintain an outer membrane (OM with lipopolysaccharide similar to classic Gram-negative (diderm bacteria. The OMs of Negativicutes have unique characteristics including the replacement of Braun's lipoprotein by OmpM for tethering the OM to the peptidoglycan. Through phylogenomic analysis, we have recently provided bioinformatic annotation of the Negativicutes diderm cell envelope. We showed that it is a unique type of envelope that was present in the ancestor of present-day Firmicutes and lost multiple times independently in this phylum, giving rise to the monoderm architecture; however, little experimental data is presently available for any Negativicutes cell envelope. Here, we performed the first experimental proteomic characterization of the cell envelope of a diderm Firmicute, producing an OM proteome of V. parvula. We initially conducted a thorough bioinformatics analysis of all 1,844 predicted proteins from V. parvula DSM 2008's genome using 12 different localization prediction programs. These results were complemented by protein extraction with surface exposed (SE protein tags and by subcellular fractionation, both of which were analyzed by liquid chromatography tandem mass spectrometry. The merging of proteomics and bioinformatics results allowed identification of 78 OM proteins. These include a number of receptors for TonB-dependent transport, the main component of the BAM system for OM protein biogenesis (BamA, the Lpt system component LptD, which is responsible for

  1. Protection from hemolytic uremic syndrome by eyedrop vaccination with modified enterohemorrhagic E. coli outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    Kyoung Sub Choi

    Full Text Available We investigated whether eyedrop vaccination using modified outer membrane vesicles (mOMVs is effective for protecting against hemolytic uremic syndrome (HUS caused by enterohemorrhagic E. coli (EHEC O157:H7 infection. Modified OMVs and waaJ-mOMVs were prepared from cultures of MsbB- and Shiga toxin A subunit (STxA-deficient EHEC O157:H7 bacteria with or without an additional waaJ mutation. BALB/c mice were immunized by eyedrop mOMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB. Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs at 4 weeks. As parameters for evaluation of the OMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN/Creatinin (Cr were tested, as well as histopathology of renal tissue. In order to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice. Modified OMVs having penta-acylated lipid A moiety did not contain STxA subunit proteins but retained non-toxic Shiga toxin B (STxB subunit. Removal of the polymeric O-antigen of O157 LPS was confirmed in waaJ-mOMVs. The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses, suggesting that intact O157 LPS antigen can be a critical component for enhancing the immunogenicity of the mOMVs. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not. Collectively, for the first time, EHEC O157-derived mOMV eyedrop vaccine was experimentally evaluated as an efficient and safe means of vaccine development against EHEC O157:H7 infection-associated HUS.

  2. Improving the immunogenicity of a trivalent Neisseria meningitidis native outer membrane vesicle vaccine by genetic modification.

    Science.gov (United States)

    Zhang, Lan; Wen, Zhiyun; Lin, Jing; Xu, Hui; Herbert, Paul; Wang, Xin-Min; Mehl, John T; Ahl, Patrick L; Dieter, Lance; Russell, Ryann; Kosinski, Mike J; Przysiecki, Craig T

    2016-07-29

    Trivalent native outer membrane vesicles (nOMVs) derived from three genetically modified Neisseria meningitidis serogroup B strains have been previously evaluated immunologically in mice and rabbits. This nOMV vaccine elicited serum bactericidal activity (SBA) against multiple N. meningitidis serogroup B strains as well as strains from serogroups C, Y, W, and X. In this study, we used trivalent nOMVs isolated from the same vaccine strains and evaluated their immunogenicity in an infant Rhesus macaque (IRM) model whose immune responses to the vaccine are likely to be more predictive of the responses in human infants. IRMs were immunized with trivalent nOMV vaccines and sera were evaluated for exogenous human serum complement-dependent SBA (hSBA). Antibody responses to selected hSBA generating antigens contained within the trivalent nOMVs were also measured and we found that antibody titers against factor H binding protein variant 2 (fHbpv2) were very low in the sera from animals immunized with these original nOMV vaccines. To increase the fHbp content in the nOMVs, the vaccine strains were further genetically altered by addition of another fHbp gene copy into the porB locus. Trivalent nOMVs from the three new vaccine strains had higher fHbp antigen levels and generated higher anti-fHbp antibody responses in immunized mice and IRMs. As expected, fHbp insertion into the porB locus resulted in no PorB expression. Interestingly, higher expression of PorA, an hSBA generating antigen, was observed for all three modified vaccine strains. Compared to the trivalent nOMVs from the original strains, higher PorA levels in the improved nOMVs resulted in higher anti-PorA antibody responses in mice and IRMs. In addition, hSBA titers against other strains with PorA as the only hSBA antigen in common with the vaccine strains also increased. Copyright © 2016 Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ. Published by Elsevier Ltd.. All rights reserved.

  3. 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.

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

    Science.gov (United States)

    Belo, Elza Ft; Farhat, Calil K; Gaspari, Elizabeth N De

    2010-01-01

    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 microg 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.

  5. 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

    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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Distinct Structural Elements Govern the Folding, Stability, and Catalysis in the Outer Membrane Enzyme PagP.

    Science.gov (United States)

    Iyer, Bharat Ramasubramanian; Mahalakshmi, Radhakrishnan

    2016-09-06

    The outer membrane enzyme PagP is indispensable for lipid A palmitoylation in Gram-negative bacteria and has been implicated in resistance to host immune defenses. PagP possesses an unusual structure for an integral membrane protein, with a highly dynamic barrel domain that is tilted with respect to the membrane normal. In addition, it contains an N-terminal amphipathic helix. Recent functional and structural studies have shown that these molecular factors are critical for PagP to carry out its function in the challenging environment of the bacterial outer membrane. However, the precise contributions of the N-helix to folding and stability and residues that can influence catalytic rates remain to be addressed. Here, we identify a sequence-dependent stabilizing role for the N-terminal helix of PagP in the measured thermodynamic stability of the barrel. Using chimeric barrel sequences, we show that the Escherichia coli PagP N-terminal helix confers 2-fold greater stability to the Salmonella typhimurium barrel. Further, we find that the W78F substitution in S. typhimurium causes a nearly 20-fold increase in the specific activity in vitro for the phospholipase reaction, compared to that of E. coli PagP. Here, phenylalanine serves as a key regulator of catalysis, possibly by increasing the reaction rate. Through coevolution analysis, we detect an interaction network between seemingly unrelated segments of this membrane protein. Exchanging the structural and functional features between homologous PagP enzymes from E. coli and S. typhimurium has provided us with an understanding of the molecular factors governing PagP stability and function.

  12. 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

  13. Identification and characterization of a novel stress-responsive outer membrane protein Lip40 from Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Hu, Xuehe; Yan, Hao; Liu, Ke; Hu, Jiansheng; Qi, Chao; Yang, Jihong; Liu, Yanli; Zhao, Jin; Liu, Jinlin

    2015-11-25

    Actinobacillus pleuropneumoniae, a Gram-negative bacterium, is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal disease. Because current vaccines confer limited protection against A. pleuropneumoniae infection, the development of more effective vaccines is urgently required. The identification of immunogenic and protective antigens, such as an outer-membrane lipoprotein, will advance this purpose. Sixty putative lipoproteins were predicted from the genomic sequence of A. pleuropneumoniae using multiple algorithms. Here, we focused on the characteristics of the putative lipoprotein Lip40 from A. pleuropneumoniae strain SLW01 (serovar 1). Lip40 shares sequence similarity with many bacterial lipoproteins, and the structural prediction of Lip40 suggests that it is similar to A. pleuropneumoniae TbpB. The N-terminus of Lip40 contains an interesting tandemly repeated sequence, Q(E/D/P)QPK. Real-time RT-PCR indicated that the expression of lip40 was significantly upregulated at 42 °C, at 16 °C, and under anaerobic conditions. Recombinant Lip40 (rLip40) produced in Escherichia coli BL21(DE3) was specifically recognized by porcine convalescent serum directed against A. pleuropneumoniae. Lip40 was confirmed to localize at the bacterial outer membrane, and its expression was significantly stimulated when A. pleuropneumoniae was cultured under various stress conditions. Lip40 also protected 75% of mice from fatal virulent A. pleuropneumoniae infection. The immunogenic outer-membrane protein Lip40 is stress responsive, protects mice against infection, and might be a virulence determinant. Further investigation of Lip40 should expedite vaccine development and provide insight into the pathogenesis of A. pleuropneumoniae.

  14. Repetitive methylene blue-mediated photoantimicrobial chemotherapy changes the susceptibility and expression of the outer membrane proteins of Pseudomonas aeruginosa.

    Science.gov (United States)

    Shih, Min-Hsiu; Huang, Fu-Chin

    2013-12-01

    Since bacterial multidrug efflux pumps mediate intracellular photosensitizer methylene blue, a change in the expression alters the susceptibility to photoantimicrobial chemotherapy (PACT) of Pseudomonas aeruginosa, which may occur following repetitive sublethal challenges. We performed 10 consecutive, methylene blue-mediated PACT on one antibiotic-sensitive strain and three antibiotic-resistant strains of P. aeruginosa. Following each therapy, the surviving bacteria were collected for subsequent PACT. The susceptibility was compared for the pre- and the post-treated strains following repetitive PACT. To explore the existence of efflux pumps, one of the inhibitors, namely Phe-Arg β-naphthylamide dihydrochloride (PAβN 25 μg/ml), was added. Profiles of outer membrane proteins were obtained for the pre-treated and the post-treated strains. The susceptibility of PACT did not correlate with the antibiotic sensitivity. Following ten PACT, there was no significant change in susceptibility for three tested strains, except for one antibiotic-resistant strain, for which the 10th generation became less susceptible than the original one. With 2-D electrophoresis, a change in the expression of outer membrane proteins was observed. PAβN successfully increased the phototoxicity in all tested strains, especially the less PACT-susceptible 10th generation of the antibiotic-resistant strain. Following repetitive challenges, PACT had a consistent antimicrobial effect on three strains; however, one antibiotic-resistant strain, which was the most vulnerable to PACT, became more resistant after consecutive challenges. In addition, the post-PACT strain had different expression of outer membrane proteins, providing further evidence view that repetitive PACT with methylene blue could change the expression of efflux pumps. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. 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.

  16. Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane Translocon Receptor Tom71 for Preprotein Loading

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jingzhi; Qian, Xinguo; Hu, Junbin; Sha, Bingdong; (UAB)

    2010-11-03

    The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading.

  17. Immunogenicity of Nontypeable Haemophilus influenzae Outer Membrane Vesicles and Protective Ability in the Chinchilla Model of Otitis Media.

    Science.gov (United States)

    Winter, Linda E; Barenkamp, Stephen J

    2017-10-01

    Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are enriched in several outer membrane components, including major and minor outer membrane proteins and lipooligosaccharide. We assessed the functional activity of nontypeable Haemophilus influenzae (NTHi) OMV-specific antisera and the protective ability of NTHi OMVs as vaccine antigens in the chinchilla otitis media model. OMVs were purified from three HMW1/HMW2-expressing NTHi strains, two of which were also engineered to overexpress Hia proteins. OMV-specific antisera raised in guinea pigs were assessed for their ability to mediate killing of representative NTHi in an opsonophagocytic assay. The three OMV-specific antisera mediated killing of 18 of 65, 24 of 65, and 30 of 65 unrelated HMW1/HMW2-expressing NTHi strains. Overall, they mediated killing of 39 of 65 HMW1/HMW2-expressing strains. The two Hia-expressing OMV-specific antisera mediated killing of 17 of 25 and 14 of 25 unrelated Hia-expressing NTHi strains. Overall, they mediated killing of 20 of 25 Hia-expressing strains. OMVs from prototype NTHi strain 12 were used to immunize chinchillas and the course of middle ear infection was monitored following intrabullar challenge with the homologous strain. All control animals developed culture-positive otitis media, as did two of three HMW1/HMW2-immunized animals. All OMV-immunized animals, with or without supplemental HMW1/HMW2 immunization, were completely protected against otitis media. NTHi OMVs are the first immunogens examined in this model that provided complete protection with sterile immunity after NTHi strain 12 challenge. These data suggest that NTHi OMVs hold significant potential as components of protective NTHi vaccines, possibly in combination with HMW1/HMW2 proteins. Copyright © 2017 American Society for Microbiology.

  18. F Plasmid TraF and TraH Are Components of an Outer Membrane Complex Involved in Conjugation▿

    Science.gov (United States)

    Arutyunov, Denis; Arenson, Barbara; Manchak, Jan; Frost, Laura S.

    2010-01-01

    F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH. PMID:20081027

  19. F plasmid TraF and TraH are components of an outer membrane complex involved in conjugation.

    Science.gov (United States)

    Arutyunov, Denis; Arenson, Barbara; Manchak, Jan; Frost, Laura S

    2010-03-01

    F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.

  20. 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.

  1. 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...

  2. 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). ...

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

    OpenAIRE

    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 vaccines, since they are exposed to the outside of the bacterium. OmpT is present in Escherichia coli, a bacterium that is commensally present in the intestines, and which is the main cause of urinar...

  4. 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...

  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. 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.

  7. 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

  8. 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

  9. The location of redox-sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli

    NARCIS (Netherlands)

    Poolman, Bert; Konings, Wil N.; Robillard, George T.

    1983-01-01

    Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane. Treatment of right-side-out membrane vesicles from E. col

  10. Defects in the Outer Limiting Membrane Are Associated with Rosette Development in the Nrl−/− Retina

    Science.gov (United States)

    Stuck, Michael W.; Conley, Shannon M.; Naash, Muna I.

    2012-01-01

    The neural retinal leucine zipper (Nrl) knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl−/− retina, rods are converted into functional cone-like cells. The Nrl−/− retina is characterized by large undulations of the outer nuclear layer (ONL) commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl−/− retina. We report that rosettes first appear at postnatal day (P)8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM) are not uniformly formed in the Nrl−/− retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl−/− retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM. PMID:22427845

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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...

  17. 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.

  18. 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.

  19. 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.

  20. 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

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

    Science.gov (United States)

    Afkar, Eman; Reguera, Gemma; Schiffer, Marianne; Lovley, Derek R

    2005-01-01

    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 reduction. Conclusion Omp

  2. Effect of toluene on Pseudomonas stutzeri ST-9 morphology - plasmolysis, cell size, and formation of outer membrane vesicles.

    Science.gov (United States)

    Michael, Esti; Nitzan, Yeshayahu; Langzam, Yakov; Luboshits, Galia; Cahan, Rivka

    2016-08-01

    Isolated toluene-degrading Pseudomonas stutzeri ST-9 bacteria were grown in a minimal medium containing toluene (100 mg·L(-1)) (MMT) or glucose (MMG) as the sole carbon source, with specific growth rates of 0.019 h(-1) and 0.042 h(-1), respectively. Scanning (SEM) as well as transmission (TEM) electron microscope analyses showed that the bacterial cells grown to mid-log phase in the presence of toluene possess a plasmolysis space. TEM analysis revealed that bacterial cells that were grown in MMT were surrounded by an additional "material" with small vesicles in between. Membrane integrity was analyzed by leakage of 260 nm absorbing material and demonstrated only 7% and 8% leakage from cultures grown in MMT compared with MMG. X-ray microanalysis showed a 4.3-fold increase in Mg and a 3-fold increase in P in cells grown in MMT compared with cells grown in MMG. Fluorescence-activated cell sorting (FACS) analysis indicated that the permeability of the membrane to propidium iodide was 12.6% and 19.6% when the cultures were grown in MMG and MMT, respectively. The bacterial cell length increased by 8.5% ± 0.1% and 17% ± 2%, as measured using SEM images and FACS analysis, respectively. The results obtained in this research show that the presence of toluene led to morphology changes, such as plasmolysis, cell size, and formation of outer membrane vesicles. However, it does not cause significant damage to membrane integrity.

  3. 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.

  4. 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...

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

    2013-01-01

    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 PMID:23991283

  10. 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.

  11. 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.

  12. 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.

  13. [Cloning of major outer membrane protein gene of Legionella pneumophila and detection of its expression in prokaryotic cell].

    Science.gov (United States)

    Zhang, Lei; Chen, Jianping; Wang, Tao; Zhang, Li; Tian, Yu

    2006-04-01

    In this study, the ompS gene, a major outer membrane protein gene of Legionella pneumophila, was obtained from the DNA of Legionella pneumophila by PCR. The gene was cloned into prokaryotic expressional plasmid pUC18 to construct recombinant plasmid. The recombinant plasmid was transformed into E. coli strain BL21. The identification was made by means of restriction enzyme analysis, polymerase chain reaction, DNA sequencing analysis, SDS--polyacrylamine gel electrophoresis analysis and Western blot. The results showed that the ompS gene of 914 bp was amplified from Legionella pneumophila DNA, the recombinant plasmid pLPompS was constructed and its expression in prokaryotic cell was detected successfully.

  14. 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/