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Sample records for outer membrane polysaccharide

  1. Pivotal Roles of the Outer Membrane Polysaccharide Export and Polysaccharide Copolymerase Protein Families in Export of Extracellular Polysaccharides in Gram-Negative Bacteria

    PubMed Central

    Cuthbertson, Leslie; Mainprize, Iain L.; Naismith, James H.; Whitfield, Chris

    2009-01-01

    Summary: Many bacteria export extracellular polysaccharides (EPS) and capsular polysaccharides (CPS). These polymers exhibit remarkably diverse structures and play important roles in the biology of free-living, commensal, and pathogenic bacteria. EPS and CPS production represents a major challenge because these high-molecular-weight hydrophilic polymers must be assembled and exported in a process spanning the envelope, without compromising the essential barrier properties of the envelope. Emerging evidence points to the existence of molecular scaffolds that perform these critical polymer-trafficking functions. Two major pathways with different polymer biosynthesis strategies are involved in the assembly of most EPS/CPS: the Wzy-dependent and ATP-binding cassette (ABC) transporter-dependent pathways. They converge in an outer membrane export step mediated by a member of the outer membrane auxiliary (OMA) protein family. OMA proteins form outer membrane efflux channels for the polymers, and here we propose the revised name outer membrane polysaccharide export (OPX) proteins. Proteins in the polysaccharide copolymerase (PCP) family have been implicated in several aspects of polymer biogenesis, but there is unequivocal evidence for some systems that PCP and OPX proteins interact to form a trans-envelope scaffold for polymer export. Understanding of the precise functions of the OPX and PCP proteins has been advanced by recent findings from biochemistry and structural biology approaches and by parallel studies of other macromolecular trafficking events. Phylogenetic analyses reported here also contribute important new insight into the distribution, structural relationships, and function of the OPX and PCP proteins. This review is intended as an update on progress in this important area of microbial cell biology. PMID:19258536

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

    PubMed

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

    2014-06-03

    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.

  3. Biophysical characterization of the outer membrane polysaccharide export protein and the polysaccharide co-polymerase protein from Xanthomonas campestris.

    PubMed

    Bianco, M I; Jacobs, M; Salinas, S R; Salvay, A G; Ielmini, M V; Ielpi, L

    2014-09-01

    This study investigated the structural and biophysical characteristics of GumB and GumC, two Xanthomonas campestris membrane proteins that are involved in xanthan biosynthesis. Xanthan is an exopolysaccharide that is thought to be a virulence factor that contributes to bacterial in planta growth. It also is one of the most important industrial biopolymers. The first steps of xanthan biosynthesis are well understood, but the polymerization and export mechanisms remain unclear. For this reason, the key proteins must be characterized to better understand these processes. Here we characterized, by biochemical and biophysical techniques, GumB, the outer membrane polysaccharide export protein, and GumC, the polysaccharide co-polymerase protein of the xanthan biosynthesis system. Our results suggested that recombinant GumB is a tetrameric protein in solution. On the other hand, we observed that both native and recombinant GumC present oligomeric conformation consistent with dimers and higher-order oligomers. The transmembrane segments of GumC are required for GumC expression and/or stability. These initial results provide a starting point for additional studies that will clarify the roles of GumB and GumC in the xanthan polymerization and export processes and further elucidate their functions and mechanisms of action.

  4. Outer eggshell membrane as delivery vehicle for polysaccharide/protein microcapsules incorporated with vitamin E.

    PubMed

    Chai, Zhi; Li, Yuanyuan; Liu, Fei; Du, Bingjian; Jiao, Tong; Zhang, Chunyue; Leng, Xiaojing

    2013-01-23

    This study investigates the features of a new type of delivery system prepared by combining a natural outer eggshell membrane (OESM) with emulsified microcapsules. The loading efficiency, controlled release properties, and forming mechanisms of the prepared system were studied. The polysaccharide/protein microcapsules incorporated with vitamin E can be attached to highly cross-linked protein fiber networks of OESM. This attachment could be reinforced more than 2-fold using glutaraldehyde as a cross-linking agent. The combined OESM/microcapsule delivery system significantly exhibited better controlled release properties than the microcapsules alone because of the steric blocking effect. Moreover, the OESM delivery system incorporated with microcapsules formed by pectin/protein as wall material showed more resistance against enzymatic attacks because of the formation of compact aggregates promoted by electrostatic effects.

  5. Influence of O polysaccharides on biofilm development and outer membrane vesicle biogenesis in Pseudomonas aeruginosa PAO1.

    PubMed

    Murphy, Kathleen; Park, Amber J; Hao, Youai; Brewer, Dyanne; Lam, Joseph S; Khursigara, Cezar M

    2014-04-01

    Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA(-) and OSA(-) cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA(-) cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development.

  6. Lambda Receptor in the Outer Membrane of Escherichia coli as a Binding Protein for Maltodextrins and Starch Polysaccharides

    PubMed Central

    Ferenci, Thomas; Schwentorat, Marina; Ullrich, Susanne; Vilmart, Jeannine

    1980-01-01

    The starch polysaccharides amylose and amylopectin are not utilized by Escherichia coli, but are bound by the bacteria. The following evidence supports the view that the outer membrane λ receptor protein, a component of the maltose/ maltodextrin transport system is responsible for the binding. (i) Amylose and amylopectin both inhibit the transport of maltose into E. coli. (ii) Both polysaccharides prevent binding of non-utilizable maltodextrins by the intact bacterium, a process previously shown to be dependent on components of the maltose transport system (T. Ferenci, Eur. J. Biochem., in press). (iii) A fluorescent amylopectin derivative, O-(fluoresceinyl thiocarbamoyl)-amylopectin, has been synthesized and shown to bind to E. coli in a reversible, saturable manner. Binding of O-(fluoresceinyl thiocarbamoyl)-amylopectin is absent in mutants lacking the λ receptor, but mutations in any of the other components of the maltose transport system do not affect binding as long as λ receptor is present. (iv) Using the inhibition of λ receptor-dependent O-(fluoresceinyl thiocarbamoyl)-amylopectin binding as an assay, the affinities of the λ receptor for maltodextrins and other sugars have been estimated. The affinity for dextrins increases with increasing degree of polymerization (Kd for maltose, 14 mM; for maltotetraose, 0.3 mM; for maltodecaose, 0.075 mM). Maltose and some other di- and trisaccharides are inhibitory to amylopectin binding, but only at concentrations above 1 mM. PMID:6445892

  7. A comparison of multiple regimens of pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine and pneumococcal polysaccharide vaccine in toddlers.

    PubMed

    Blum, M D; Dagan, R; Mendelman, P M; Pinsk, V; Giordani, M; Li, S; Bohidar, N; McNeely, T B

    2000-05-08

    Children who had been randomized to receive one dose of either heptavalent pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine (PCV) or 23-valent pneumococcal polysaccharide vaccine (PN23) at 12, 15, or 18 months of age were subsequently randomized to receive a booster injection of either PCV or PN23 12 months later. For those children who received a priming dose of PCV (N=75) compared to PN23 (N=48) at 12, 15, or 18 months of age, higher serum antibody concentrations were achieved 1 month following a booster injection of either PCV or PN23 for all serotypes tested (p<0.001). Within the group of children receiving a priming dose of PCV, those children who received a booster dose of PN23 developed higher serum antibody concentrations for four of the seven serotypes tested and similar opsonic antibody titers to serotype 6B, yet more frequent erythema (p=0.030) and pain or soreness (p=0.024) at the injection site compared to those boosted with PCV. In conclusion, a single dose of PCV at 12-18 months of age primed for responses to booster doses of either PCV or PN23 12 months later. For those children who received a priming dose of PCV, boosting with PN23 resulted in more frequent injection site pain and erythema than boosting with PCV, yet higher antibody concentrations for most of the serotypes tested.

  8. Xanthan chain length is modulated by increasing the availability of the polysaccharide copolymerase protein GumC and the outer membrane polysaccharide export protein GumB.

    PubMed

    Galván, Estela M; Ielmini, María V; Patel, Yamini N; Bianco, María I; Franceschini, Esteban A; Schneider, Jane C; Ielpi, Luis

    2013-02-01

    Xanthan is a polysaccharide secreted by Xanthomonas campestris that contains pentameric repeat units. The biosynthesis of xanthan involves an operon composed of 12 genes (gumB to gumM). In this study, we analyzed the proteins encoded by gumB and gumC. Membrane fractionation showed that GumB was mainly associated with the outer membrane, whereas GumC was an inner membrane protein. By in silico analysis and specific globomycin inhibition, GumB was characterized as a lipoprotein. By reporter enzyme assays, GumC was shown to contain two transmembrane segments flanking a large periplasmic domain. We confirmed that gumB and gumC mutant strains uncoupled the synthesis of the lipid-linked repeat unit from the polymerization process. We studied the effects of gumB and gumC gene amplification on the production, composition and viscosity of xanthan. Overexpression of GumB, GumC or GumB and GumC simultaneously did not affect the total amount or the chemical composition of the polymer. GumB overexpression did not affect xanthan viscosity; however, a moderate increase in xanthan viscosity was achieved when GumC protein levels were increased 5-fold. Partial degradation of GumC was observed when only that protein was overexpressed; but co-expression of GumB and GumC diminished GumC degradation and resulted in higher xanthan viscosity than individual GumB or GumC overexpression. Compared with xanthan from the wild-type strain, longer polymer chains from the strain that simultaneously overexpressed GumB and GumC were observed by atomic force microscopy. Our results suggest that GumB-GumC protein levels modulate xanthan chain length, which results in altered polymer viscosity.

  9. Mutant Native Outer Membrane Vesicles Combined with a Serogroup A Polysaccharide Conjugate Vaccine for Prevention of Meningococcal Epidemics in Africa

    PubMed Central

    Pajon, Rolando; Fergus, Andrew M.; Granoff, Dan M.

    2013-01-01

    Background The meningococcal serogroup A (MenA) polysaccharide conjugate vaccine used in Sub-Saharan Africa does not prevent disease caused by MenW or MenX strains, which also cause epidemics in the region. We investigated the vaccine-potential of native outer membrane vesicles with over-expressed factor H-binding protein (NOMV-fHbp), which targeted antigens in African meningococcal strains, and was combined with a MenA polysaccharide conjugate vaccine. Methodology/Principal Findings The NOMV-fHbp vaccine was prepared from a mutant African MenW strain with PorA P1.5,2, attenuated endotoxin (ΔLpxL1), deleted capsular genes, and over-expressed fHbp in variant group 1. The NOMV-fHbp was adsorbed with Al(OH)3 and used to reconstitute a lyophilized MenA conjugate vaccine, which normally is reconstituted with liquid MenC, Y and W conjugates in a meningococcal quadrivalent conjugate vaccine (MCV4-CRM, Novartis). Mice immunized with the NOMV-fHbp vaccine alone developed serum bactericidal (human complement) activity against 13 of 15 African MenA strains tested; 10 of 10 African MenX strains, 7 of 7 African MenW strains, and 6 of 6 genetically diverse MenB strains with fHbp variant group 1 (including 1 strain from The Gambia). The combination NOMV-fHbp/MenA conjugate vaccine elicited high serum bactericidal titers against the two MenA strains tested that were resistant to bactericidal antibodies elicited by the NOMV-fHbp alone; the combination elicited higher titers against the MenA and MenW strains than those elicited by a control MCV4-CRM vaccine (P<0.05); and high titers against MenX and MenB strains. For most strains, the titers elicited by a control NOMV-fHbp knock out vaccine were <1∶10 except when the strain PorA matched the vaccine (titers >1∶000). Conclusion/Significance The NOMV-fHbp/MenA conjugate vaccine provided similar or higher coverage against MenA and MenW strains than a quadrivalent meningococcal conjugate vaccine, and extended protection against Men

  10. Immune response to Vi polysaccharide, heat-killed whole cells, and outer membrane protein of Salmonella typhi.

    PubMed

    Hosny, Alaa El-Din Shawky; Diab, Mohamed Reda; Khattab, Rania Abdelmonem; Awad, Heba Osama

    2015-07-04

    Salmonella typhiVi capsular polysaccharide (ViCPS) is a licensed vaccine against typhoid fever in many countries; in Egypt, the killed whole-cell vaccine is still used. In this study, mice were used as an animal model to evaluate the immune response to ViCPS and other S. typhi antigens such as heat-killed whole cells and outer membrane protein (OMP). The three antigens were laboratory prepared, injected into mice groups, and the humoral response was evaluated using the indirect whole-cell enzyme-linked immunosorbent assay (ELISA). The sensitivity of this assay was investigated using in situ or pre-heated whole cells as coating antigens. In addition, the effect of the immunization route for ViCPS was examined. Immunizing doses of heat-killed whole cells as well as ViCPS, 2 and 4 µg given subcutaneously (SC) and 4 µg given intraperitoneally (IP), showed significant immune response compared to controls. However, the responses to these doses were not significantly different from each other. The OMP showed a higher significant response. The sensitivity of indirect whole-cell ELISA was enhanced significantly by in situ heat treatment of the coating antigen rather than the pre-heated coating antigen. The three antigens showed significant immune response. The immune response to OMP was higher. Though heat-killed whole cells and ViCPS are almost similar in immunizing level, ViCPS is recommended. The SC route was more immunizing than the IP one. Furthermore, the sensitivity of the indirect whole-cell ELISA technique could be enhanced by in situ heat inactivation of the coating cells.

  11. Outer membrane protein complex of Meningococcus enhances the antipolysaccharide antibody response to pneumococcal polysaccharide-CRM₁₉₇ conjugate vaccine.

    PubMed

    Lai, Zengzu; Schreiber, John R

    2011-05-01

    Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM₁₉₇ conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM₁₉₇ conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands.

  12. Sensor Kinase RscS Induces the Production of Antigenically Distinct Outer Membrane Vesicles That Depend on the Symbiosis Polysaccharide Locus in Vibrio fischeri

    PubMed Central

    Shibata, Satoshi

    2012-01-01

    Robust biofilm formation by Vibrio fischeri depends upon activation of the symbiosis polysaccharide (syp) locus, which is achieved by overexpressing the RscS sensor kinase (RscS+). Other than the Syp polysaccharide, however, little is known about V. fischeri biofilm matrix components. In other bacteria, biofilms contain polysaccharides, secreted proteins, and outer membrane vesicles (OMVs). Here, we asked whether OMVs are part of V. fischeri biofilms. Transmission electron microscopy revealed OMV-like particles between cells within colonies. In addition, OMVs could be purified from culture supernatants of both RscS+ and control cells, with the former releasing 2- to 3-fold more OMVs. The increase depended upon the presence of an intact syp locus, as an RscS+ strain deleted for sypK, which encodes a putative oligosaccharide translocase, exhibited reduced production of OMVs; it also showed a severe defect in biofilm formation. Western immunoblot analyses revealed that the RscS+ strain, but not the control strain or the RscS+ sypK mutant, produced a distinct set of nonproteinaceous molecules that could be detected in whole-cell extracts, OMV preparations, and lipopolysaccharide (LPS) extracts. Finally, deletion of degP, which in other bacteria influences OMV production, decreased OMV production and reduced the ability of the cells to form biofilms. We conclude that overexpression of RscS induces OMV production in a manner that depends on the presence of the syp locus and that OMVs produced under these conditions contain antigenically distinct molecules, possibly representing a modified form of lipopolysaccharide (LPS). Finally, our data indicate a correlation between OMV production and biofilm formation by V. fischeri. PMID:22020639

  13. Sensor kinase RscS induces the production of antigenically distinct outer membrane vesicles that depend on the symbiosis polysaccharide locus in Vibrio fischeri.

    PubMed

    Shibata, Satoshi; Visick, Karen L

    2012-01-01

    Robust biofilm formation by Vibrio fischeri depends upon activation of the symbiosis polysaccharide (syp) locus, which is achieved by overexpressing the RscS sensor kinase (RscS(+)). Other than the Syp polysaccharide, however, little is known about V. fischeri biofilm matrix components. In other bacteria, biofilms contain polysaccharides, secreted proteins, and outer membrane vesicles (OMVs). Here, we asked whether OMVs are part of V. fischeri biofilms. Transmission electron microscopy revealed OMV-like particles between cells within colonies. In addition, OMVs could be purified from culture supernatants of both RscS(+) and control cells, with the former releasing 2- to 3-fold more OMVs. The increase depended upon the presence of an intact syp locus, as an RscS(+) strain deleted for sypK, which encodes a putative oligosaccharide translocase, exhibited reduced production of OMVs; it also showed a severe defect in biofilm formation. Western immunoblot analyses revealed that the RscS(+) strain, but not the control strain or the RscS(+) sypK mutant, produced a distinct set of nonproteinaceous molecules that could be detected in whole-cell extracts, OMV preparations, and lipopolysaccharide (LPS) extracts. Finally, deletion of degP, which in other bacteria influences OMV production, decreased OMV production and reduced the ability of the cells to form biofilms. We conclude that overexpression of RscS induces OMV production in a manner that depends on the presence of the syp locus and that OMVs produced under these conditions contain antigenically distinct molecules, possibly representing a modified form of lipopolysaccharide (LPS). Finally, our data indicate a correlation between OMV production and biofilm formation by V. fischeri.

  14. The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis.

    PubMed

    Cosme, Ana M; Becker, Anke; Santos, Mário R; Sharypova, Larissa A; Santos, Pedro M; Moreira, Leonilde M

    2008-07-01

    Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.

  15. Preclinical immunogenicity and functional activity studies of an A+W meningococcal outer membrane vesicle (OMV) vaccine and comparisons with existing meningococcal conjugate- and polysaccharide vaccines.

    PubMed

    Tunheim, G; Arnemo, M; Næss, L M; Fjeldheim, Å K; Nome, L; Bolstad, K; Aase, A; Mandiarote, A; González, H; González, D; García, L; Cardoso, D; Norheim, G; Rosenqvist, E

    2013-12-09

    Meningococci of serogroups A and W (MenA and MenW) are the main causes of epidemic bacterial meningitis outbreaks in sub-Saharan Africa. In this study we prepared a detergent extracted outer membrane vesicle (dOMV) vaccine from representative African MenA and MenW strains, and compared the immunogenicity of this vaccine with existing meningococcal conjugate and polysaccharide (PS) vaccines in mice. NMRI mice were immunized with preclinical batches of the A+W dOMV vaccine, or with commercially available vaccines; a MenA conjugate vaccine (MenAfriVac(®), Serum Institute of India), ACYW conjugate vaccine (Menveo(®), Novartis) or ACYW PS vaccine (Mencevax(®), GlaxoSmithKline). The mice received 2 doses of 1/10 or 1/50 of a human dose with a three week interval. Immune responses were tested in ELISA, serum bactericidal activity (SBA) and opsonophagocytic activity (OPA) assays. High levels of IgG antibodies against both A and W dOMV were detected in mice receiving the A+W dOMV vaccine. High SBA titers against both MenA and MenW vaccine strains were detected after only one dose of the A+W dOMV vaccine, and the titers were further increased after the second dose. The SBA and OPA titers in mice immunized with dOMV vaccine were significantly higher than in mice immunized with the ACYW-conjugate vaccine or the PS vaccine. Furthermore, the A+W dOMV vaccine was shown to induce SBA and OPA titers against MenA of the same magnitude as the titers induced by the A-conjugate vaccine. In conclusion, the A+W dOMV vaccine induced high levels of functional antibodies to both MenA and MenW strains, levels that were shown to be higher or equal to the levels induced by licensed meningococcal vaccines. Thus, an A+W dOMV vaccine could potentially serve as an alternative or a supplement to existing conjugate and PS vaccines in the African meningitis belt. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Biochemical characteristics of the outer membranes of plant mitochondria.

    PubMed

    Mannella, C A; Bonner, W D

    1975-12-01

    Like the outer membranes of liver mitochondria, those of plant mitochondria are impermeable to cytochrome c when intact and can be ruptured by osmotic shock. Isolated plant outer mitochondrial membranes are also similar to the corresponding liver membranes in terms of phospholipid and sterol content. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis experiments indicate that a single class of proteins (apparent molecular weight 30 000) comprises the bulk of the plant outer membrane protein. There are also considerable amounts of polysaccharide associated with these membranes, which may contribute to their osmotic stability.

  17. Combined meningococcal serogroup A and W135 outer-membrane vesicles activate cell-mediated immunity and long-term memory responses against non-covalent capsular polysaccharide A.

    PubMed

    Romeu, Belkis; Lastre, Miriam; García, Luis; Cedré, Bárbara; Mandariote, Aleida; Fariñas, Mildrey; Oliva, Reynaldo; Rosenqvist, Einar; Pérez, Oliver

    2014-01-01

    Outer-membrane vesicles (OMVs) have inherent adjuvant properties, and many vaccines use OMV as vaccine components. Utilizing the adjuvant properties of OMV could lead to the formulation of vaccines that are less expensive and potentially more immunogenic than covalently conjugated polysaccharide vaccines. We evaluated the adjuvant effect in Balb/c mice of combinations of OMV from Neisseria meningitidis serogroup A and W135 as compared to that of the non-covalently conjugated capsular polysaccharide A. Both antigens were adsorbed onto aluminum hydroxide. The mice were given a booster dose of plain polysaccharide A to stimulate an immunologic memory response. Subclasses determination and cytokine assays demonstrated the capacity of OMV to induce a IgG2a/IgG2b isotype profile and IFN-γ production, suggesting the induction of a Th1 pattern immune response. Lymphoproliferative responses to OMVs were high, with affinity maturation of antibodies observed. Bactericidal titers after the booster dose were also observed. Memory B cells and long-term memory T cells were also detected. The results of this study indicate that combined meningococcal serogroup A and W135 OMV can activate cell-mediated immunity and induce a long-term memory response.

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

    PubMed Central

    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. PMID:26859882

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

    PubMed

    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.

  20. Interactions between magainin 2 and Salmonella typhimurium outer membranes: Effect of lipopolysaccharide structure

    SciTech Connect

    Rana, F.R.; Macias, E.A.; Sultany, C.M.; Modzrakowski, M.C.; Blazyk, J. )

    1991-06-18

    The role of the outer membrane and lipopolysaccharide (LPS) in the interaction between the small cationic antimicrobial peptide magainin 2 and the Gram-negative cell envelope was studied by FT-IR spectroscopy. Magainin 2 alters the thermotropic properties of the outer membrane-peptidoglycan complexes from wild-type Salmonella typhimurium and a series of LPS mutants which display differential susceptibility to the bactericidal activity of cationic antibiotics. These results are correlated with the LPS phosphorylation pattern and charge (characterized by high-resolution {sup 31}P NMR) and outer membrane lipid composition, and are compared to the bactericidal susceptibility. LPS mutants show a progressive loss of resistance to killing by magainin 2 as the length of the LPS polysaccharide moiety decreases. Disordering of the outer membrane lipid fatty acyl chains by magainin 2, however, depends primarily upon the magnitude of PLS charge rather than the length of the LPS polysaccharide. While disruption of outer membrane structure most likely is not the primary factor leading to cell death, the susceptibility of Gram-negative cells to magainin 2 is associated with factors that facilitate the transport of the peptide across the outer membrane, such as the magnitude and location of LPS charge, and concentration of LPS in the outer membrane, outer membrane molecular architecture, and the presence or absence of the O-antigen side chain.

  1. Ultrafine polysaccharide nanofibrous membranes for water purification.

    PubMed

    Ma, Hongyang; Burger, Christian; Hsiao, Benjamin S; Chu, Benjamin

    2011-04-11

    Ultrafine polysaccharide nanofibers (i.e., cellulose and chitin) with 5-10 nm diameters were employed as barrier layers in a new class of thin-film nanofibrous composite (TFNC) membranes for water purification. In addition to concentration, the viscosity of the polysaccharide nanofiber coating suspension was also found to be affected by the pH value and ionic strength. When compared with two commercial UF membranes (PAN10 and PAN400), 10-fold higher permeation flux with above 99.5% rejection ratio were achieved by using ultrafine cellulose nanofibers-based TFNC membranes for ultrafiltration of oil/water emulsions. The very high surface-to-volume ratio and negatively charged surface of cellulose nanofibers, which lead to a high virus adsorption capacity as verified by MS2 bacteriophage testing, offer further opportunities in drinking water applications. The low cost of raw cellulose/chitin materials, the environmentally friendly fabrication process, and the impressive high-flux performance indicate that such ultrafine polysaccharide nanofibers-based TFNC membranes can surpass conventional membrane systems in many different water applications.

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

    PubMed

    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.

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

    SciTech Connect

    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.

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

    SciTech Connect

    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.

  5. Outer membrane proteins of pathogenic spirochetes

    PubMed Central

    Cullen, Paul A.; Haake, David A.; Adler, Ben

    2009-01-01

    Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. PMID:15449605

  6. Improved membrane fluidity of ionic polysaccharide bead-supported phospholipid bilayer membrane systems.

    PubMed

    Haratake, Mamoru; Takahira, Ekuko; Yoshida, Sakura; Osei-Asante, Samuel; Fuchigami, Takeshi; Nakayama, Morio

    2013-07-01

    Supported phospholipid bilayer membranes on polysaccharide-based cationic polymer beads (cationic group: -[OCH2CH(OH)CH2]2N(+)(CH3)3·X(-), 45-165 μm in diameter) were prepared using small unilamellar vesicles from mixtures of phosphatidylserine (PS) and phosphatidylcholine (PC). Confocal fluorescence microscopic observations with a fluorescent membrane probe (N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine) revealed that the phospholipid molecules in the phospholipid-bead complexes were along the outer surface of the beads. The fluidity of the phospholipid bilayer membranes in the PS/PC-bead complexes was investigated by the fluorescence recovery after photobleaching (FRAP) technique. The lateral diffusion coefficients (D) for the PS/PC-bead complexes were lower than that for the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles without solid supports. Such less fluid membranes in the complexes appeared to be due to the immobilization of the phospholipid bilayer membranes by electrostatic attractive forces between PS and the bead. The D values for the PS/PC-bead complexes were dependent on the phospholipid composition; the PS(100 mol%)/PC(0 mol%)-bead complex had the least fluid membranes among the PS/PC-bead complexes tested in this study. The phospholipid bilayer membranes formed on the polysaccharide-based cationic polymer beads were much more fluid than those on a polystyrene-based one. Furthermore, such fluid phospholipid bilayer membranes formed on the polysaccharide-based cationic polymer bead were maintained for 10 days, even though the complex sample was stood in plain buffer (pH 8.5) at ambient temperature. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. The outer mitochondrial membrane in higher plants.

    PubMed

    Duncan, Owen; van der Merwe, Margaretha J; Daley, Daniel O; Whelan, James

    2013-04-01

    The acquisition and integration of intracellular organelles, such as mitochondria and plastids, were important steps in the emergence of complex multicellular life. Although the outer membranes of these organelles have lost many of the functions of their free-living bacterial ancestor, others were acquired during organellogenesis. To date, the biological roles of these proteins have not been systematically characterized. In this review, we discuss the evolutionary origins and functions of outer membrane mitochondrial (OMM) proteins in Arabidopsis thaliana. Our analysis, using phylogenetic inference, indicates that several OMM proteins either acquired novel functional roles or were recruited from other subcellular localizations during evolution in Arabidopsis. These observations suggest the existence of novel communication routes and functions between organelles within plant cells.

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

    PubMed

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

    2015-11-01

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

  9. Separate fusion of outer and inner mitochondrial membranes

    PubMed Central

    Malka, Florence; Guillery, Olwenn; Cifuentes-Diaz, Carmen; Guillou, Emmanuelle; Belenguer, Pascale; Lombès, Anne; Rojo, Manuel

    2005-01-01

    Mitochondria are enveloped by two closely apposed boundary membranes with different properties and functions. It is known that they undergo fusion and fission, but it has remained unclear whether outer and inner membranes fuse simultaneously, coordinately or separately. We set up assays for the study of inner and outer membrane fusion in living human cells. Inner membrane fusion was more sensitive than outer membrane fusion to inhibition of glycolysis. Fusion of the inner membrane, but not of the outer membrane, was abolished by dissipation of the inner membrane potential with K+ (valinomycin) or H+ ionophores (cccp). In addition, outer and inner membrane fusion proceeded separately in the absence of any drug. The separate fusion of outer and inner membranes and the different requirements of these fusion reactions point to the existence of fusion machineries that can function separately. PMID:16113651

  10. Conserved outer membrane protein of Neisseria meningitidis involved in capsule expression.

    PubMed Central

    Frosch, M; Müller, D; Bousset, K; Müller, A

    1992-01-01

    In Neisseria meningitidis, translocation of capsular polysaccharides to the cell surface is mediated by a transport system that fits the characteristics of ABC (ATP-binding cassette) transporters. One protein of this transport system, termed CtrA, is located in the outer membrane. By use of a CtrA-specific monoclonal antibody, we could demonstrate that CtrA occurs exclusively in N. meningitidis and not in other pathogenic or nonpathogenic Neisseria species. Nucleotide sequence comparison of the ctrA gene from different meningococcal serogroups indicated that CtrA is strongly conserved in all meningococcal serogroups, independent of the chemical composition of the capsular polysaccharide. Secondary structure analysis revealed that CtrA is anchored in the outer membrane by eight membrane-spanning amphipathic beta strands, a structure of proteins that function as porins. Images PMID:1371768

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

    PubMed

    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. Acinetobacter baumannii outer membrane protein A modulates the biogenesis of outer membrane vesicles.

    PubMed

    Moon, Dong Chan; Choi, Chul Hee; Lee, Jung Hwa; Choi, Chi-Won; Kim, Hye-Yeon; Park, Jeong Soon; Kim, Seung Il; Lee, Je Chul

    2012-02-01

    Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.

  13. Outer membrane vesicles as platform vaccine technology

    PubMed Central

    Stork, Michiel; van der Ley, Peter

    2015-01-01

    Abstract 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. PMID:26912077

  14. Bacterial outer membrane vesicles and vaccine applications.

    PubMed

    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.

  15. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    PubMed

    Kieselbach, Thomas; Zijnge, Vincent; Granström, 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 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.

  16. Bacterial Outer Membrane Vesicles and Vaccine Applications

    PubMed Central

    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. PMID:24715891

  17. Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis.

    PubMed

    Moriyón, I; López-Goñi, I

    1998-03-01

    The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance. This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B. melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes. On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids. This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes.

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

    PubMed

    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.

  19. Outer membrane vesicles as platform vaccine technology.

    PubMed

    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.

  20. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    PubMed

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

  1. Isolation and Characterization of the Outer Membrane of Borrelia hermsii

    PubMed Central

    Shang, Ellen S.; Skare, Jonathan T.; Exner, Maurice M.; Blanco, David R.; Kagan, Bruce L.; Miller, James N.; Lovett, Michael A.

    1998-01-01

    The outer membrane of Borrelia hermsii has been shown by freeze-fracture analysis to contain a low density of membrane-spanning outer membrane proteins which have not yet been isolated or identified. In this study, we report the purification of outer membrane vesicles (OMV) from B. hermsii HS-1 and the subsequent identification of their constituent outer membrane proteins. The B. hermsii outer membranes were released by vigorous vortexing of whole organisms in low-pH, hypotonic citrate buffer and isolated by isopycnic sucrose gradient centrifugation. The isolated OMV exhibited porin activities ranging from 0.2 to 7.2 nS, consistent with their outer membrane origin. Purified OMV were shown to be relatively free of inner membrane contamination by the absence of measurable β-NADH oxidase activity and the absence of protoplasmic cylinder-associated proteins observed by Coomassie blue staining. Approximately 60 protein spots (some of which are putative isoelectric isomers) with 25 distinct molecular weights were identified as constituents of the OMV enrichment. The majority of these proteins were also shown to be antigenic with sera from B. hermsii-infected mice. Seven of these antigenic proteins were labeled with [3H]palmitate, including the surface-exposed glycerophosphodiester phosphodiesterase, the variable major proteins 7 and 33, and proteins of 15, 17, 38, 42, and 67 kDa, indicating that they are lipoprotein constituents of the outer membrane. In addition, immunoblot analysis of the OMV probed with antiserum to the Borrelia garinii surface-exposed p66/Oms66 porin protein demonstrated the presence of a p66 (Oms66) outer membrane homolog. Treatment of intact B. hermsii with proteinase K resulted in the partial proteolysis of the Oms66/p66 homolog, indicating that it is surface exposed. This identification and characterization of the OMV proteins should aid in further studies of pathogenesis and immunity of tick-borne relapsing fever. PMID:9488399

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

    PubMed

    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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26627837

  4. Decreasing Outer Hair Cell Membrane Cholesterol Increases Cochlear Electromechanics

    NASA Astrophysics Data System (ADS)

    Brownell, William E.; Jacob, Stefan; Hakizimana, Pierre; Ulfendahl, Mats; Fridberger, Anders

    2011-11-01

    The effect of decreasing membrane cholesterol on the mechanical response of the cochlea to acoustic and/or electrical stimulation was monitored using laser interferometry. In contrast to pharmacological interventions that typically decrease cochlear electromechanics, reducing membrane cholesterol increased the response. The electromechanical response in untreated preparations was asymmetric with greater displacements in response to positive currents and cholesterol depletion increased the asymmetry. The results confirm that outer hair cell electromotility is enhanced by low membrane cholesterol. The asymmetry of the response indicates the outer hair cell resting membrane potential is hyperpolarized relative to the voltage of maximum gain for the outer hair cell voltage-displacement function. The magnitude of the response increase suggests a non-uniform distribution of cholesterol along the lateral wall of normal adult outer hair cells.

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

    PubMed Central

    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

  6. Proteome analysis of mitochondrial outer membrane from Neurospora crassa

    SciTech Connect

    Schmitt, Simone; Prokisch, Holger; Schlunk, Tilman; Camp, David G.; Ahting, Uwe; Waizenegger, Thomas; Scharfe, Curt M.; Meitinger, Thomas; Imhof, Axel; Neupert, Walter; Oefner, Peter J.; Rapaport, Doron

    2006-01-01

    The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of liquid chromatography tandem mass spectrometry of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS peptide fingerprinting. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (Porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.

  7. Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

    PubMed Central

    Martorana, Alessandra M.; Motta, Sara; Di Silvestre, Dario; Falchi, Federica; Dehò, Gianni; Mauri, Pierluigi; Sperandeo, Paola; Polissi, Alessandra

    2014-01-01

    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. PMID:24967819

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

    PubMed Central

    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

  9. Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies.

    PubMed

    Chen, Linxiao; Valentine, Jenny L; Huang, Chung-Jr; Endicott, Christine E; Moeller, Tyler D; Rasmussen, Jed A; Fletcher, Joshua R; Boll, Joseph M; Rosenthal, Joseph A; Dobruchowska, Justyna; Wang, Zhirui; Heiss, Christian; Azadi, Parastoo; Putnam, David; Trent, M Stephen; Jones, Bradley D; DeLisa, Matthew P

    2016-06-28

    The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

  10. Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

    PubMed Central

    Valentine, Jenny L.; Huang, Chung-Jr; Endicott, Christine E.; Moeller, Tyler D.; Rasmussen, Jed A.; Fletcher, Joshua R.; Boll, Joseph M.; Rosenthal, Joseph A.; Dobruchowska, Justyna; Wang, Zhirui; Heiss, Christian; Azadi, Parastoo; Putnam, David; Trent, M. Stephen; Jones, Bradley D.; DeLisa, Matthew P.

    2016-01-01

    The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens. PMID:27274048

  11. Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

    DOE PAGES

    Chen, Linxiao; Valentine, Jenny L.; Huang, Chung-Jr; ...

    2016-06-06

    The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. In this paper, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Usingmore » this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Finally, given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.« less

  12. Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Shang, E S; Foley, D M; Blanco, D R; Champion, C I; Mirzabekov, T; Sokolov, Y; Kagan, B L; Miller, J N; Lovett, M A

    1995-01-01

    We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease. Images PMID:7593626

  13. Agents that increase the permeability of the outer membrane.

    PubMed Central

    Vaara, M

    1992-01-01

    The outer membrane of gram-negative bacteria provides the cell with an effective permeability barrier against external noxious agents, including antibiotics, but is itself a target for antibacterial agents such as polycations and chelators. Both groups of agents weaken the molecular interactions of the lipopolysaccharide constituent of the outer membrane. Various polycations are able, at least under certain conditions, to bind to the anionic sites of lipopolysaccharide. Many of these disorganize and cross the outer membrane and render it permeable to drugs which permeate the intact membrane very poorly. These polycations include polymyxins and their derivatives, protamine, polymers of basic amino acids, compound 48/80, insect cecropins, reptilian magainins, various cationic leukocyte peptides (defensins, bactenecins, bactericidal/permeability-increasing protein, and others), aminoglycosides, and many more. However, the cationic character is not the sole determinant required for the permeabilizing activity, and therefore some of the agents are much more effective permeabilizers than others. They are useful tools in studies in which the poor permeability of the outer membrane poses problems. Some of them undoubtedly have a role as natural antibiotic substances, and they or their derivatives might have some potential as pharmaceutical agents in antibacterial therapy as well. Also, chelators (such as EDTA, nitrilotriacetic acid, and sodium hexametaphosphate), which disintegrate the outer membrane by removing Mg2+ and Ca2+, are effective and valuable permeabilizers. PMID:1406489

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

    PubMed

    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.

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

    PubMed

    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.

  16. Production of Outer Membrane Vesicles and Outer Membrane Tubes by Francisella novicida

    PubMed Central

    McCaig, William D.; Koller, Antonius

    2013-01-01

    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. PMID:23264574

  17. Cross-linking analysis of antigenic outer membrane protein complexes of Neisseria meningitidis.

    PubMed

    Sánchez, Sandra; Abel, Ana; Arenas, Jesús; Criado, María Teresa; Ferreirós, Carlos M

    2006-03-01

    Polysaccharide-based approaches have not enabled the development of effective vaccines against meningococci of serogroup B, and the most promising current research is focused on the use of outer membrane vesicles. Due to the toxicity of the outer membrane oligosaccharides, new vaccines based on purified proteins are being sought, but despite the application of advanced techniques, they remain elusive, perhaps due to the fact that standard techniques for analysis of antigens overlook conformational epitopes located in membrane complexes. Membrane complex antigens have been analyzed in Neisseria gonorrhoeae, and a study published on Neisseria meningitidis has reported the in vitro formation of 800-kD complexes by deposition of a purified protein (MSP63) onto synthetic lipid layers; however, no studies to date have attempted to identify membrane complexes present in vivo in N. meningitidis. In the present study, cross-linking with formaldehyde was used to identify outer membrane protein associations in various N. meningitidis and Neisseria lactamica strains. In N. meningitides, complexes of about 450 kD (also present in N. lactamica), 165 and 95 kD were detected and shown to be made up of the proteins MSP63, PorA/PorB/RmpM/FetA, and PorA/PorB/RmpM, respectively. In western blots, the 450-kD complex was identified by mouse antibodies raised against outer membrane vesicles, but not by antibodies raised against the purified complex, demonstrating the importance of conformational epitopes, and thus suggesting that the analysis of antigens in their native conformation may be useful or even essential for the design of effective vaccines against meningococci.

  18. Substrate Specificity within a Family of Outer Membrane Carboxylate Channels

    SciTech Connect

    Eren, Elif; Vijayaraghavan, Jagamya; Liu, Jiaming; Cheneke, Belete R.; Touw, Debra S.; Lepore, Bryan W.; Indic, Mridhu; Movileanu, Liviu; van den Berg, Bert; Dutzler, Raimund

    2012-01-17

    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.

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

    PubMed

    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.

  20. A Molecularly Complete Planar Bacterial Outer Membrane Platform

    PubMed Central

    Hsia, Chih-Yun; Chen, Linxiao; Singh, Rohit R.; DeLisa, Matthew P.; Daniel, Susan

    2016-01-01

    The bacterial outer membrane (OM) is a barrier containing membrane proteins and liposaccharides that fulfill crucial functions for Gram-negative bacteria. With the advent of drug-resistant bacteria, it is necessary to understand the functional role of this membrane and its constituents to enable novel drug designs. Here we report a simple method to form an OM-like supported bilayer (OM-SB), which incorporates native lipids and membrane proteins of gram-negative bacteria from outer membrane vesicles (OMVs). We characterize the formation of OM-SBs using quartz crystal microbalance with dissipation (QCM-D) and fluorescence microscopy. We show that the orientation of proteins in the OM-SB matches the native bacterial membrane, preserving the characteristic asymmetry of these membranes. As a demonstration of the utility of the OM-SB platform, we quantitatively measure antibiotic interactions between OM-SBs and polymyxin B, a cationic peptide used to treat Gram-negative infections. This data enriches understanding of the antibacterial mechanism of polymyxin B, including disruption kinetics and changes in membrane mechanical properties. Combining OM-SBs with microfluidics will enable higher throughput screening of antibiotics. With a broader view, we envision that a molecularly complete membrane-scaffold could be useful for cell-free applications employing engineered membrane proteins in bacterial membranes for myriad technological purposes. PMID:27600663

  1. Molecular Basis of Bacterial Outer Membrane Permeability Revisited

    PubMed Central

    Nikaido, Hiroshi

    2003-01-01

    Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions. PMID:14665678

  2. Inner/Outer Nuclear Membrane Fusion in Nuclear Pore Assembly

    PubMed Central

    Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon

    2010-01-01

    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. PMID:20926687

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

    PubMed

    Elhenawy, Wael; Bording-Jorgensen, Michael; Valguarnera, Ezequiel; Haurat, M Florencia; Wine, Eytan; Feldman, Mario F

    2016-07-12

    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. The role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis in S Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. An S Typhimurium ΔpagL strain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are

  4. Heterogeneity in Lipid Composition of the Outer Membrane and Cytoplasmic Membrane of Pseudomonas BAL-31

    PubMed Central

    Diedrich, D. L.; Cota-Robles, E. H.

    1974-01-01

    The outer membranes and cytoplasmic membranes of the marine bacterium Pseudomonas BAL-31 were separated by washing the cells three times in 0.5 M NaCl and twice in 0.5 M sucrose. Electron microscopy during the removal of membranes revealed that the outer membranes fragmented in a regular manner to give rise to fairly uniform vesicles measuring approximately 140 nm in diameter. Isolated outer membranes had a buoyant density in sucrose of 1.230 g per cm3, whereas the cytoplasmic membranes had a density of 1.194 g per cm3. The removal of the outer membrane during the application of this procedure was monitored by measuring the release of 2-keto-3-deoxyoctulosonic acid and phospholipid. The cells lost 85.5% of their 2-keto-3-deoxyoctulosonic acid and 47.3% of their phospholipid during this treatment. Complete recovery of outer membrane material could be achieved. The removal of 25.5% of the 2-keto-3-deoxyoctulosonic acid and 0.9% of the phospholipid rendered the cells sensitive to lysis with Triton X-100. The phospholipid composition of the outer membrane was calculated to be 78.9% phosphatidylethanolamine and 16.1% phosphatidylglycerol. The phospholipid composition of the cytoplasmic membrane proved to be 71.5% phosphatidylethanolamine and 23.5% phosphatidylglycerol. The fatty acid composition was also found to be quantitatively heterogeneous between the two membranes. Images PMID:4852262

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

    PubMed Central

    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

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

    PubMed Central

    2011-01-01

    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. PMID:22133164

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

    PubMed Central

    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

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

    PubMed

    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

  9. Electrospun cellulose acetate membrane for size separating and antibacterial screening of crude polysaccharides.

    PubMed

    Chumpol, Jiraporn; Siri, Sineenat

    2016-12-01

    This study aims to produce electrospun cellulose acetate (CA) membrane as the alternative supporting medium for a separation of crude polysaccharides by electrophoresis and a screening of their antibacterial activity. Among the tested conditions of fabrication, electrospun CA membrane at 57% porosity showed the best separation of each polysaccharide from the standard mixture and the crude extract of Aloe vera via electrophoresis. As compared with the commercial CA membrane, the produced electrospun CA membrane demonstrated more separated spots of polysaccharides. The antibacterial activity of the electrophoretic polysaccharide was also determined against Escherichia coli and Staphylococcus aureus as the inhibition zone after the bacterial culture agar was overlaid on the membrane and incubated for 24 h. The results of this study suggested the potential application of electrospun CA membrane combining with electrophoresis as a simple method for separating crude polysaccharides and screening for their antibacterial activity.

  10. Changes in the cell-wall polysaccharides of outer pericarp tissues of kiwifruit during development.

    PubMed

    Li, Xingjun; Nakagawa, Naoki; Nevins, Donald J; Sakurai, Naoki

    2006-01-01

    Changes in pectin, hemicelluloses and cellulose in the cell walls of outer pericarp tissues of kiwifruit (Actinidia deliciosa cv. Hayward) were determined during development. An extensive amylase digestion was employed to remove possible contaminating starch before and after fractionation of wall polysaccharides. An initial treatment of crude cell walls with alpha-amylase and iso-amylase or DMSO, was found to be insufficient removing the contaminating starch from wall polysaccharides. After EDTA and alkaline extraction, the pectic and hemicellulose fractions were again treated with the combination of alpha-amylase and iso-amylase. The amounts of predominant pectic sugars Gal, Rha and Ara, unaffected by the first and second amylase digestion, decreased markedly during the early fruit enlargement (8-12 weeks after anthesis, WAA), then increased during 16-20 WAA, and finally declined during fruit maturity (20-25 WAA). The molecular-mass of pectic polysaccharides decreased during fruit enlargement (8-16 WAA), and then changed little during fruit maturity. The higher molecular-mass components of hemicelluloses in HC-I and HC-II fractions detected at the early stage of fruit enlargement (8-12 WAA) were degraded at the late stage of fruit enlargement (16 WAA), but then remained stable at the much lower molecular-mass till fruit maturity. The amount of Xyl in the HC-II fraction decreased during the early fruit enlargement and fruit maturity, an observation that was consistent with xyloglucan (XG) content. The gel permeation profiles of XG showed a slight increase in higher molecular-mass components during 8-12 WAA, but thereafter there was no significant down-shift of molecular-mass until harvest time. The cellulose fraction increased steadily during fruit enlargement through maturity, but the XG contents in HC-I and HC-II fractions remained at a low level during these stages. Methylation analysis of HC-I and HC-II fractions confirmed the low level of XG in the

  11. A membrane bending model of outer hair cell electromotility.

    PubMed Central

    Raphael, R M; Popel, A S; Brownell, W E

    2000-01-01

    We propose a new mechanism for outer hair cell electromotility based on electrically induced localized changes in the curvature of the plasma membrane (flexoelectricity). Electromechanical coupling in the cell's lateral wall is modeled in terms of linear constitutive equations for a flexoelectric membrane and then extended to nonlinear coupling based on the Langevin function. The Langevin function, which describes the fraction of dipoles aligned with an applied electric field, is shown to be capable of predicting the electromotility voltage displacement function. We calculate the electrical and mechanical contributions to the force balance and show that the model is consistent with experimentally measured values for electromechanical properties. The model rationalizes several experimental observations associated with outer hair cell electromotility and provides for constant surface area of the plasma membrane. The model accounts for the isometric force generated by the cell and explains the observation that the disruption of spectrin by diamide reduces force generation in the cell. We discuss the relation of this mechanism to other proposed models of outer hair cell electromotility. Our analysis suggests that rotation of membrane dipoles and the accompanying mechanical deformation may be the molecular mechanism of electromotility. PMID:10827967

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

    PubMed

    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.

  13. Outer membrane protein biogenesis in Gram-negative bacteria

    PubMed Central

    Rollauer, Sarah E.; Sooreshjani, Moloud A.; Noinaj, Nicholas; Buchanan, Susan K.

    2015-01-01

    Gram-negative bacteria contain a double membrane which serves for both protection and for providing nutrients for viability. The outermost of these membranes is called the outer membrane (OM), and it contains a host of fully integrated membrane proteins which serve essential functions for the cell, including nutrient uptake, cell adhesion, cell signalling and waste export. For pathogenic strains, many of these outer membrane proteins (OMPs) also serve as virulence factors for nutrient scavenging and evasion of host defence mechanisms. OMPs are unique membrane proteins in that they have a β-barrel fold and can range in size from 8 to 26 strands, yet can still serve many different functions for the cell. Despite their essential roles in cell survival and virulence, the exact mechanism for the biogenesis of these OMPs into the OM has remained largely unknown. However, the past decade has witnessed significant progress towards unravelling the pathways and mechanisms necessary for moulding a nascent polypeptide into a functional OMP within the OM. Here, we will review some of these recent discoveries that have advanced our understanding of the biogenesis of OMPs in Gram-negative bacteria, starting with synthesis in the cytoplasm to folding and insertion into the OM. PMID:26370935

  14. Mitochondria and cell death: outer membrane permeabilization and beyond.

    PubMed

    Tait, Stephen W G; Green, Douglas R

    2010-09-01

    Mitochondrial outer membrane permeabilization (MOMP) is often required for activation of the caspase proteases that cause apoptotic cell death. Various intermembrane space (IMS) proteins, such as cytochrome c, promote caspase activation following their mitochondrial release. As a consequence, mitochondrial outer membrane integrity is highly controlled, primarily through interactions between pro- and anti-apoptotic members of the B cell lymphoma 2 (BCL-2) protein family. Following MOMP by pro-apoptotic BCL-2-associated X protein (BAX) or BCL-2 antagonist or killer (BAK), additional regulatory mechanisms govern the mitochondrial release of IMS proteins and caspase activity. MOMP typically leads to cell death irrespective of caspase activity by causing a progressive decline in mitochondrial function, although cells can survive this under certain circumstances, which may have pathophysiological consequences.

  15. The outer membrane vesicles: Secretion system type zero.

    PubMed

    Guerrero-Mandujano, Andrea; Hernández-Cortez, Cecilia; Ibarra, Jose Antonio; Castro-Escarpulli, Graciela

    2017-07-01

    Gram-negative bacteria have mechanisms through which they can colonize and survive in different environments, such as the secretion systems types (1-6) that have been widely studied and characterized. Nowadays, some authors have proposed extracellular structures, such as the outer membrane vesicles (OMVs), to be considered as an additional and independent secretion system. The OMVs are spherical particles of 50-250 nm in diameter; they originate in the outer membrane, and therefore they have a very similar composition to the latter. These particles can transport an important variety of biomolecules: enzymes, toxins, antigenic determinants and even nucleic acids. Thus, it is of great interest to collect data describing the advantages of the transport of biomolecules through the OMVs and, thus, determine their role as a potential secretion system. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Localized translation near the mitochondrial outer membrane: An update.

    PubMed

    Lesnik, Chen; Golani-Armon, Adi; Arava, Yoav

    2015-01-01

    Local synthesis of proteins near their activity site has been demonstrated in many biological systems, and has diverse contributions to cellular functions. Studies in recent years have revealed that hundreds of mitochondria-destined proteins are synthesized by cytosolic ribosomes near the mitochondrial outer membrane, indicating that localized translation also occurs at this cellular locus. Furthermore, in the last year central factors that are involved in this process were identified in yeast, Drosophila, and human cells. Herein we review the experimental evidence for localized translation on the cytosolic side of the mitochondrial outer membrane; in addition, we describe the factors that are involved in this process and discuss the conservation of this mechanism among various species. We also describe the relationship between localized translation and import into the mitochondria and suggest avenues of study that look beyond cotranslational import. Finally we discuss future challenges in characterizing the mechanisms for localized translation and its physiological significance.

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

    PubMed

    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.

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

    PubMed

    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. © 2015 The Authors Molecular Oral Microbiology Published by John Wiley & Sons Ltd.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23731905

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

    PubMed

    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.

  1. Structural basis of the Escherichi coli outer-membrane permeability

    NASA Astrophysics Data System (ADS)

    Amro, Nabil A.; Kotra, Lakshmi P.; Wadu-Mesthrige, Kapila; Bulychev, Alexy; Mobashery, Shahriar; Liu, Gang-yu

    1999-06-01

    We have studied, using AFM, the structural basis of the outer membrane permeability for the bacterium E. col. The surface of the bacteria is visualized with an unprecedented details. Our AFM images clearly reveal that the outer membrane exhibits protrusions, which correspond to patches of LPS containing hundreds to thousands of LPS molecules. The packing of the nearest neighbor patches is tight, and as such the LPS layer provides an effective permeability barrier for the Gram-negative bacteria. We have also studied the mechanism of their permeability increase upon metal depletion. Our AFM images reveal that LPS molecules are released from the boundaries of some patches during the initial EDTA treatment. Further metal depletion produces a very distinct structure at the outer membrane: appearance of irregularly shaped pits. The pits are likely formed as a result of liberation of LPS patches and lipoproteins, exposing areas of peptidoglacan surface. Our study has proven AFM to be a very useful technique in providing structural basis for the functions of organisms.

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

    PubMed

    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.

  3. PelC is a Pseudomonas aeruginosa outer membrane lipoprotein of the OMA family of proteins involved in exopolysaccharide transport.

    PubMed

    Vasseur, Perrine; Soscia, Chantal; Voulhoux, Romé; Filloux, Alain

    2007-08-01

    Pseudomonas aeruginosa is a gram-negative bacterium, opportunistic pathogen, which causes severe acute or chronic infections, as is the case with cystic fibrosis patients. Chronic infections are frequently accompanied by the development of the bacterial population into a specialized community called biofilm. The pelA-G gene cluster of P. aeruginosa has been shown to be involved in pellicle production and biofilm formation. The pel genes have been proposed to contribute to the formation of the exopolysaccharide-containing pellicle. However, the function and the subcellular localization of the seven different Pel proteins are poorly understood. Based on bioinformatics analysis, we have previously considered that PelF is a putative glycosyltransferase (GT4 family), whereas PelG is a Wzx-like polysaccharide transporter from the PST family. In this study we have further characterized the PelC protein. We have shown that PelC is an outer membrane lipoprotein. The N-terminal signal peptide of the PelC lipoprotein is sufficient to target the protein into the membranes. However, by constructing various PelC hybrid proteins we also proposed that efficient and functional outer membrane insertion of PelC requires not only the signal peptide and the lipid modification, but also requires the C-terminal domain of PelC. Because the gene encoding the outer membrane lipoprotein PelC is part of a putative gene cluster involved in exopolysaccharide biogenesis, we suggest that PelC is a new member of the outer membrane auxiliary (OMA) family of lipoprotein whose Wza, involved in Escherichia coli capsular polysaccharide transport, is an archetype.

  4. Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

    PubMed Central

    Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

    1995-01-01

    The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

  5. Application of glyco-blotting for identification of structures of polysaccharides causing membrane fouling in a pilot-scale membrane bioreactor treating municipal wastewater.

    PubMed

    Kimura, Katsuki; Nishimura, Shin-Ichiro; Miyoshi, Risho; Hoque, Asiful; Miyoshi, Taro; Watanabe, Yoshimasa

    2015-03-01

    A new approach for the analysis of polysaccharides in membrane bioreactor (MBR) is proposed in this study. Enrichment of polysaccharides by glyco-blotting, in which polysaccharides are specifically collected via interactions between the aldehydes in the polysaccharides and aminooxy groups on glycoblotting beads, enabled MALDI-TOF/MS analysis at a high resolution. Structures of polysaccharides extracted from fouled membranes used in a pilot-scale MBR treating municipal wastewater and those in the supernatant of the mixed liquor suspension in the MBR were investigated. It was found that the overlap between polysaccharides found in the supernatants and those extracted from the fouled membrane was rather limited, suggesting that polysaccharides that dominate in supernatants may not be important in membrane fouling in MBRs. Analysis using a bacterial carbohydrate database suggested that capsular polysaccharides (CPS) and/or lipo-polysaccharides (LPS) produced by gram-negative bacteria are key players in the evolution of membrane fouling in MBRs.

  6. Ligand-gated Diffusion Across the Bacterial Outer Membrane

    SciTech Connect

    B Lepore; M Indic; H Pham; E Hearn; D Patel; B van den Berg

    2011-12-31

    Ligand-gated channels, in which a substrate transport pathway is formed as a result of the binding of a small-molecule chemical messenger, constitute a diverse class of membrane proteins with important functions in prokaryotic and eukaryotic organisms. Despite their widespread nature, no ligand-gated channels have yet been found within the outer membrane (OM) of Gram-negative bacteria. Here we show, using in vivo transport assays, intrinsic tryptophan fluorescence and X-ray crystallography, that high-affinity (submicromolar) substrate binding to the OM long-chain fatty acid transporter FadL from Escherichia coli causes conformational changes in the N terminus that open up a channel for substrate diffusion. The OM long-chain fatty acid transporter FadL from E. coli is a unique paradigm for OM diffusion-driven transport, in which ligand gating within a {beta}-barrel membrane protein is a prerequisite for channel formation.

  7. C1q binding and activation of the complement classical pathway by Klebsiella pneumoniae outer membrane proteins.

    PubMed Central

    Albertí, S; Marqués, G; Camprubí, S; Merino, S; Tomás, J M; Vivanco, F; Benedí, V J

    1993-01-01

    The mechanisms of killing of Klebsiella pneumoniae serum-sensitive strains in nonimmune serum by the complement classical pathway have been studied. The bacterial cell surface components that bind C1q more efficiently were identified as two major outer membrane proteins, presumably the porins of this bacterial species. These two outer membrane proteins were isolated from a representative serum-sensitive strain. We have demonstrated that in their purified form, they bind C1q and activate the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted in factor D). Binding of C1q to other components of the bacterial outer membrane, in particular the rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the two outer membrane proteins. The antibody-independent binding of C1q to serum-sensitive strains was independent of the presence of capsular polysaccharide, while strains possessing lipopolysaccharide O antigen bind less C1q and are resistant to complement-mediated killing. Images PMID:8432605

  8. Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.

    PubMed

    Bahar, Ofir; Mordukhovich, Gideon; Luu, Dee Dee; Schwessinger, Benjamin; Daudi, Arsalan; Jehle, Anna Kristina; Felix, Georg; Ronald, Pamela C

    2016-05-01

    Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.

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

    PubMed

    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.

  10. Detection of outer membrane vesicles in Synechocystis PCC 6803

    PubMed Central

    Pardo, Yehudah A.; Florez, Catalina; Baker, Kristopher M.; Schertzer, Jeffrey W.; Mahler, Gretchen J.

    2015-01-01

    It has been well established that many species of Gram-negative bacteria release nanoscale outer membrane vesicles (OMVs) during normal growth. Furthermore, the roles of these structures in heterotrophic bacteria have been extensively characterized. However, little is known about the existence or function of OMVs in photoautotrophs. In the present study, we report for the first time the production of OMVs by the model photosynthetic organism Synechocystis sp. PCC 6803, a species of biotechnological importance. We detected extracellular proteins and lipids in cell-free supernatants derived from Synechocystis culture, yet the cytoplasmic and thylakoid membrane markers NADH oxidase and chlorophyll were absent. This indicated that the extracellular proteins and lipids derived from the outer membrane, and not from cell lysis. Furthermore, we identified spherical structures within the expected size range of OMVs in Synechocystis culture using scanning electron microscopy. Taken together, these results suggest that the repertoire of Gram-negative bacteria that are known to produce OMVs may be expanded to include Synechocystis PCC6803. Because of the considerable genetic characterization of Synechocystis in particular, our discovery has the potential to support novel biotechnological applications as well. PMID:26363014

  11. Characterization of Francisella tularensis Outer Membrane Proteins▿ †

    PubMed Central

    Huntley, Jason F.; Conley, Patrick G.; Hagman, Kayla E.; Norgard, Michael V.

    2007-01-01

    Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs. PMID:17114266

  12. Iron-Associated Outer Membrane Proteins of Magnetic Bacteria

    DTIC Science & Technology

    1989-06-16

    AD-A210 088 _ _ _ _ _ _ _ _ _ _ _ _ Form Approved WrMN PAGE0MB No ()704-0188 la RPORTSECQ!TY -AssF.(L; i RES’C it MA %CS ()NA 14 J 1 ILL 2a SECURITY...NUMBERS 800N. uicy t.PROGRAM PROiECT rASK P T’O ~80NQunyS.EiLEVE T NO NO NO jACCES ON NO Arlington, VA 22217-5000 61153N IRR 4106 4413-009 1 1 TITLE...include Security Classification) (u) Iron Associated Outer Membrane Proteins of Magnetic Bacteria 12 PERSONAL AuTHOR(S) Blakemore, Richard Peter 1 3a

  13. Membrane tether formation from outer hair cells with optical tweezers.

    PubMed Central

    Li, Zhiwei; Anvari, Bahman; Takashima, Masayoshi; Brecht, Peter; Torres, Jorge H; Brownell, William E

    2002-01-01

    Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasma membrane by pulling tethers with 4.5-microm polystyrene beads. Tether formation force and tether force were measured in static and dynamic conditions. A greater force was required for tether formations from OHC lateral wall (499 +/- 152 pN) than from OHC basal end (142 +/- 49 pN). The difference in the force required to pull tethers is consistent with an extensive cytoskeletal framework associated with the lateral wall known as the cortical lattice. The apparent plasma membrane stiffness, estimated under the static conditions by measuring tether force at different tether length, was 3.71 pN/microm for OHC lateral wall and 4.57 pN/microm for OHC basal end. The effective membrane viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and estimated in the range of 2.39 to 5.25 pN x s/microm. The viscous force most likely results from the viscous interactions between plasma membrane lipids and the OHC cortical lattice and/or integral membrane proteins. The information these studies provide on the mechanical properties of the OHC lateral wall is important for understanding the mechanism of OHC electromotility. PMID:11867454

  14. Antibody response of swine to outer membrane components of Haemophilus pleuropneumoniae during infection.

    PubMed Central

    Rapp, V J; Ross, R F

    1986-01-01

    Sera from pigs infected with Haemophilus (Actinobacillus) pleuropneumoniae were tested for antibodies to outer membrane proteins (OMPs) of the organism by immunoblotting. Convalescent sera were produced in naturally born, colostrum-fed pigs and in cesarean-derived, colostrum-deprived pigs given H. pleuropneumoniae serotype 5 intranasally twice at 5-week intervals. Sera, collected at weekly intervals, were reacted with Sarkosyl-insoluble, OMP-enriched preparations of H. pleuropneumoniae which had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Antibodies were detected to OMPs with an apparent molecular weight of 16,500 (16.5K OMP); to 29K, 38.5K, 43.5K, 45K, 49.5K, and 66.5K OMPs; and to several high-molecular-weight (greater than or equal to 94,000) OMPs, but not to the major 42K OMP. Antibodies to the heat-modifiable OMP (29K/43.5K) and the 38.5K OMP were detected in sera from noninfected pigs. Antibodies were also detected to two broad 54,000- and 95,000-molecular-weight bands which did not stain with Coomassie blue, stained with silver nitrate, resisted proteinase K digestion, and were eliminated by oxidation with sodium metaperiodate. This indicates that the 54,000- and 95,000-molecular-weight bands represent polysaccharide, possibly capsular or lipopolysaccharide immunogens. Adsorption of sera with cells from the homologous serotype 5 strain removed antibodies to the 45K, 49.5K, 66.5K, and greater than or equal to 94K OMPs and to the two polysaccharide bands, indicating that these antibodies were directed primarily to surface-exposed epitopes. When tested with OMP preparations from other serotype 5 strains, heterogeneity was apparent, both in the reactions with OMPs and with the polysaccharide bands. Silver staining of proteinase K-treated, whole-cell lysates from serotype 5 strains also indicated variable expression of the polysaccharide bands. Sera also reacted with OMPs from

  15. A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria.

    PubMed

    Wang, Yan; Wang, Rui; Jin, Feng; Liu, Yang; Yu, Jiayu; Fu, Xinmiao; Chang, Zengyi

    2016-08-05

    β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. HHomp—prediction and classification of outer membrane proteins

    PubMed Central

    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

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

    PubMed

    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.

  18. Protein selection and export via outer membrane vesicles

    PubMed Central

    Bonnington, K. E.; Kuehn, M. J.

    2014-01-01

    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 300 nm 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. PMID:24370777

  19. Protein selection and export via outer membrane vesicles.

    PubMed

    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.

  20. Outer membrane vesicles of Pasteurella multocida contain virulence factors

    PubMed Central

    Fernández-Rojas, Miguel A; Vaca, Sergio; Reyes-López, Magda; de la Garza, Mireya; Aguilar-Romero, Francisco; Zenteno, Edgar; Soriano-Vargas, Edgardo; Negrete-Abascal, Erasmo

    2014-01-01

    Pasteurella multocida (Pm) is a gram-negative bacterium able to infect different animal species, including human beings. This bacterium causes economic losses to the livestock industry because of its high morbidity and mortality in animals. In this work, we report the characterization of outer membrane vesicles (OMVs) released into the culture medium by different Pm serogroups. Purified OMVs in the range of 50–300 nm were observed by electron microscopy. Serum obtained from chickens infected with Pm recognized several proteins from Pm OMVs. Additionally, rabbit antiserum directed against a secreted protease from Actinobacillus pleuropneumoniae recognized a similar protein in the Pm OVMs, suggesting that OMVs from these bacterial species contain common immunogenic proteins. OmpA, a multifunctional protein, was identified in OMVs from different Pm serogroups, and its concentration was twofold higher in OMVs from Pm serogroups B and D than in OMVs from other serogroups. Three outer membrane proteins were also identified: OmpH, OmpW, and transferrin-binding protein. Three bands of 65, 110, and 250 kDa with proteolytic activity were detected in Pm OMVs of serogroups A and E. Additionally, β-lactamase activity was detected only in OMVs from Pm 12945 Ampr (serogroup A). Pm OMVs may be involved in different aspects of disease pathogenesis. PMID:25065983

  1. Outer membrane proteome and antigens of Tannerella forsythia.

    PubMed

    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.

  2. Outer membrane vesicles of Pasteurella multocida contain virulence factors.

    PubMed

    Fernández-Rojas, Miguel A; Vaca, Sergio; Reyes-López, Magda; de la Garza, Mireya; Aguilar-Romero, Francisco; Zenteno, Edgar; Soriano-Vargas, Edgardo; Negrete-Abascal, Erasmo

    2014-10-01

    Pasteurella multocida (Pm) is a gram-negative bacterium able to infect different animal species, including human beings. This bacterium causes economic losses to the livestock industry because of its high morbidity and mortality in animals. In this work, we report the characterization of outer membrane vesicles (OMVs) released into the culture medium by different Pm serogroups. Purified OMVs in the range of 50-300 nm were observed by electron microscopy. Serum obtained from chickens infected with Pm recognized several proteins from Pm OMVs. Additionally, rabbit antiserum directed against a secreted protease from Actinobacillus pleuropneumoniae recognized a similar protein in the Pm OVMs, suggesting that OMVs from these bacterial species contain common immunogenic proteins. OmpA, a multifunctional protein, was identified in OMVs from different Pm serogroups, and its concentration was twofold higher in OMVs from Pm serogroups B and D than in OMVs from other serogroups. Three outer membrane proteins were also identified: OmpH, OmpW, and transferrin-binding protein. Three bands of 65, 110, and 250 kDa with proteolytic activity were detected in Pm OMVs of serogroups A and E. Additionally, β-lactamase activity was detected only in OMVs from Pm 12945 Amp(r) (serogroup A). Pm OMVs may be involved in different aspects of disease pathogenesis. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  3. Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

    PubMed Central

    Costello, Shawn M.; Plummer, Ashlee M.; Fleming, Patrick J.; Fleming, Karen G.

    2016-01-01

    Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed “Outer Membrane Protein Biogenesis Model” (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway. PMID:27482090

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

    PubMed

    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.

  5. Cell wall polysaccharide biosynthesis by membrane fragments from Streptococcus pyogenes and stabilized L-form.

    PubMed

    Cohen, M; Panos, C

    1971-05-01

    The formation and composition of a cell wall rhamnose-containing polysaccharide by membrane fragments from Streptococcus pyogenes and its stabilized L-form were compared. Also, the effect of prior treatment on the ability of coccal whole-cell and membrane fragments to incorporate radioactivity from thymidine diphosphate-(14)C-rhamnose, and the results of subsequent attempts to remove labeled polysaccharide from such membranes are given. L-form membrane fragments were capable of only 10% uptake of (14)C-rhamnose from this nucleotide as compared with streptococcal membranes. However, once bound, both membrane fragments polymerized rhamnose to the same extent. These findings tend to negate the almost complete lack of polymeric rhamnose within the intact L-form as being due to the absence of membrane enzymes necessary for the transfer of rhamnose from a suitable precursor to membrane acceptor sites or enzymes responsible for rhamnose polymerization. Degradation of labeled rhamnose polysaccharide after isolation from coccal membranes by mild acid hydrolysis showed muramic acid and glucosamine to be attached. This same polysaccharide from L-form membrane fragments was devoid of amino sugars. These data suggest the possible involvement of amino sugars in the attachment of cell wall polymeric rhamnose to the streptococcal cytoplasmic membrane. The absence of attached amino sugars to rhamnose polysaccharide from L-form membrane fragments is discussed in terms of this organism's continued inability for new cell wall formation. The isolation, from streptococcal membrane fragments, of a polysaccharide containing rhamnose and amino sugars common to at least two different streptococcal cell wall-type polymers was demonstrated.

  6. Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles.

    PubMed

    Kahnt, Jörg; Aguiluz, Kryssia; Koch, Jürgen; Treuner-Lange, Anke; Konovalova, Anna; Huntley, Stuart; Hoppert, Michael; Søgaard-Andersen, Lotte; Hedderich, Reiner

    2010-10-01

    Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

  7. Major outer membrane proteins unique to reproductive cells of Hyphomonas jannaschiana.

    PubMed Central

    Shen, N; Dagasan, L; Sledjeski, D; Weiner, R M

    1989-01-01

    Separation on the basis of molecular weight resolved three proteins specific to the swarmer cell of Hyphomonas jannaschiana. In the reproductive cell, 4 major proteins were identified as cytoplasmic and 10 were identified as envelope. Of these envelope proteins, one was common to both the inner and outer membranes, four were common to the inner membrane, and five were common to the outer membrane. Four of these outer membrane proteins were specific to the reproductive cell, and two of these proteins, with apparent molecular weights of 116,000 and 29,000, constituted 19% of the total cell protein and 54% of the outer membrane protein. Images PMID:2703471

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

    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).

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

    PubMed

    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.

  12. Helicobacter pylori Outer Membrane Protein-Related Pathogenesis

    PubMed Central

    Matsuo, Yuichi; Kido, Yasutoshi; Yamaoka, Yoshio

    2017-01-01

    Helicobacter pylori colonizes the human stomach and induces inflammation, and in some cases persistent infection can result in gastric cancer. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and outer membrane proteins (OMPs) play a pivotal role in binding to human cells. Some OMP interaction molecules are known in H. pylori, and their associated host cell responses have been gradually clarified. Many studies have demonstrated that OMPs are essential to CagA translocation into gastric cells via the Type IV secretion system of H. pylori. This review summarizes the mechanisms through which H. pylori utilizes OMPs to colonize the human stomach and how OMPs cooperate with the Type IV secretion system. PMID:28287480

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

    PubMed

    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.

  14. Helicobacter pylori Outer Membrane Protein-Related Pathogenesis.

    PubMed

    Matsuo, Yuichi; Kido, Yasutoshi; Yamaoka, Yoshio

    2017-03-11

    Helicobacter pylori colonizes the human stomach and induces inflammation, and in some cases persistent infection can result in gastric cancer. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and outer membrane proteins (OMPs) play a pivotal role in binding to human cells. Some OMP interaction molecules are known in H. pylori, and their associated host cell responses have been gradually clarified. Many studies have demonstrated that OMPs are essential to CagA translocation into gastric cells via the Type IV secretion system of H. pylori. This review summarizes the mechanisms through which H. pylori utilizes OMPs to colonize the human stomach and how OMPs cooperate with the Type IV secretion system.

  15. Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles

    PubMed Central

    Xie, H

    2015-01-01

    Porphyromonas gingivalis is one of the keystone pathogens associated with chronic periodontitis. All P. gingivalis strains examined thus far produce outer membrane vesicles. Recent studies have found that vesicles possess some well-known virulence factors of P. gingivalis such as adhesins, toxins and proteolytic enzymes. Carrying most of the characteristic features of their parent P. gingivalis cells, vesicles communicate with host cells and other members of microbial biofilms, resulting in the transmission of virulence factors into these host cells and the formation of pathogenic bacteria-dominated microbial communities. An in-depth understanding of both the nature and role of vesicles in the pathogenicity of P. gingivalis is both important and timely, particularly when speaking of periodontitis and its related systemic effects. PMID:26343879

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

    PubMed

    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.

  17. Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

    PubMed Central

    Jacobson, Kurt H.; Gunsolus, Ian L.; Kuech, Thomas R.; Troiano, Julianne M.; Melby, Eric S.; Lohse, Samuel E.; Hu, Dehong; Chrisler, William B.; Murphy, Catherine J.; Orr, Galya; Geiger, Franz M.; Haynes, Christy L.; Pedersen, Joel A.

    2015-01-01

    Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides. PMID:26207769

  18. The lethal cargo of Myxococcus xanthus outer membrane vesicles

    PubMed Central

    Berleman, James E.; Allen, Simon; Danielewicz, Megan A.; Remis, Jonathan P.; Gorur, Amita; Cunha, Jack; Hadi, Masood Z.; Zusman, David R.; Northen, Trent R.; Witkowska, H. Ewa; Auer, Manfred

    2014-01-01

    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 the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus. PMID:25250022

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

    PubMed Central

    Konovalova, Anna; Silhavy, Thomas J.

    2015-01-01

    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. PMID:26370942

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

    PubMed

    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. © 2015 The Author(s).

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

    PubMed

    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.

  2. Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane.

    PubMed

    Yoshii, Saori R; Kishi, Chieko; Ishihara, Naotada; Mizushima, Noboru

    2011-06-03

    Upon mitochondrial depolarization, Parkin, a Parkinson disease-related E3 ubiquitin ligase, translocates from the cytosol to mitochondria and promotes their degradation by mitophagy, a selective type of autophagy. Here, we report that in addition to mitophagy, Parkin mediates proteasome-dependent degradation of outer membrane proteins such as Tom20, Tom40, Tom70, and Omp25 of depolarized mitochondria. By contrast, degradation of the inner membrane and matrix proteins largely depends on mitophagy. Furthermore, Parkin induces rupture of the outer membrane of depolarized mitochondria, which also depends on proteasomal activity. Upon induction of mitochondrial depolarization, proteasomes are recruited to mitochondria in the perinuclear region. Neither proteasome-dependent degradation of outer membrane proteins nor outer membrane rupture is required for mitophagy. These results suggest that Parkin regulates degradation of outer and inner mitochondrial membrane proteins differently through proteasome- and mitophagy-dependent pathways.

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

    PubMed

    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.

  4. The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

    PubMed Central

    Wenz, Lena-Sophie; Opaliński, Łukasz; Schuler, Max-Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; van der Laan, Martin; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

    2014-01-01

    The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane. PMID:24781695

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

    PubMed

    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.

  6. A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis

    PubMed Central

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

    2014-01-01

    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. PMID:24671550

  7. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda

    PubMed Central

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-bin; Ye, Jin-zhou; Yang, Man-jun; Jiang, Ming; Peng, Xuan-xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication. PMID:28210241

  8. Some Characteristics of the Outer Membrane Material Released by Growing Enterotoxigenic Escherichia coli

    PubMed Central

    Gankema, Henk; Wensink, Jan; Guinée, Pieter A. M.; Jansen, Wim H.; Witholt, Bernard

    1980-01-01

    The high-molecular-weight material released into the medium by Escherichia coli AP1, an enterotoxigenic strain of porcine origin, has been isolated and resolved into two clearly distinct fractions, based on sucrose density gradient and differential centrifugation, chemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and freeze-fracture electron microscopy. These two fractions, referred to as “medium vesicles” and “medium lipopolysaccharides”, were compared with the cellular outer and cytoplasmic membranes, the periplasmic fraction, and the cytoplasmic fraction. The medium vesicles closely resembled outer membrane and accounted for 3 to 5% of the total cellular outer membrane. They contained most of the heat-labile enterotoxin (LT) activity released into the medium by E. coli AP1. The medium lipopolysaccharide consisted mostly of lipopolysaccharide and a small amount of outer membrane and contained relatively little LT activity. Based on experiments with E. coli K-12 strains, in which about 5% of the newly synthesized outer membrane is lost from areas of outer membrane synthesis, it is proposed that enterotoxigenic E. coli strains release LT as part of such newly synthesized outer membrane fragments and that released outer membrane fragments may function as physiologically significant LT carriers. ImagesFig. 4Fig. 1Fig. 2Fig. 2Fig. 3 PMID:7011982

  9. Bacterial tail anchors can target to the mitochondrial outer membrane.

    PubMed

    Lutfullahoğlu-Bal, Güleycan; Keskin, Abdurrahman; Seferoğlu, Ayşe Bengisu; Dunn, Cory D

    2017-07-24

    During the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was re-fashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol. Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM. Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins. This article was reviewed by Michael Ryan and Thomas Simmen.

  10. Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

    SciTech Connect

    Jacobson, Kurt H.; Gunsolus, Ian L.; Kuech, Thomas R.; Troiano, Julianne M.; Melby, Eric S.; Lohse, Samuel E.; Hu, Dehong; Chrisler, William B.; Murphy, Catherine J.; Orr, Galya; Geiger, Franz M.; Haynes, Christy L.; Pedersen, Joel A.

    2015-07-24

    We report that design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations, and assessment of the potential implications of nanoparticle release into the environment require understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the lipid-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. Association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Lastly, our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.

  11. Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

    DOE PAGES

    Jacobson, Kurt H.; Gunsolus, Ian L.; Kuech, Thomas R.; ...

    2015-07-24

    We report that design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations, and assessment of the potential implications of nanoparticle release into the environment require understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the lipid-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) andmore » second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. Association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Lastly, our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.« less

  12. Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery

    PubMed Central

    Zakharzhevskaya, Natalya B.; Tsvetkov, Vladimir B.; Vanyushkina, Anna A.; Varizhuk, Anna M.; Rakitina, Daria V.; Podgorsky, Victor V.; Vishnyakov, Innokentii E.; Kharlampieva, Daria D.; Manuvera, Valentin A.; Lisitsyn, Fedor V.; Gushina, Elena A.; Lazarev, Vassili N.; Govorun, Vadim M.

    2017-01-01

    The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to host cells. However, recent studies of gram-negative bacteria have shown that outer membrane vesicles (OMVs) could be an essential mechanism for the spread of a large number of virulence factors. Here, we show for the first time that BFT is not a freely secreted protease but is associated with OMVs. Our findings indicate that only outer surface-exposed BFT causes epithelial cell contact disruption. According to our in silico models confirmed by Trp quenching assay and NMR, BFT has special interactions with outer membrane components such as phospholipids and is secreted during vesicle formation. Moreover, the strong cooperation of BFT with polysaccharides is similar to the behavior of lectins. Understanding the molecular mechanisms of BFT secretion provides new perspectives for investigating intestinal inflammation pathogenesis and its prevention. PMID:28144586

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

    PubMed Central

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

    2016-01-01

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

  14. Function of the mitochondrial outer membrane as a diffusion barrier in health and diseases.

    PubMed

    Gellerich, F N; Trumbeckaite, S; Opalka, J R; Seppet, E; Rasmussen, H N; Neuhoff, C; Zierz, S

    2000-02-01

    The mitochondrial outer membrane separates the intermembrane space from the cytosol. The whole exchange of metabolites, cations and information between mitochondria and the cell occurs through the outer membrane. Experimental evidence is reviewed supporting the hypothesis of dynamic ADP compartmentation within the intermembrane space. The outer membrane creates a diffusion barrier for small molecules (adenine nucleotides, creatine phosphate, creatine etc.) causing rate-dependent concentration gradients as a prerequisite for the action of ADP shuttles via creatine kinases or adenylate kinases. If the outer membrane becomes leaky, cytochrome c and apoptosis-inducing factor can be released, leading to apoptosis, and as a bioenergetic consequence the cytosolic phosphorylation potential decreases. Leaky outer membranes can be detected in saponin-skinned fibres with spectrophotometric and oxygraphic methods. This is of special interest in respect to acute impairment of mitochondria during ischaemia/reperfusion.

  15. Proteomic analysis of Vibrio cholerae outer membrane vesicles

    PubMed Central

    Altindis, Emrah; Fu, Yang; Mekalanos, John J.

    2014-01-01

    Outer membrane vesicles (OMVs) produced by Gram-negative bacteria provide an interesting research material for defining cell-envelope proteins without experimental cell disruption. OMVs are also promising immunogenic platforms and may play important roles in bacterial survival and pathogenesis. We used in-solution trypsin digestion coupled to mass spectrometry to identify 90 proteins present in OMVs of Vibrio cholerae when grown under conditions that activate the TCP pilus virulence regulatory protein (ToxT) virulence regulon. The ToxT expression profile and potential contribution to virulence of these proteins were assessed using ToxT and in vivo RNA-seq, Tn-seq, and cholera stool proteomic and other genome-wide data sets. Thirteen OMV-associated proteins appear to be essential for cell growth, and therefore may represent antibacterial drug targets. Another 12 nonessential OMV proteins, including DegP protease, were required for intestinal colonization in rabbits. Comparative proteomics of a degP mutant revealed the importance of DegP in the incorporation of nine proteins into OMVs, including ones involved in biofilm matrix formation and various substrates of the type II secretion system. Taken together, these results suggest that DegP plays an important role in determining the content of OMVs and also affects phenotypes such as intestinal colonization, proper function of the type II secretion system, and formation of biofilm matrix. PMID:24706774

  16. Myxobacteria Produce Outer Membrane-Enclosed Tubes in Unstructured Environments

    PubMed Central

    Wei, Xueming; Vassallo, Christopher N.; Pathak, Darshankumar T.

    2014-01-01

    Myxobacteria are social microbes that exhibit complex multicellular behaviors. By use of fluorescent reporters, we show that Myxococcus xanthus isolates produce long narrow filaments that are enclosed by the outer membrane (OM) and contain proteins. We show that these OM tube (OMT) structures are produced at surprisingly high levels when cells are placed in liquid medium or buffer without agitation. OMTs can be long and easily exceed multiple cell lengths. When viewed by transmission electron microscopy, their morphology varies between tubes and chain-like structures. Intermediate-like structures are also found, suggesting that OMTs may transition between these two morphotypes. In support of this, video epifluorescence microscopy found that OMTs in solution dynamically twist and jiggle. On hard surfaces, myxobacteria glide, and upon cell-cell contact, they can efficiently exchange their OM proteins and lipids by a TraAB-dependent mechanism. Although the structure of OMTs hints at a possible role as conduits for exchange, evidence is presented to the contrary. For example, abundant OMT production occurs in traA or traB mutants and when cells are grown in liquid medium, yet transfer cannot occur under these conditions. Thus, genetic and environmental conditions that promote OMT production are incongruent with OM exchange. PMID:24391054

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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 program 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 hemocytes 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 (TCT) release by the symbionts, which can induce hemocyte 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 hemocyte 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 detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis. PMID:26399913

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:25840612

  1. Mechanisms of outer membrane vesicle entry into host cells

    PubMed Central

    O'Donoghue, Eloise J.

    2016-01-01

    Abstract 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. PMID:27529760

  2. Structural Basis for Outer Membrane Sugar Uptake in Pseudomonads*

    PubMed Central

    van den Berg, Bert

    2012-01-01

    Substrate-specific outer membrane channels of Gram-negative bacteria mediate uptake of many small molecules, including carbohydrates. The mechanism of sugar uptake by enterobacterial channels, such as Escherichia coli LamB (maltoporin), has been characterized in great detail. In pseudomonads and related organisms, sugar uptake is not mediated by LamB but by OprB channels. Beyond the notion that OprB channels seem to prefer monosaccharides as substrates, very little is known about OprB-mediated sugar uptake. Here I report the X-ray crystal structure of an OprB channel from Pseudomonas putida F1. The structure shows that OprB forms a monomeric, 16-stranded β-barrel with a constriction formed by extracellular loops L2 and L3. The side chains of two highly conserved arginine residues (Arg83 and Arg110) and a conserved glutamate (Glu106) line the channel constriction and interact with a bound glucose molecule. Liposome swelling uptake assays show a strong preference for monosaccharide transport over disaccharides. Moreover, substrates with a net negative charge are disfavored by the channel, probably due to the negatively charged character of the constriction. The architecture of the eyelet and the absence of a greasy slide provide an explanation for the observed specificity of OprB for monosaccharides rather than the oligosaccharides preferred by LamB and related enterobacterial channels. PMID:23066028

  3. Structural basis for outer membrane sugar uptake in pseudomonads.

    PubMed

    van den Berg, Bert

    2012-11-30

    Substrate-specific outer membrane channels of gram-negative bacteria mediate uptake of many small molecules, including carbohydrates. The mechanism of sugar uptake by enterobacterial channels, such as Escherichia coli LamB (maltoporin), has been characterized in great detail. In pseudomonads and related organisms, sugar uptake is not mediated by LamB but by OprB channels. Beyond the notion that OprB channels seem to prefer monosaccharides as substrates, very little is known about OprB-mediated sugar uptake. Here I report the X-ray crystal structure of an OprB channel from Pseudomonas putida F1. The structure shows that OprB forms a monomeric, 16-stranded β-barrel with a constriction formed by extracellular loops L2 and L3. The side chains of two highly conserved arginine residues (Arg(83) and Arg(110)) and a conserved glutamate (Glu(106)) line the channel constriction and interact with a bound glucose molecule. Liposome swelling uptake assays show a strong preference for monosaccharide transport over disaccharides. Moreover, substrates with a net negative charge are disfavored by the channel, probably due to the negatively charged character of the constriction. The architecture of the eyelet and the absence of a greasy slide provide an explanation for the observed specificity of OprB for monosaccharides rather than the oligosaccharides preferred by LamB and related enterobacterial channels.

  4. Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases.

    PubMed

    Paradis-Bleau, Catherine; Markovski, Monica; Uehara, Tsuyoshi; Lupoli, Tania J; Walker, Suzanne; Kahne, Daniel E; Bernhardt, Thomas G

    2010-12-23

    Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer in vivo. Here, we identify the Escherichia coli outer-membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function in vivo. We further show that LpoB promotes PG synthesis by its partner PBP in vitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis, and like the PBPs they work with, these factors are attractive targets for antibiotic development. Copyright © 2010 Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  6. Polysaccharide-Based Membranes in Food Packaging Applications

    PubMed Central

    Ferreira, Ana R. V.; Alves, Vítor D.; Coelhoso, Isabel M.

    2016-01-01

    Plastic packaging is essential nowadays. However, the huge environmental problem caused by landfill disposal of non-biodegradable polymers in the end of life has to be minimized and preferentially eliminated. The solution may rely on the use of biopolymers, in particular polysaccharides. These macromolecules with film-forming properties are able to produce attracting biodegradable materials, possibly applicable in food packaging. Despite all advantages of using polysaccharides obtained from different sources, some drawbacks, mostly related to their low resistance to water, mechanical performance and price, have hindered their wider use and commercialization. Nevertheless, with increasing attention and research on this field, it has been possible to trace some strategies to overcome the problems and recognize solutions. This review summarizes some of the most used polysaccharides in food packaging applications. PMID:27089372

  7. Inner- and outer-membrane enzymes of mitochondria during liver regeneration

    PubMed Central

    Gear, A. R. L.

    1970-01-01

    1. Marker enzymes for the mitochondrial matrix, inner membrane, inter-membrane space and outer membrane were measured in mitochondria isolated from control and regenerating rat liver. The specific activity of these enzymes was then followed for up to 30 days after operation. 2. The specific activity of marker enzymes for the matrix, inner membrane and inter-membrane space remained constant during liver regeneration. 3. However, the specific activities of monoamine oxidase and kynurenine hydroxylase, both outer-membrane markers, fell by 67% and 49% respectively from their control values at 4 days after operation, and returned to normal by about 3 weeks. 4. The repression of kynurenine hydroxylase activity was shown to be unrelated to any independent variation in tryptophan catabolism, based on tryptophan pyrrolase assays. 5. These results are considered to indicate that enzymes of the inner and outer mitochondrial membranes are synthesized asynchronously during morphogenesis. 6. The enzyme complement of purified outer membrane at 4 days after operation was about 50% of that of the appropriate control. Thus the composition of the outer membrane itself may vary dramatically, and supports the concept that constitutive enzymes may turn over independently of a membrane's existence. 7. The behaviour of the rotenone-insensitive, NADH cytochrome c reductase did not parallel the other outer-membrane enzymes for intact mitochondria, but did so when assayed in highly purified fractions of outer membrane. This suggests a labile binding to the outer membrane during the early stages of morphogenesis. 8. Electrophoresis of inner- and outer-membrane proteins revealed little difference between control and experimental mitochondria at 4 days, except for an increase in several, high-molecular-weight components of the outer membrane. These bands closely correspond to similar bands derived from smooth endoplasmic reticulum. 9. The results are discussed in relation to the biogenesis and

  8. Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes

    PubMed Central

    1981-01-01

    Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane

  9. Voltage- and Tension-Dependent Lipid Mobility in the Outer Hair Cell Plasma Membrane

    NASA Astrophysics Data System (ADS)

    Oghalai, John S.; Zhao, Hong-Bo; Kutz, J. Walter; Brownell, William E.

    2000-01-01

    The mechanism responsible for electromotility of outer hair cells in the ear is unknown but is thought to reside within the plasma membrane. Lipid lateral diffusion in the outer hair cell plasma membrane is a sigmoidal function of transmembrane potential and bathing media osmolality. Cell depolarization or hyposmotic challenge shorten the cell and reduce membrane fluidity by half. Changing the membrane tension with amphipathic drugs results in similar reductions. These dynamic changes in membrane fluidity represent the modulation of membrane tension by lipid-protein interactions. The voltage dependence may be associated with the force-generating motors that contribute to the exquisite sensitivity of mammalian hearing.

  10. Physicochemical properties and membrane biofouling of extra-cellular polysaccharide produced by a Micrococcus luteus strain.

    PubMed

    Feng, Lei; Li, Xiufen; Song, Ping; Du, Guocheng; Chen, Jian

    2014-07-01

    The physicochemical properties of the extra-cellular polysaccharide (EPS) produced by a Micrococcus luteus strain, a dominating strain isolated from membrane biofouling layer, were determined in this study. The EPS isolated from this strain was measured to have an average molecular weight of 63,540 Da and some typical polysaccharide absorption peaks in Fourier transform infrared spectrum. Monosaccharide components of the EPS contained rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose in a molar ratio of 0.2074:0.0454:0.0262:0.0446:1.7942:1.2086:0.4578. Pseudo plastic properties were also observed for the EPS through the rheological measurement. The EPS was further characterized for its behavior to cause membrane flux decline. The results showed that both flux declines for polyvinylidenefluoride (PVDF) and polypropylene membranes became more severe as EPS feed concentration increased. A higher irreversible fouling for the PVDF membrane suggested that the EPS had the larger fouling potential to this microfiltration membrane.

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

    PubMed

    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.

  12. Stress-Induced Outer Membrane Vesicle Production by Pseudomonas aeruginosa

    PubMed Central

    MacDonald, Ian A.

    2013-01-01

    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. PMID:23625841

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

    PubMed

    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.

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

    PubMed

    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.

  15. Protein–protein interactions and the spatiotemporal dynamics of bacterial outer membrane proteins

    PubMed Central

    Kleanthous, Colin; Rassam, Patrice; Baumann, Christoph G

    2015-01-01

    It has until recently been unclear whether outer membrane proteins (OMPs) of Gram-negative bacteria are organized or distributed randomly. Studies now suggest promiscuous protein–protein interactions (PPIs) between β-barrel OMPs in Escherichia coli govern their local and global dynamics, engender spatiotemporal patterning of the outer membrane into micro-domains and are the basis of β-barrel protein turnover. We contextualize these latest advances, speculate on areas of bacterial cell biology that might be influenced by the organization of OMPs into supramolecular assemblies, and highlight the new questions and controversies this revised view of the bacterial outer membrane raises. PMID:26629934

  16. Interaction between Polyamines and Bacterial Outer Membranes as Investigated with Ion-Selective Electrodes

    PubMed Central

    Katsu, Takashi; Nakagawa, Hideki; Yasuda, Keiko

    2002-01-01

    We analyzed the interaction between polyamines and the outer membrane of Escherichia coli cells using potentiometric measurements with Ca2+, tetraphenylphosphonium (TPP+), and K+ electrodes. The Ca2+ electrode was used to examine the ability of the polyamines to release Ca2+ from the outer membrane. The TPP+ electrode was used to examine the ability to permeabilize the outer membrane, since the uptake of TPP+ was enhanced when the permeability barrier of the outer membrane was disrupted. The K+ electrode was used to examine permeabilization in the cytoplasmic membrane by monitoring the efflux of K+ in cytosol. Although Ca2+ release was remarkably enhanced by increasing the number of amino groups in polyamines, no TPP+ uptake was observed with polyamines of a simple structure, such as ethylenediamine, spermidine, and spermine. TPP+ uptake was observed when appropriate lipophilic moieties were further attached to the polyamines with three or four amino groups, indicating that the existence of bulky moieties as well as the number of amino groups is important to induce outer membrane permeabilization. Thus, 1-naphthylacetylspermine and N,N′-bis[6-[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octanediamine (methoctramine) were especially effective in increasing the permeability of the outer membrane of E. coli cells, being comparable to polymyxin B nonapeptide, a well-known cationic peptide showing such action. PMID:11897592

  17. Carbohydrate-reactive, pore-forming outer membrane proteins of Aeromonas hydrophila.

    PubMed Central

    Quinn, D M; Atkinson, H M; Bretag, A H; Tester, M; Trust, T J; Wong, C Y; Flower, R L

    1994-01-01

    Two outer membrane proteins of Aeromonas hydrophila A6, isolated in a one-step affinity chromatography process based on carbohydrate reactivity, were found to be pore-forming molecules in artificial planar bilayer membranes. These carbohydrate-reactive outer membrane proteins (CROMPs; M(r)s, 40,000 and 43,000) were subjected to amino acid analysis. The amino acid profiles for these two outer membrane proteins were almost identical. A partial protein sequence of a 14-amino-acid fragment of the 40,000-Da protein revealed homology with outer membrane porins of Escherichia coli and A. hydrophila. CROMPs were compared with carbohydrate-reactive porins also extracted from outer membranes of A. hydrophila A6. These porins were isolated by using standard porin purification techniques (insolubility in 2% sodium dodecyl sulfate, solubility in 0.4 M NaCl, and Sephacryl S-200 gel filtration), and then Synsorb H type 2 affinity chromatography was done. The physical and functional properties of the carbohydrate-reactive porins and CROMPs were found to be identical. On the basis of pore-forming properties in planar lipid bilayers and channel inhibition with maltotriose solutions, a nonspecific, general diffusion porin and a LamB-like maltoporin were identified in both CROMP and carbohydrate-reactive porin preparations. To our knowledge, the use of carbohydrate reactivity to isolate channel-forming proteins from bacterial outer membranes has not been reported previously. Images PMID:7520425

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  20. Functional definition of outer membrane proteins involved in preprotein import into mitochondria.

    PubMed

    Lister, Ryan; Carrie, Chris; Duncan, Owen; Ho, Lois H M; Howell, Katharine A; Murcha, Monika W; Whelan, James

    2007-11-01

    The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.

  1. Studies of the Outer Membrane Proteins of Campylobacter Jejuni for Vaccine Development

    DTIC Science & Technology

    1991-11-26

    acute phase, and 2 and 4 weeks after the diarrheal episode. By 2 ELISA , children infected with Campylobacter but not Shigella showed a significant...AD-A245 442 AD___1111111i1i11l 01 li[i ] i 1 I1 STUDIES OF THE OUTER MEMBRANE PROTEINS OF CAMPYLOBACTER JEJUNI FOR VACCINE DEVELOPMENT MIDTERM...the Outer Membrane Proteins of Campylobacter 90PP0820 Jejuni for Vaccine Development ____ ___ ___ ____ _ _ ___ ___ ___ ____ ___ ___61102A .1 6

  2. Role of Pseudomonas aeruginosa Peptidoglycan-Associated Outer Membrane Proteins in Vesicle Formation

    PubMed Central

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

    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. PMID:23123904

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

    PubMed

    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.

  4. A Bilayer-Couple Model of Bacterial Outer Membrane Vesicle Biogenesis

    PubMed Central

    Schertzer, Jeffrey W.; Whiteley, Marvin

    2012-01-01

    ABSTRACT 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. PMID:22415005

  5. PagP activation in the outer membrane triggers R3 core oligosaccharide truncation in the cytoplasm of Escherichia coli O157:H7.

    PubMed

    Smith, Abigail E; Kim, Sang-Hyun; Liu, Feng; Jia, Wenyi; Vinogradov, Evgeny; Gyles, Carlton L; Bishop, Russell E

    2008-02-15

    The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the first outer core glucose unit of the R3 core oligosaccharide. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagPDelta5-14 allele, which mostly eliminates a periplasmic amphipathic alpha-helical domain but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier.

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

    PubMed

    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

  7. Membrane Composition Tunes the Outer Hair Cell Motor

    NASA Astrophysics Data System (ADS)

    Rajagopalan, L.; Sfondouris, J.; Oghalai, J. S.; Pereira, F. A.; Brownell, W. E.

    2009-02-01

    Cholesterol and docosahexaenoic acid (DHA), an ω-3 fatty acid, affect membrane mechanical properties in different ways and modulate the function of membrane proteins. We have probed the functional consequence of altering cholesterol and DHA levels in the membranes of OHCs and prestin expressing HEK cells. Large, dynamic and reversible changes in prestin-associated charge movement and OHC motor activity result from altering the concentration of membrane cholesterol. Increasing membrane cholesterol shifts the q/V function ~ 50 mV in the hyperpolarizing direction, possibly a response related to increases in membrane stiffness. The voltage shift is linearly related to total membrane cholesterol. Increasing cholesterol also decreases the total charge moved in a linear fashion. Decreasing membrane cholesterol shifts the q/V function ~ 50 mV in the depolarizing direction with little or no effect on the amount of charge moved. In vivo increases in membrane cholesterol transiently increase but ultimately lead to decreases in DPOAE. Docosahexaenoic acid shifts the q/V function in the hyperpolarizing direction < 15 mV and increases total charge moved. Tuning of cochlear function by membrane cholesterol contributes to the exquisite temporal and frequency processing of mammalian hearing by optimizing the cochlear amplifier.

  8. Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: lateral reorganization of LPS monolayer, model of the Escherichia coli outer membrane.

    PubMed

    Derde, Melanie; Nau, Françoise; Lechevalier, Valérie; Guérin-Dubiard, Catherine; Paboeuf, Gilles; Jan, Sophie; Baron, Florence; Gautier, Michel; Vié, Véronique

    2015-01-01

    Lysozyme is mainly described active against Gram-positive bacteria, but is also efficient against some Gram-negative species. Especially, it was recently demonstrated that lysozyme disrupts Escherichia coli membranes. Moreover, dry-heating changes the physicochemical properties of the protein and increases the membrane activity of lysozyme. In order to elucidate the mode of insertion of lysozyme into the bacterial membrane, the interaction between lysozyme and a LPS monolayer mimicking the E. coli outer membrane has been investigated by tensiometry, ellipsometry, Brewster angle microscopy and atomic force microscopy. It was thus established that lysozyme has a high affinity for the LPS monolayer, and is able to insert into the latter as long as polysaccharide moieties are present, causing reorganization of the LPS monolayer. Dry-heating increases the lysozyme affinity for the LPS monolayer and its insertion capacity; the resulting reorganization of the LPS monolayer is different and more drastic than with the native protein. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Mutation of the Sensor Kinase chvG in Rhizobium leguminosarum Negatively Impacts Cellular Metabolism, Outer Membrane Stability, and Symbiosis

    PubMed Central

    Vanderlinde, Elizabeth M.

    2012-01-01

    Two-component signal transduction systems (TCS) are a main strategy used by bacteria to sense and adapt to changes in their environment. In the legume symbiont Rhizobium leguminosarum biovar viciae VF39, mutation of chvG, a histidine kinase, caused a number of pleiotropic phenotypes. ChvG mutants are unable to grow on proline, glutamate, histidine, or arginine as the sole carbon source. The chvG mutant secreted smaller amounts of acidic and neutral surface polysaccharides and accumulated abnormally large amounts of poly-ß-hydroxybutyrate. Mutation of chvG caused symbiotic defects on peas, lentils, and vetch; nodules formed by the chvG mutant were small and white and contained only a few cells that had failed to differentiate into bacteroids. Mutation of chvG also destabilized the outer membrane of R. leguminosarum, resulting in increased sensitivity to membrane stressors. Constitutive expression of ropB, the outer membrane protein-encoding gene, restored membrane stability and rescued the sensitivity phenotypes described above. Similar phenotypes have been described for mutations in other ChvG-regulated genes encoding a conserved operon of unknown function and in the fabXL genes required for synthesis of the lipid A very-long-chain fatty acid, suggesting that ChvG is a key component of the envelope stress response in Rhizobium leguminosarum. Collectively, the results of this study demonstrate the important and unique role the ChvG/ChvI TCS plays in the physiology, metabolism, and symbiotic competency of R. leguminosarum. PMID:22155778

  10. Acinetobacter baumannii Outer Membrane Vesicles Elicit a Potent Innate Immune Response via Membrane Proteins

    PubMed Central

    Jun, So Hyun; Lee, Jung Hwa; Kim, Bo Ra; Kim, Seung Il; Park, Tae In

    2013-01-01

    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 19606T 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. PMID:23977136

  11. Genomic Analysis Indicates the Presence of an Asymmetric Bilayer Outer Membrane in Planctomycetes and Verrucomicrobia

    PubMed Central

    Speth, Daan R.; van Teeseling, Muriel C. F.; Jetten, Mike S. M.

    2012-01-01

    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. These analyses provide a strong indication that an asymmetrical outer membrane may be present in bacteria of both phyla. However, previous experiments have made obvious that the cell envelope of Planctomycetes is clearly divergent from both the Gram-negative and Gram-positive cell types. Thus, the functional implications of the presence of an outer membrane for the Planctomycete cell plan

  12. Myo-conductive and osteo-inductive free-standing polysaccharide membranes

    PubMed Central

    Caridade, Sofia G.; Monge, Claire; Almodóvar, Jorge; Guillot, Raphael; Lavaud, Jonathan; Josserand, Véronique; Coll, Jean-Luc; Mano, João F.; Picart, Catherine

    2015-01-01

    Free-standing (FS) membranes have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we studied the potential of free-standing membranes made by the layer-by-layer assembly of chitosan and alginate to be used as a simple biomimetic system of the periosteum. The design of a periosteum-like membrane implies the elaboration of a thick membrane suitable for both muscle and bone formation. Our aim was to produce well defined ~50 μm thick polysaccharide membranes that could be easily manipulated, be mechanically resistant, and enable both myogenesis and osteogenesis in vitro and in vivo. The membranes were chemically crosslinked to improve their mechanical properties. Crosslinking chemistry was followed via FTIR and the mechanical properties of the membranes were assessed using dynamic mechanical analysis. The loading and release of the potent osteoinductive growth factor bone morphogenetic protein 2 (BMP-2) inside and outside of the FS membrane was followed by fluorescence spectroscopy in a physiological buffer over one month. The myogenic and osteogenic potential of the membranes in vitro was assessed using BMP-2 responsive skeletal myoblasts. Finally, their osteoinductive properties in vivo were studied in a preliminary experiment using a mouse ectopic model. Our results showed that the more crosslinked FS membranes enabled a more efficient myoblast differentiation in myotubes. In addition, we showed that a tunable amount of BMP-2 can be loaded in and subsequently released from the membranes depending on the crosslinking degree and BMP-2 initial concentration in solution. Only the more crosslinked membranes were found to be osteoinductive in vivo. These polysaccharide-based membranes have strong potential as a periosteum-mimetic scaffold for bone tissue regeneration. PMID:25575853

  13. Nanodomain organization of rhodopsin in native human and murine rod outer segment disc membranes

    PubMed Central

    Whited, Allison M.; Park, Paul S.-H.

    2014-01-01

    Biological membranes display distinct domains that organize membrane proteins and signaling molecules to facilitate efficient and reliable signaling. The organization of rhodopsin, a G protein-coupled receptor, in native rod outer segment disc membranes was investigated by atomic force microscopy. Atomic force microscopy revealed that rhodopsin is arranged into domains of variable size, which we refer to herein as nanodomains, in native membranes. Quantitative analysis of 150 disc membranes revealed that the physical properties of nanodomains are conserved in humans and mice and that the properties of individual disc membranes can be variable. Examining the variable properties of disc membranes revealed some of the factors contributing to the size of rod outer segment discs and the formation of nanodomains in the membrane. The diameter of rod outer segment discs was dependent on the number of rhodopsin molecules incorporated into the membrane but independent of the spatial density of rhodopsin. The number of nanodomains present in a single disc was also dependent on the number of rhodopsin molecules incorporated into the membrane. The size of the nanodomains was largely independent of the number or spatial density of rhodopsin in the membrane. PMID:25305340

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    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

  17. The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

    PubMed

    Pierce, Brittany K; Voegel, Tanja; Kirkpatrick, Bruce C

    2014-01-01

    Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa.

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

    SciTech Connect

    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.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27872310

  1. Phellinus linteus polysaccharide extracts increase the mitochondrial membrane potential and cause apoptotic death of THP-1 monocytes

    PubMed Central

    2013-01-01

    Background The differentiation resp. death of human monocytic THP-1 cells induced by polysaccharide extracts of the medicinal mushrooms Phellinus linteus, Agaricus bisporus and Agaricus brasiliensis have been studied. This study aims to identify leads for the causal effects of these mushroom components on cell differentiation and death. Methods THP-1 cells were treated with different polysaccharide extracts of mushrooms and controls. Morphological effects were observed by light microscopy. Flow cytometry was applied to follow the cell differentiation by cell cycle shifts after staining with propidium iodide, changes of mitochondrial membrane potential after incubation with JC-1, and occurrence of intracellular reactive oxygen species after incubation with hydroethidine. Principal component analysis of the data was performed to evaluate the cellular effects of the different treatments. Results P. linteus polysaccharide extracts induced dose-dependent apoptosis of THP-1 cells within 24 h, while A. bisporus and A. brasiliensis polysaccharide extracts caused differentiation into macrophages. A pure P. linteus polysaccharide had no effect. Apoptosis was inhibited by preincubating THP-1 cells with human serum. The principal component analysis revealed that P. linteus, A. bisporus and A. brasiliensis polysaccharide extracts increased reactive oxygen species production. Both A. bisporus and A. brasiliensis polysaccharide extracts decreased the mitochondrial membrane potential, while this was increased by P. linteus polysaccharide extracts. Conclusions P. linteus polysaccharide extracts caused apoptosis of THP-1 monocytes while A. bisporus and A. brasiliensis polysaccharide extracts caused these cells to differentiate into macrophages. The protective effects of human serum suggested that P. linteus polysaccharide extract induced apoptosis by extrinsic pathway, i.e. by binding to the TRAIL receptor. The mitochondrial membrane potential together with reactive oxygen species

  2. Phellinus linteus polysaccharide extracts increase the mitochondrial membrane potential and cause apoptotic death of THP-1 monocytes.

    PubMed

    van Griensven, Leo Jld; Verhoeven, Harrie A

    2013-12-18

    The differentiation resp. death of human monocytic THP-1 cells induced by polysaccharide extracts of the medicinal mushrooms Phellinus linteus, Agaricus bisporus and Agaricus brasiliensis have been studied. This study aims to identify leads for the causal effects of these mushroom components on cell differentiation and death. THP-1 cells were treated with different polysaccharide extracts of mushrooms and controls. Morphological effects were observed by light microscopy. Flow cytometry was applied to follow the cell differentiation by cell cycle shifts after staining with propidium iodide, changes of mitochondrial membrane potential after incubation with JC-1, and occurrence of intracellular reactive oxygen species after incubation with hydroethidine. Principal component analysis of the data was performed to evaluate the cellular effects of the different treatments. P. linteus polysaccharide extracts induced dose-dependent apoptosis of THP-1 cells within 24 h, while A. bisporus and A. brasiliensis polysaccharide extracts caused differentiation into macrophages. A pure P. linteus polysaccharide had no effect. Apoptosis was inhibited by preincubating THP-1 cells with human serum. The principal component analysis revealed that P. linteus, A. bisporus and A. brasiliensis polysaccharide extracts increased reactive oxygen species production. Both A. bisporus and A. brasiliensis polysaccharide extracts decreased the mitochondrial membrane potential, while this was increased by P. linteus polysaccharide extracts. P. linteus polysaccharide extracts caused apoptosis of THP-1 monocytes while A. bisporus and A. brasiliensis polysaccharide extracts caused these cells to differentiate into macrophages. The protective effects of human serum suggested that P. linteus polysaccharide extract induced apoptosis by extrinsic pathway, i.e. by binding to the TRAIL receptor. The mitochondrial membrane potential together with reactive oxygen species seems to play an important role in cell

  3. Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane of Escherichia coli

    PubMed Central

    Ghosh, Anindya S.; Young, Kevin D.

    2005-01-01

    In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen of E. coli 2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane of E. coli has a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod. PMID:15743937

  4. Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment.

    PubMed

    Tsuchido, T; Katsui, N; Takeuchi, A; Takano, M; Shibasaki, I

    1985-08-01

    Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.

  5. Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis.

    PubMed

    Bessueille, Laurence; Sindt, Nicolas; Guichardant, Michel; Djerbi, Soraya; Teeri, Tuula T; Bulone, Vincent

    2009-04-28

    Detergent-resistant plasma membrane microdomains [DRMs (detergent-resistant membranes)] were isolated recently from several plant species. As for animal cells, a large range of cellular functions, such as signal transduction, endocytosis and protein trafficking, have been attributed to plant lipid rafts and DRMs. The data available are essentially based on proteomics and more approaches need to be undertaken to elucidate the precise function of individual populations of DRMs in plants. We report here the first isolation of DRMs from purified plasma membranes of a tree species, the hybrid aspen Populus tremula x tremuloides, and their biochemical characterization. Plasma membranes were solubilized with Triton X-100 and the resulting DRMs were isolated by flotation in sucrose density gradients. The DRMs were enriched in sterols, sphingolipids and glycosylphosphatidylinositol-anchored proteins and thus exhibited similar properties to DRMs from other species. However, they contained key carbohydrate synthases involved in cell wall polysaccharide biosynthesis, namely callose [(1-->3)-beta-D-glucan] and cellulose synthases. The association of these enzymes with DRMs was demonstrated using specific glucan synthase assays and antibodies, as well as biochemical and chemical approaches for the characterization of the polysaccharides synthesized in vitro by the isolated DRMs. More than 70% of the total glucan synthase activities present in the original plasma membranes was associated with the DRM fraction. In addition to shedding light on the lipid environment of callose and cellulose synthases, our results demonstrate the involvement of DRMs in the biosynthesis of important cell wall polysaccharides. This novel concept suggests a function of plant membrane microdomains in cell growth and morphogenesis.

  6. Insights into the Structure and Assembly of Escherichia coli Outer Membrane Protein A

    PubMed Central

    Reusch, Rosetta N.

    2012-01-01

    Outer membrane protein A (OmpA) of Escherichia coli is a paradigm for the biogenesis of outer membrane proteins; however, the structure and assembly of OmpA have remained controversial. A review of studies to date supports the hypothesis that native OmpA is a single-domain large pore, while a two-domain narrow pore structure is a folding intermediate or minor conformer. The in vitro refolding of OmpA to the large pore conformation requires that the protein be isolated from outer membranes with an intact disulfide bond and then adequately incubated in lipids at temperatures ≥ 26 °C to overcome the high energy of activation for refolding. The in vivo maturation of the protein involves covalent modification of serines in the eighth β-barrel of the N-terminal domain by oligo-(R)-3-hydroxybutyrates as the protein is escorted across the cytoplasm by SecB for post-translational secretion across the SEC translocase in the inner membrane. After cleavage of the signal sequence, protein chaperones, such a Skp, DegP and SurA, guide OmpA across the periplasm to the BAM complex in the outer membrane. During this passage, a disulfide bond is formed between C290 and C302 by DsbA, and the hydrophobicity of segments of the C-terminal domain which are destined for incorporation as β-barrels in the outer membrane bilayer is increased by covalent attachment of oligo-(R)-3-hydroxybutyrates. With the aid of the BAM complex, OmpA is then assembled into the outer membrane as a single-domain large pore. PMID:22251410

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

    PubMed

    Rakshit, Tatini; Senapati, Subhadip; Sinha, Satyabrata; Whited, A M; Park, Paul S-H

    2015-01-01

    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.

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

    PubMed

    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. Structure, function and binding selectivity and stereoselectivity of siderophore-iron outer membrane transporters.

    PubMed

    Schalk, Isabelle J; Mislin, Gaëtan L A; Brillet, Karl

    2012-01-01

    To get access to iron, microorganisms produce and release into their environment small organic metal chelators called siderophores. In parallel, they produce siderophore-iron outer membrane transporters (also called TonB-Dependent Transporters or TBDT) embedded in the outer membrane; these proteins actively reabsorb the siderophore loaded with iron from the extracellular medium. This active uptake requires energy in the form of the proton motive force transferred from the inner membrane to the outer membrane transporter via the inner membrane TonB complex. Siderophores produced by microorganisms are structurally very diverse with molecular weights of 150 up to 2000Da. Siderophore-iron uptake from the extracellular medium by TBDTs is a highly selective and sometimes even stereoselective process, with each siderophore having a specific TBDT. Unlike the siderophores, all TBDTs have similar structures and belong to the outer membrane β-barrel protein superfamily. The way in which the siderophore-iron complex passes through the TBDT is still unclear. In some bacteria, TBDTs are also partners of signaling cascades regulating the expression of proteins involved in siderophore biosynthesis and siderophore-iron acquisition.

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

    PubMed

    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. High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

    PubMed Central

    2012-01-01

    Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

  12. High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

    PubMed

    Walian, Peter J; Allen, Simon; Shatsky, Maxim; Zeng, Lucy; Szakal, Evelin D; Liu, Haichuan; Hall, Steven C; Fisher, Susan J; Lam, Bonita R; Singer, Mary E; Geller, Jil T; Brenner, Steven E; Chandonia, John-Marc; Hazen, Terry C; Witkowska, H Ewa; Biggin, Mark D; Jap, Bing K

    2012-12-07

    Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

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

    PubMed Central

    Valeru, Soni Priya; Shanan, Salah; Alossimi, Haifa; Sandström, Gunnar

    2014-01-01

    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. PMID:24799908

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

    NASA Astrophysics Data System (ADS)

    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.

  15. Cold Stress Makes Escherichia coli Susceptible to Glycopeptide Antibiotics by Altering Outer Membrane Integrity.

    PubMed

    Stokes, Jonathan M; French, Shawn; Ovchinnikova, Olga G; Bouwman, Catrien; Whitfield, Chris; Brown, Eric D

    2016-02-18

    A poor understanding of the mechanisms by which antibiotics traverse the outer membrane remains a considerable obstacle to the development of novel Gram-negative antibiotics. Herein, we demonstrate that the Gram-negative bacterium Escherichia coli becomes susceptible to the narrow-spectrum antibiotic vancomycin during growth at low temperatures. Heterologous expression of an Enterococcus vanHBX vancomycin resistance cluster in E. coli confirmed that the mechanism of action was through inhibition of peptidoglycan biosynthesis. To understand the nature of vancomycin permeability, we screened for strains of E. coli that displayed resistance to vancomycin at low temperature. Surprisingly, we observed that mutations in outer membrane biosynthesis suppressed vancomycin activity. Subsequent chemical analysis of lipopolysaccharide from vancomycin-sensitive and -resistant strains confirmed that suppression was correlated with truncations in the core oligosaccharide of lipopolysaccharide. These unexpected observations challenge the current understanding of outer membrane permeability, and provide new chemical insights into the susceptibility of E. coli to glycopeptide antibiotics.

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

    PubMed

    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.

  17. The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes.

    PubMed

    Yuan, C; Chen, H; Anderson, R E; Kuwata, O; Ebrey, T G

    1998-08-01

    This study investigated the lipid and fatty acid composition of gecko photoreceptor outer segment membranes which contain the P521 cone-type pigment. The lipids of gecko photoreceptor outer segment membranes were first extracted and separated by thin layer chromatography (TLC) and then analyzed by gas chromatography (GC). Our results show that gecko photoreceptor outer segment membranes contain less phosphatidylethanolamine (PE) and more phosphatidylcholine (PC) and phosphatidylserine (PS) compared with those of bovine and frog. The content of the polyunsaturated fatty acid, docosahexaenoic acid (DHA), in PC and PS is also the highest yet reported (55 and 63%, respectively). These lipid differences may provide some insight into the specific lipid requirements of cone-type pigments.

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

    PubMed Central

    Kulp, Adam J.; Sun, Bo; Ai, Teresa; Manning, Andrew J.; Orench-Rivera, Nichole

    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. PMID:26406465

  19. Crystal structure of the open state of the Neisseria gonorrhoeae MtrE outer membrane channel.

    PubMed

    Lei, Hsiang-Ting; Chou, Tsung-Han; Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Do, Sylvia V; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2014-01-01

    Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel.

  20. The heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans: relationship to OmpA proteins.

    PubMed Central

    Wilson, M E

    1991-01-01

    The outer membrane of Actinobacillus actinomycetemcomitans contains a 29-kDa protein which exhibits heat modifiability on sodium dodecyl sulfate-polyacrylamide gels and represents a major target for immunoglobulin G antibody in sera of periodontitis patients colonized by this organism. In the present study, the N-terminal amino acid sequence of the 29-kDa outer membrane protein was determined and compared with reported sequences for other known proteins. The heat-modifiable outer membrane protein of A. actinomycetemcomitans was found to exhibit significant N-terminal homology with the OmpA proteins of other gram-negative bacteria. Moreover, this protein reacted with antiserum raised against the purified OmpA protein of Escherichia coli K-12. Whether the heat-modifiable OMP of A. actinomycetemcomitans also shares functional properties of OmpA proteins, particularly with respect to bacteriophage receptor activity, is presently under investigation. Images PMID:2050416

  1. Characterisation of cell-wall polysaccharides from mandarin segment membranes.

    PubMed

    Coll-Almela, Luis; Saura-López, Domingo; Laencina-Sánchez, José; Schols, Henk A; Voragen, Alfons G J; Ros-García, José María

    2015-05-15

    In an attempt to develop a process of enzymatic peeling of mandarin segments suitable for use on an industrial scale, the cell wall fraction of the segment membrane of Satsuma mandarin fruits was extracted to obtain a chelating agent-soluble pectin fraction (ChSS), a dilute sodium hydroxide-soluble pectin fraction (DASS), a 1M sodium hydroxide-soluble hemicellulose fraction (1MASS), a 4M sodium hydroxide-soluble hemicellulose fraction (4MASS) and a cellulose-rich residue (3.1, 0.9, 0.4, 0.7 and 1.6%w/w of fresh membrane, respectively). The ChSS pectin consisted mainly of galacturonic acid followed by arabinose and galactose. The DASS fraction contained less galacturonic acid and more neutral sugars than ChSS. Eighty-nine percent of the galacturonic acid present in the segment membranes was recovered in the above two pectin fractions. The two hemicellulosic fractions consisted of two different molecular weight populations, which also differed in their sugar composition. Arabinose, xylose, mannose, galactose and glucose were the main sugar constituents of these hemicellulose fractions. In addition to an (arabino)xylan and a xyloglucan, the presence of an arabinogalactan is suggested by the sugar composition of both hemicelluloses. The pectin fractions were also characterised by their degradability by the pectic enzymes polygalacturonase, pectinmethylesterase and rhamnogalacturonan hydrolase. However the degree of degradation of the pectin fractions by enzymes differed, and the amount of the polymeric materials resistant to further degradation and the oligomeric products also differed. Using pectic enzymes it is possible to obtain peeled mandarin segments ready to eat or for canning. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Mitochondrial Outer Membrane Proteome of Trypanosoma brucei Reveals Novel Factors Required to Maintain Mitochondrial Morphology*

    PubMed Central

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

    2013-01-01

    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. PMID:23221899

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed

    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.

  6. Cell wall polysaccharide synthases are located in detergent-resistant membrane microdomains in oomycetes.

    PubMed

    Briolay, Anne; Bouzenzana, Jamel; Guichardant, Michel; Deshayes, Christian; Sindt, Nicolas; Bessueille, Laurence; Bulone, Vincent

    2009-04-01

    The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1-->3)-beta-d-glucan synthases are located in detergent-resistant membrane microdomains (DRMs) in oomycetes, a phylum that comprises some of the most devastating microorganisms in the agriculture and aquaculture industries. Interestingly, no cellulose synthase activity was detected in the DRMs. The purified DRMs exhibited similar biochemical features as lipid rafts from animal, plant, and yeast cells, although they contained some species-specific lipids. This report sheds light on the lipid environment of the (1-->3)-beta-d-glucan and chitin synthases, as well as on the sterol biosynthetic pathways in oomycetes. The results presented here are consistent with a function of lipid rafts in cell polarization and as platforms for sorting specific sets of proteins targeted to the plasma membrane, such as carbohydrate synthases. The involvement of DRMs in the biosynthesis of major cell wall polysaccharides in eukaryotic microorganisms suggests a function of lipid rafts in hyphal morphogenesis and tip growth.

  7. Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis.

    PubMed

    Küper, Ulf; Meyer, Carolin; Müller, Volker; Rachel, Reinhard; Huber, Harald

    2010-02-16

    ATP synthase catalyzes ATP synthesis at the expense of an electrochemical ion gradient across a membrane that can be generated by different exergonic reactions. Sulfur reduction is the main energy-yielding reaction in the hyperthermophilic strictly anaerobic Crenarchaeon Ignicoccus hospitalis. This organism is unusual in having an inner and an outer membrane that are separated by a huge intermembrane compartment. Here we show, on the basis of immuno-EM analyses of ultrathin sections and immunofluorescence experiments with whole I. hospitalis cells, that the ATP synthase and H(2):sulfur oxidoreductase complexes of this organism are located in the outer membrane. These two enzyme complexes are mandatory for the generation of an electrochemical gradient and for ATP synthesis. Thus, among all prokaryotes possessing two membranes in their cell envelope (including Planctomycetes, gram-negative bacteria), I. hospitalis is a unique organism, with an energized outer membrane and ATP synthesis within the periplasmic space. In addition, DAPI staining and EM analyses showed that DNA and ribosomes are localized in the cytoplasm, leading to the conclusion that in I. hospitalis energy conservation is separated from information processing and protein biosynthesis. This raises questions regarding the function of the two membranes, the interaction between these compartments, and the general definition of a cytoplasmic membrane.

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:26752045

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

    PubMed Central

    Florez, Catalina; Raab, Julie E.; Cooke, Adam C.

    2017-01-01

    ABSTRACT 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. PMID:28790210

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

    PubMed

    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.

  12. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis

    SciTech Connect

    Caldwell, H.D.; Kromhout, J.; Schachter, J.

    1981-03-01

    Elementary bodies (EB) of Chlamydia trachomatis serotypes C, E, and L2 were extrinsically radioiodinated, and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit molecular weight of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine), and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtained after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycan-like cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60 degrees C. This protein accounted for 61% of the total COMC-associated protein, and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.

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

    PubMed

    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.

  14. The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri.

    PubMed

    Wang, Xia; Jiang, Feng; Zheng, Jianhua; Chen, Lihong; Dong, Jie; Sun, Lilian; Zhu, Yafang; Liu, Bo; Yang, Jian; Yang, Guowei; Jin, Qi

    2016-09-01

    Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.

  15. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    USDA-ARS?s Scientific Manuscript database

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>500) known and predicted TA proteins in Arabidopsis for those annotated, based on Gene Ontology, to possess mitochondrial...

  16. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    USDA-ARS?s Scientific Manuscript database

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>600) known and predicted TA proteins in Arabidopsis thaliana for those annotated, based on Gene Ontology, to possess mitoc...

  17. TonB-Dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5

    USDA-ARS?s Scientific Manuscript database

    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 ...

  18. Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

    PubMed Central

    Ripple, Maureen O.; Abajian, Michelle

    2010-01-01

    Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa3) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be ≈62% oxidized before MOMP and became ≈70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc1 with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s. PMID:20094799

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

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

    2015-12-14

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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. An essential novel component of the noncanonical mitochondrial outer membrane protein import system of trypanosomatids

    PubMed Central

    Pusnik, Mascha; Mani, Jan; Schmidt, Oliver; Niemann, Moritz; Oeljeklaus, Silke; Schnarwiler, Felix; Warscheid, Bettina; Lithgow, Trevor; Meisinger, Chris; Schneider, André

    2012-01-01

    The mitochondrial outer membrane protein Tom40 is the general entry gate for imported proteins in essentially all eukaryotes. Trypanosomatids lack Tom40, however, and use instead a protein termed the archaic translocase of the outer mitochondrial membrane (ATOM). Here we report the discovery of pATOM36, a novel essential component of the trypanosomal outer membrane protein import system that interacts with ATOM. pATOM36 is not related to known Tom proteins from other organisms and mediates the import of matrix proteins. However, there is a group of precursor proteins whose import is independent of pATOM36. Domain-swapping experiments indicate that the N-terminal presequence-containing domain of the substrate proteins at least in part determines the dependence on pATOM36. Secondary structure profiling suggests that pATOM36 is composed largely of α-helices and its assembly into the outer membrane is independent of the sorting and assembly machinery complex. Taken together, these results show that pATOM36 is a novel component associated with the ATOM complex that promotes the import of a subpopulation of proteins into the mitochondrial matrix. PMID:22787278

  4. Salmonella typhimurium contains an anion-selective outer membrane porin induced by phosphate starvation.

    PubMed Central

    Bauer, K; Benz, R; Brass, J; Boos, W

    1985-01-01

    A mutant of Salmonella typhimurium was selected that is constitutive for the pho regulon. It exhibited constitutive glycerol-3-phosphate transport activity and synthesized a new outer membrane porin. Upon measurement of porin activity in black lipid films, it exhibited anion selectivity. It therefore appears analogous to the Escherichia coli PhoE porin. Images PMID:2981826

  5. Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations

    PubMed Central

    Siskind, Leah J.; Kolesnick, Richard N.; Colombini, Marco

    2007-01-01

    Recent evidence suggests that the ability of ceramides to induce apoptosis is due to a direct action on mitochondria. Mitochondria are known to contain enzymes responsible for ceramide synthesis and hydrolysis and mitochondrial ceramide levels have been shown to be elevated prior to the mitochondrial phase of apoptosis. Ceramides have been reported to induce the release of intermembrane space proteins from mitochondria, which has been linked to their ability to form large channels in membranes. The aim of this study was to determine if the membrane concentration of ceramide required for the formation of protein permeable channels is within the range that is present in mitochondria during the induction phase of apoptosis. Only a very small percentage of the ceramide actually inserts into the mitochondrial membranes. The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane. Importantly, the concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (4 pmol ceramide/nanomole phospholipid). Similar results were obtained with short- and long-chain ceramide. Ceramide channel formation is specific to mitochondrial membranes in that no channel formation occurs in the plasma membranes of erythrocytes even at concentrations 20 times higher than those required for channel formation in mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway by which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis. PMID:16713754

  6. Molecular Structure of the Outer Bacterial Membrane of Pseudomonas aeruginosa via Classical Simulation

    SciTech Connect

    Shroll, Robert M.; Straatsma, TP

    2002-10-23

    A detailed structural analysis has been performed of the outer bacterial membrane of Pseudomonas aeruginosa using a parameterized classical simulation model [R. D. Lins and T. P. Straatsma, Biophys. J. 81:1037-1046, (2001)] with modest modifications. The structural analysis of the membrane is presented and newly discovered characteristics of the membrane are discussed. Simulations indicate that the relative contribution of different ligands to calcium ion coordination varies across the membrane, while maintaining a constant average coordination number of 6.1. Water penetrates the surface of the membrane to a depth of about 30?. The hydration of ions and phosphate groups is shown to depend on location within the membrane. A measure of saccharide residue orientation is defined and average orientations are presented. Saccharide residues possess varying degrees of motion with a trend of greater mobility at the membrane surface. However, their motion is limited and even in the membrane outer core region the average structure appears fairly rigid over a period of 1 ns.

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

    NASA Astrophysics Data System (ADS)

    Haarmann, Raimund; Ibrahim, Mohamed; Stevanovic, Mara; Bredemeier, Rolf; Schleiff, Enrico

    2010-11-01

    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 β-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.

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

    PubMed Central

    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

  9. Freezing tolerance in plants requires lipid remodeling at the outer chloroplast membrane.

    PubMed

    Moellering, Eric R; Muthan, Bagyalakshmi; Benning, Christoph

    2010-10-08

    Plants show complex adaptations to freezing that prevent cell damage caused by cellular dehydration. Lipid remodeling of cell membranes during dehydration is one critical mechanism countering loss of membrane integrity and cell death. SENSITIVE TO FREEZING 2 (SFR2), a gene essential for freezing tolerance in Arabidopsis, encodes a galactolipid remodeling enzyme of the outer chloroplast envelope membrane. SFR2 processively transfers galactosyl residues from the abundant monogalactolipid to different galactolipid acceptors, forming oligogalactolipids and diacylglycerol, which is further converted to triacylglycerol. The combined activity of SFR2 and triacylglycerol-biosynthetic enzymes leads to the removal of monogalactolipids from the envelope membrane, changing the ratio of bilayer- to non-bilayer-forming membrane lipids. This SFR2-based mechanism compensates for changes in organelle volume and stabilizes membranes during freezing.

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

    PubMed

    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.

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

    PubMed

    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.

  12. Single-point single-molecule FRAP distinguishes inner and outer nuclear membrane protein distribution

    PubMed Central

    Mudumbi, Krishna C; Schirmer, Eric C; Yang, Weidong

    2016-01-01

    The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM). Quantitative determination of the distribution of NETs on the ONM and INM is limited in available approaches, which moreover provide no information about translocation rates in the two membranes. Here we demonstrate a single-point single-molecule FRAP microscopy technique that enables determination of distribution and translocation rates for NETs in vivo. PMID:27558844

  13. RND transporters protect Corynebacterium glutamicum from antibiotics by assembling the outer membrane.

    PubMed

    Yang, Liang; Lu, Shuo; Belardinelli, Juan; Huc-Claustre, Emilie; Jones, Victoria; Jackson, Mary; Zgurskaya, Helen I

    2014-08-01

    Corynebacterium-Mycobacterium-Nocardia (CMN) group are the causative agents of a broad spectrum of diseases in humans. A distinctive feature of these Gram-positive bacteria is the presence of an outer membrane of unique structure and composition. Recently, resistance-nodulation-division (RND) transporters (nicknamed MmpLs, Mycobacterial membrane protein Large) have emerged as major contributors to the biogenesis of the outer membranes in mycobacteria and as promising drug targets. In this study, we investigated the role of RND transporters in the physiology of Corynebacterium glutamicum and analyzed properties of these proteins. Our results show that in contrast to Gram-negative species, in which RND transporters actively extrude antibiotics from cells, in C. glutamicum and relatives these transporters protect cells from antibiotics by playing essential roles in the biogenesis of the low-permeability barrier of the outer membrane. Conditional C. glutamicum mutants lacking RND proteins and with the controlled expression of either NCgl2769 (CmpL1) or NCgl0228 (CmpL4) are hypersusceptible to multiple antibiotics, have growth deficiencies in minimal medium and accumulate intracellularly trehalose monocorynomycolates, free corynomycolates, and the previously uncharacterized corynomycolate-containing lipid. Our results also suggest that similar to other RND transporters, Corynebacterial membrane proteins Large (CmpLs) functions are dependent on a proton-motive force.

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

    PubMed

    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.

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

    PubMed

    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, <0.9). For the first time, we have demonstrated that mitochondrial 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.

  16. Ribosomes specifically bind to mammalian mitochondria via protease-sensitive proteins on the outer membrane.

    PubMed

    MacKenzie, James A; Payne, R Mark

    2004-03-12

    The interaction of ribosomes with specific components of membranes is one of the central themes to the co-translational targeting and import of proteins. To examine ribosome binding to mammalian mitochondria, we used ribosome-nascent chain complexes (RNCs) to follow the in vitro binding of ribosomes that correspond to the initial targeting stage of proteins. Mitochondria were found to contain a limited number of RNC binding sites on the outer membrane. It required more than twice the amount of non-translating ribosomes to inhibit RNC binding by one-half, indicating that RNCs have a competitive binding advantage. In addition, we found that RNCs bind mainly through the ribosomal component and not the nascent chain. RNCs bind via protease-sensitive proteins on the outer membrane, as well as by protease-insensitive components suggesting that two classes of receptors exist. We also show that binding is sensitive to cation conditions. Nearly all of the binding was inhibited in 0.5 m KCl, indicating that they interact with the membrane primarily through electrostatic interactions. In addition, disruption of RNC structure by removing magnesium causes the complete inhibition of binding under normal binding conditions indicating that it is the intact ribosome that is crucial for binding and not the nascent chain. These findings support the hypothesis that the outer mitochondrial membrane contains receptors specific for ribosomes, which would support the conditions necessary for co-translational import.

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

    PubMed

    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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  19. Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli.

    PubMed

    Abdeljabbar, Diya M; Song, Hank J; Link, A James

    2012-01-01

    Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.

  20. Outer membrane proteins can be simply identified using secondary structure element alignment

    PubMed Central

    2011-01-01

    Background Outer membrane proteins (OMPs) are frequently found in the outer membranes of gram-negative bacteria, mitochondria and chloroplasts and have been found to play diverse functional roles. Computational discrimination of OMPs from globular proteins and other types of membrane proteins is helpful to accelerate new genome annotation and drug discovery. Results Based on the observation that almost all OMPs consist of antiparallel β-strands in a barrel shape and that their secondary structure arrangements differ from those of other types of proteins, we propose a simple method called SSEA-OMP to identify OMPs using secondary structure element alignment. Through intensive benchmark experiments, the proposed SSEA-OMP method is better than some well-established OMP detection methods. Conclusions The major advantage of SSEA-OMP is its good prediction performance considering its simplicity. The web server implements the method is freely accessible at http://protein.cau.edu.cn/SSEA-OMP/index.html. PMID:21414186

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

    PubMed

    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.

  2. The protein import channel in the outer mitosomal membrane of Giardia intestinalis.

    PubMed

    Dagley, Michael J; Dolezal, Pavel; Likic, Vladimir A; Smid, Ondrej; Purcell, Anthony W; Buchanan, Susan K; Tachezy, Jan; Lithgow, Trevor

    2009-09-01

    The identification of mitosomes in Giardia generated significant debate on the evolutionary origin of these organelles, whether they were highly reduced mitochondria or the product of a unique endosymbiotic event in an amitochondrial organism. As the protein import pathway is a defining characteristic of mitochondria, we sought to discover a TOM (translocase in the outer mitochondrial membrane) complex in Giardia. A Hidden Markov model search of the Giardia genome identified a Tom40 homologous sequence (GiTom40), where Tom40 is the protein translocation channel of the TOM complex. The GiTom40 protein is located in the membrane of mitosomes in a approximately 200-kDa TOM complex. As Tom40 was derived in the development of mitochondria to serve as the protein import channel in the outer membrane, its presence in Giardia evidences the mitochondrial ancestry of mitosomes.

  3. Mathematical Modeling of Plate–gap Biosensors with an Outer Porous Membrane

    PubMed Central

    Baronas, Romas; Ivanauskas, Feliksas; Kaunietis, Irmantas; Laurinavicius, Valdas

    2006-01-01

    A plate–gap model of a porous enzyme doped electrode covered by a porous inert membrane has been proposed and analyzed. The two–dimensional–in–space mathematical model of the plate–gap biosensors is based on the reaction–diffusion equations containing a nonlinear term related to the Michaelis–Menten kinetics. Using numerical simulation of the biosensor action, the influence of the geometry of the outer membrane on the biosensor response was investigated at wide range of analyte concentrations as well as of the reaction rates. The numerical simulation was carried out using finite–difference technique. The behavior of the plate–gap biosensors was compared with that of a flat electrode deposited with a layer of enzyme and covered with the same outer membrane.

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

    SciTech Connect

    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.

  5. Major outer membrane protein of Legionella pneumophila carries a species-specific epitope.

    PubMed Central

    Nolte, F S; Conlin, C A

    1986-01-01

    A monoclonal antibody (LP3IIG2) directed against a species-specific epitope of Legionella pneumophila is available from Genetic Systems Corp., Seattle, Wash., for use as a diagnostic reagent. Outer membrane protein-rich fractions were prepared from L. pneumophila serogroups 1 to 8 by treatment of cell envelopes with 2% Triton X-100. Immunoblots of sodium dodecyl sulfate-polyacrylamide gels demonstrated that each membrane fraction contained two bands that reacted with LP3IIG2. The monoclonal antibody bound preferentially to a 26,000-molecular-weight band that appears to result from modification of the 29,000-molecular-weight major outer membrane protein. Images PMID:2420824

  6. Surface-Localized Spermidine Protects the Pseudomonas aeruginosa Outer Membrane from Antibiotic Treatment and Oxidative Stress

    PubMed Central

    Johnson, Lori; Mulcahy, Heidi; Kanevets, Uliana; Shi, Yan

    2012-01-01

    Extracellular DNA acts as a cation chelator and induces the expression of antibiotic resistance genes regulated by Mg2+ levels. Here we report the characterization of novel DNA-induced genes in Pseudomonas aeruginosa that are annotated as homologs of the spermidine synthesis genes speD (PA4773) and speE (PA4774). The addition of sublethal concentrations of DNA and membrane-damaging antibiotics induced expression of the genes PA4773 to PA4775, as shown using transcriptional lux fusions and quantitative RT-PCR. Exogenous polyamine addition prevented DNA- and peptide-mediated gene induction. Mutation of PA4774 resulted in an increased outer membrane (OM) susceptibility phenotype upon polymyxin B, CP10A, and gentamicin treatment. When the membrane-localized fluorescent probe C11-BODIPY581/591 was used as an indicator of peroxidation of membrane lipids, the PA4774::lux mutant demonstrated an increased susceptibility to oxidative membrane damage from H2O2 treatment. Addition of exogenous polyamines protected the membranes of the PA4774::lux mutant from polymyxin B and H2O2 treatment. Polyamines from the outer surface were isolated and shown to contain putrescine and spermidine by using high-performance liquid chromatography and mass spectrometry. The PA4774::lux mutant did not produce spermidine on the cell surface, but genetic complementation restored surface spermidine production as well as the antibiotic and oxidative stress resistance phenotypes of the membrane. We have identified new functions for spermidine on the cell surface and propose that polyamines are produced under Mg2+-limiting conditions as an organic polycation to bind lipopolysaccharide (LPS) and to stabilize and protect the outer membrane against antibiotic and oxidative damage. PMID:22155771

  7. Human antibody responses after vaccination with the Norwegian group B meningococcal outer membrane vesicle vaccine: results from ELISA studies.

    PubMed

    Rosenqvist, E; Høiby, E A; Bjune, G; Bryn, K; Closs, O; Feiring, B; Klem, A; Nøkleby, H; Frølm, L O

    1991-12-01

    Antibody responses after vaccination with three different formulations of a new meningococcal group B outer membrane vesicle (OMV) vaccine have been studied with the ELISA technique using four different antigens. Sera from about 1200 vaccinees participating in steps 1, 2, 3 and 6 of the phase II clinical trials in Norway were analysed. The effects of non-covalently complexing the OMV antigen to group C polysaccharide (C-PS) and of adsorbing OMV (with and without C-PS) to aluminium hydroxide (AH) were studied. All three vaccine formulations were highly immunogenic in humans. Adsorption of the vaccine to AH had a relatively small effect on the immune response, but the results indicated that the booster response was stronger with the adsorbed than with the unadsorbed vaccines. Some increase in the immune response against OMV was also observed by non-covalent complexing OMV with C-PS, particularly after the second dose. In most of the vaccinees the antibody levels were significantly reduced 6 to 12 months after vaccination. Adsorption of the vaccine to AH had no effect on the antibody response against C-PS. Comparison with bactericidal activity of the same sera was done. A highly significant correlation was observed between the bactericidal titres and the levels of IgG antibodies against OMV and class 5C protein, whereas the correlation between antibody levels against lipopolysaccharide and the bactericidal activity was poor.

  8. Destabilization of the Outer and Inner Mitochondrial Membranes by Core and Linker Histones

    PubMed Central

    Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove

    2012-01-01

    Background Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. Methodology/Principal Findings We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. Conclusions We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage. PMID:22523586

  9. Destabilization of the outer and inner mitochondrial membranes by core and linker histones.

    PubMed

    Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove

    2012-01-01

    Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage.

  10. Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response

    PubMed Central

    McBroom, Amanda J; Kuehn, Meta J

    2007-01-01

    Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism. PMID:17163978

  11. Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae

    SciTech Connect

    Hirst, T.R.; Holmgren, J.

    1987-11-01

    The secretion of enterotoxin by Vibrio cholerae is punctuated by the transient entry of the toxin subunits into the periplasm. In this paper, the authors show that the subunits oligomerize into an assembled holotoxin within the periplasm prior to their secretion across the outer membrane. The rate of toxin assembly was studied by pulse-labeling cells with (/sup 35/S)-methionine and then monitoring the turnover of radiolabeled subunits as they assembled within the periplasm. The subunits entered the periplasm as monomers and assembled into oligomers with a half-time of approx. = 1 min. Since assembly was a rapid event compared to the rate of toxin efflux from the periplasm, which had a half-time of approx. = 13 min, they conclude that all of the subunits that pass through the periplasm assemble before they traverse the outer membrane. The average concentration of subunit monomers and assembled holotoxin within the periplasm was calculated to be approx. = 20 and approx. = 260 ..mu..g/ml, respectively. This indicates that the periplasm is a suitably concentrated milieu where spontaneous toxin assembly can occur. These findings suggest that protein movement across bacterial outer membranes, in apparent contrast to export across other biological membranes, involves translocation of polypeptides that have already folded into tertiary and even quaternary conformations.

  12. Influence of Core Oligosaccharide of Lipopolysaccharide to Outer Membrane Behavior of Escherichia coli

    PubMed Central

    Wang, Zhou; Wang, Jianli; Ren, Ge; Li, Ye; Wang, Xiaoyuan

    2015-01-01

    Lipopolysaccharides, major molecules in the outer membrane of Gram-negative bacteria, play important roles on membrane integrity of the cell. However, how the core oligosaccharide of lipopolysaccharide affect the membrane behavior is not well understood. In this study, the relationship between the core oligosaccharide of lipopolysaccharide and the membrane behavior was investigated using a series of Escherichia coli mutants defective in genes to affect the biosynthesis of core oligosaccharide of lipopolysaccharide. Cell surface hydrophobicity, outer membrane permeability, biofilm formation and auto-aggregation of these mutant cells were compared. Compared to the wild type W3110, cell surface hydrophobicities of mutant ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaP, ΔwaaY and ΔwaaB were enhanced, outer membrane permeabilities of ΔwaaC, ΔwaaF, ΔwaaG and ΔwaaP were significantly increased, abilities of biofilm formation by ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaP, ΔwaaQ and ΔwaaY decreased, and auto-aggregation abilities of ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaU, ΔwaaP and ΔwaaY were strongly enhanced. These results give new insight into the influence of core oligosaccharide of lipopolysaccharide on bacterial cell membrane behavior. PMID:26023839

  13. Matriglycan: a novel polysaccharide that links dystroglycan to the basement membrane

    PubMed Central

    Yoshida-Moriguchi, Takako; Campbell, Kevin P

    2015-01-01

    Associations between cells and the basement membrane are critical for a variety of biological events including cell proliferation, cell migration, cell differentiation and the maintenance of tissue integrity. Dystroglycan is a highly glycosylated basement membrane receptor, and is involved in physiological processes that maintain integrity of the skeletal muscle, as well as development and function of the central nervous system. Aberrant O-glycosylation of the α subunit of this protein, and a concomitant loss of dystroglycan's ability to function as a receptor for extracellular matrix (ECM) ligands that bear laminin globular (LG) domains, occurs in several congenital/limb-girdle muscular dystrophies (also referred to as dystroglycanopathies). Recent genetic studies revealed that mutations in DAG1 (which encodes dystroglycan) and at least 17 other genes disrupt the ECM receptor function of dystroglycan and cause disease. Here, we summarize recent advances in our understanding of the enzymatic functions of two of these disease genes: the like-glycosyltransferase (LARGE) and protein O-mannose kinase (POMK, previously referred to as SGK196). In addition, we discuss the structure of the glycan that directly binds the ECM ligands and the mechanisms by which this functional motif is linked to dystroglycan. In light of the fact that dystroglycan functions as a matrix receptor and the polysaccharide synthesized by LARGE is the binding motif for matrix proteins, we propose to name this novel polysaccharide structure matriglycan. PMID:25882296

  14. Proteomic Analysis of the Yeast Mitochondrial Outer Membrane Reveals Accumulation of a Subclass of PreproteinsD⃞

    PubMed Central

    Zahedi, Rene P.; Sickmann, Albert; Boehm, Andreas M.; Winkler, Christiane; Zufall, Nicole; Schönfisch, Birgit; Guiard, Bernard; Pfanner, Nikolaus; Meisinger, Chris

    2006-01-01

    Mitochondria consist of four compartments–outer membrane, intermembrane space, inner membrane, and matrix—with crucial but distinct functions for numerous cellular processes. A comprehensive characterization of the proteome of an individual mitochondrial compartment has not been reported so far. We used a eukaryotic model organism, the yeast Saccharomyces cerevisiae, to determine the proteome of highly purified mitochondrial outer membranes. We obtained a coverage of ∼85% based on the known outer membrane proteins. The proteome represents a rich source for the analysis of new functions of the outer membrane, including the yeast homologue (Hfd1/Ymr110c) of the human protein causing Sjögren–Larsson syndrome. Surprisingly, a subclass of proteins known to reside in internal mitochondrial compartments were found in the outer membrane proteome. These seemingly mislocalized proteins included most top scorers of a recent genome-wide analysis for mRNAs that were targeted to mitochondria and coded for proteins of prokaryotic origin. Together with the enrichment of the precursor form of a matrix protein in the outer membrane, we conclude that the mitochondrial outer membrane not only contains resident proteins but also accumulates a conserved subclass of preproteins destined for internal mitochondrial compartments. PMID:16407407

  15. Preparation and characterization of polysaccharides/PVA blend nanofibrous membranes by electrospinning method.

    PubMed

    Santos, Carla; Silva, Carla J; Büttel, Zsófia; Guimarães, Rodrigo; Pereira, Sara B; Tamagnini, Paula; Zille, Andrea

    2014-01-01

    A series of polyvinyl alcohol (PVA), PVA/chitosan (CS) and PVA/cyanobacterial extracellular polymeric substances (EPS) blended nanofibrous membranes were produced by electrospinning using a microfiltration poly(vinylidene fluoride) (PVDF) basal membrane, for potential applications in water filtration. Nanofibres were obtained from solutions of 20% (w/w) PVA with 1% (w/w) CS or EPS, using a weight ratio of 60/40. Blended nanofibres have shown a smooth morphology, no beads formation and diameters between 50 and 130 nm. Thermo-mechanical analysis demonstrated that there were inter and/or intramolecular hydrogen bonds between the molecules of PVA/CS and PVA/EPS in the blends. The electrospun blended PVA/EPS membrane showed better tensile mechanical properties when compared with PVA and PVA/CS, and resisted more against disintegration in the temperature range between 10 and 50 °C. Finally, the blended membranes have shown an increase in chromium binding capacity of 5%. This is the first successful report of a blended membrane of electrospinned cyanobacterial polysaccharide with PVA.

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

    PubMed

    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

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

    PubMed

    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.

  18. Screening of the Salmonella paratyphi A CMCC 50973 strain outer membrane proteins for the identification of potential vaccine targets.

    PubMed

    Yang, Tian-Ci; Ma, Xian-Cang; Liu, Fan; Lin, Li-Rong; Liu, Li-Li; Liu, Gui-Li; Tong, Man-Li; Fu, Zuo-Gen; Zhou, Lei

    2012-01-01

    Outer membrane protein antigens usually have strong immunogenicities, closely interact with the immune system and play a significant role in the development of new vaccines. The outer membrane proteins of Salmonella paratyphi A (S. paratyphi A) were screened for immunogenicity and immunoprotection for potential vaccine targets. In this study, the bactericidal effect of antiserum against the total outer membrane proteins of S. paratyphi A CMCC 50973 strain was determined, and their immunoprotection was detected with a challenge experiment on vaccinated mice. The immunogenic outer membrane proteins were identified via immunoproteomic technology, and recombinant outer membrane proteins were expressed and purified. The immunoprotection provided by the immunogenic membrane proteins was verified through active and passive immunity challenge experiments. The result revealed a number of S. paratyphi A outer membrane proteins that were proven as strong protective antigens. Twelve immunogenic outer membrane proteins were located and identified. Five recombinant proteins (LamB, pagC, TolC, nmpC and fadL) with strong immunoprotective abilities were found via the active immunity challenge experiment, with protection rates of 95, 95, 85, 80 and 70%, respectively. They were also proven to induce good immunoprotection via the passive immunity challenge experiment, with protection rates of 65, 55, 60, 55 and 50%, respectively. The immunoprotective rate of the five-antiserum combination was 85%. In conclusion, the LamB, pagC, TolC, nmpC and fadL outer membrane proteins, with strong immunogenicities and immunoprotection, are effective protein candidate targets for the development of new vaccines, whereas the recombinant outer membrane proteins are a promising tool for improving immunoprotection.

  19. Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

    SciTech Connect

    Clifton, Luke A.; Skoda, Maximilian W. A.; Le Brun, Anton P.; Ciesielski, Filip; Kuzmenko, Ivan; Holt, Stephen A.; Lakey, Jeremy H.

    2014-12-09

    The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg2+ and Ca2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.

  20. Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

    DOE PAGES

    Clifton, Luke A.; Skoda, Maximilian W. A.; Le Brun, Anton P.; ...

    2014-12-09

    The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg2+ and Ca2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutronmore » reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.« less

  1. Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin.

    PubMed

    Bartra, Sara Schesser; Ding, Yi; Fujimoto, L Miya; Ring, Joshua G; Jain, Vishal; Ram, Sanjay; Marassi, Francesca M; Plano, Gregory V

    2015-11-01

    Yersinia pestis, the agent of plague, requires the Ail (attachment invasion locus) outer membrane protein to survive in the blood and tissues of its mammalian hosts. Ail is important for both attachment to host cells and for resistance to complement-dependent bacteriolysis. Previous studies have shown that Ail interacts with components of the extracellular matrix, including fibronectin, laminin and heparan sulfate proteoglycans, and with the complement inhibitor C4b-binding protein. Here, we demonstrate that Ail-expressing Y. pestis strains bind vitronectin - a host protein with functions in cell attachment, fibrinolysis and inhibition of the complement system. The Ail-dependent recruitment of vitronectin resulted in efficient cleavage of vitronectin by the outer membrane Pla (plasminogen activator protease). Escherichia coli DH5α expressing Y. pestis Ail bound vitronectin, but not heat-treated vitronectin. The ability of Ail to directly bind vitronectin was demonstrated by ELISA using purified refolded Ail in nanodiscs.

  2. Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin

    PubMed Central

    Bartra, Sara Schesser; Ding, Yi; Miya Fujimoto, L.; Ring, Joshua G.; Jain, Vishal; Ram, Sanjay; Marassi, Francesca M.

    2015-01-01

    Yersinia pestis, the agent of plague, requires the Ail (attachment invasion locus) outer membrane protein to survive in the blood and tissues of its mammalian hosts. Ail is important for both attachment to host cells and for resistance to complement-dependent bacteriolysis. Previous studies have shown that Ail interacts with components of the extracellular matrix, including fibronectin, laminin and heparan sulfate proteoglycans, and with the complement inhibitor C4b-binding protein. Here, we demonstrate that Ail-expressing Y. pestis strains bind vitronectin – a host protein with functions in cell attachment, fibrinolysis and inhibition of the complement system. The Ail-dependent recruitment of vitronectin resulted in efficient cleavage of vitronectin by the outer membrane Pla (plasminogen activator protease). Escherichia coli DH5α expressing Y. pestis Ail bound vitronectin, but not heat-treated vitronectin. The ability of Ail to directly bind vitronectin was demonstrated by ELISA using purified refolded Ail in nanodiscs. PMID:26377177

  3. The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD

    NASA Astrophysics Data System (ADS)

    Calmettes, Charles; Ing, Christopher; Buckwalter, Carolyn M.; El Bakkouri, Majida; Chieh-Lin Lai, Christine; Pogoutse, Anastassia; Gray-Owen, Scott D.; Pomès, Régis; Moraes, Trevor F.

    2015-08-01

    Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.

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

    PubMed

    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.

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

    PubMed

    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.

  6. Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica.

    PubMed

    Troncoso, Gemma; Sánchez, Sandra; Criado, María Teresa; Ferreirós, Carlos

    2004-01-15

    Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.

  7. The Design and Structure of Outer Membrane Receptors from Peroxisomes, Mitochondria, and Chloroplasts.

    PubMed

    Panigrahi, Rashmi; Kubiszewski-Jakubiak, Szymon; Whelan, James; Vrielink, Alice

    2015-10-06

    The eukaryotic cell is defined by compartments that allow specialization of function. This compartmental structure generates a new concept in cell biology compared with the simpler prokaryotic cell structure, namely the specific targeting of proteins to intracellular compartments. Protein targeting is achieved by the action of specialized signals on proteins destined for organelles that are recognized by cognate receptors. An understanding of the specificity of targeting signal recognition leading to import requires an understanding of the receptor structures. Here, we focus on the structures of receptors of different import machineries located on the outer membrane of three organelles: peroxisomes, mitochondria, and chloroplasts. This review provides an overview of the structural features of outer membrane import receptors that recognize targeting signals. Finally, we briefly discuss combinatorial approaches that might aid in understanding the structural factors mediating receptor targeting signal recognition. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed

    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.

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

    PubMed

    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.

  10. The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD

    PubMed Central

    Calmettes, Charles; Ing, Christopher; Buckwalter, Carolyn M.; El Bakkouri, Majida; Chieh-Lin Lai, Christine; Pogoutse, Anastassia; Gray-Owen, Scott D.; Pomès, Régis; Moraes, Trevor F.

    2015-01-01

    Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops. PMID:26282243

  11. The major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, Pan 1, is a lipoprotein.

    PubMed Central

    Hoehn, G T; Clark, V L

    1992-01-01

    Pan 1 is an acidic outer membrane protein of Neisseria gonorrhoeae that is expressed only when gonococci are grown anaerobically. On silver-stained sodium dodecyl sulfate-polyacrylamide gels, Pan 1 migrates as an intense but diffuse 54-kDa protein. The deduced amino acid sequence of Pan 1 from the aniA (anaerobically induced protein) open reading frame reveals a lipoprotein consensus sequence, Ala-Leu-Ala-Ala-Cys, and a processed molecular mass of 39 kDa. Furthermore, there is strong homology at the N terminus and C terminus of Pan 1 to the termini of the gonococcal outer membrane lipoproteins Lip and Laz. [3H]palmitic acid labeling of gonococci grown under oxygen-limited conditions demonstrated specific incorporation of label into Pan 1, suggesting further that Pan 1 is a lipoprotein. Images PMID:1398981

  12. Escherichia coli pleiotropic mutant that reduces amounts of several periplasmic and outer membrane proteins.

    PubMed Central

    Wanner, B L; Sarthy, A; Beckwith, J

    1979-01-01

    We have isolated a mutant of Escherichia coli K-12 that is reduced from 6- to 10-fold in the amount of alkaline phosphatase found in the periplasmic space. The reduced synthesis is not due to effects at the level of transcription regulation of the phoA gene, the structural gene for the enzyme. In addition, the mutation (termed perA) responsible for this phenotype results in reduced amounts of possibly six or more other periplasmic proteins and at least three outer membrane proteins. One of the outer membrane proteins affected is protein IA (D. L. Diedrich, A. O. Summers, and C. A. Schnaitman, J. Bacteriol. 131:598-607, 1977). Although other possibilities exist, one explanation for the phenotype of the perA mutation is that it affects the cell's secretory apparatus. Images PMID:387722

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

    PubMed

    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.

  14. The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD.

    PubMed

    Calmettes, Charles; Ing, Christopher; Buckwalter, Carolyn M; El Bakkouri, Majida; Chieh-Lin Lai, Christine; Pogoutse, Anastassia; Gray-Owen, Scott D; Pomès, Régis; Moraes, Trevor F

    2015-08-18

    Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Optical tweezers study of viscoelastic properties in the outer hair cell plasma membrane

    NASA Astrophysics Data System (ADS)

    Murdock, David R.; Ermilov, Sergey A.; Qian, Feng; Brownell, William E.; Anvari, Bahman

    2004-06-01

    An optical tweezers system was used to study the mechanical characteristics of the outer hair cell (OHC) lateral wall by forming plasma membrane tethers. A 2nd order generalized Kelvin model was applied to describe the viscoelastic behavior of OHC membrane tethers. The measured parameters included equilibrium tethering force, (Feq), force relaxation times (τ), stiffness values (κ), and coefficients of friction (μ). An analysis of force relaxation in membrane tethers indicated that the force decay is a biphasic process containing both an elastic and a viscous phase. In general, we observed an overall negative trend in the measured parameters upon application of the cationic amphipath chlorpromazine (CPZ). CPZ was found to cause up to a 40 pN reduction in Feq in OHCs. A statistically significant reduction in relaxation times and coefficients of friction was also observed, suggesting an increase in rate of force decay and a decrease in plasma membrane viscosity.

  17. Studies of cochlear outer hair cell membrane mechanics using optical tweezers

    NASA Astrophysics Data System (ADS)

    Murdock, David R.; Ermilov, Sergey A.; Brownell, William E.; Anvari, Bahman

    2003-06-01

    An optical tweezers system was used to study the mechanical characteristics of outer hair cell (OHC) and human embryonic kidney (HEK) cell plasma membranes. The effect of the cationic amphipath chlorpromazine (CPZ) on the equilibrium tethering force, (Feq) force relaxation time constant,(τ) and effective membrane viscosity (ηeff) was measured. The Feq for the OHC lateral wall plasma membrane was ~60 pN and was unchanged by addition of CPZ. A significantly greater τ value was observed in CPZ-treated OHCs (30.5 +/- 12.6 s) than in control OHCs (19.0 +/- 13.2 s). The Feq and τ values for control HEK cells were >60% lower than the respective OHC values but increased by ~3 times following CPZ addition. Effective viscosity ranged between 1.49-1.81 pN•s/μm for CPZ-treated OHCs. This represents a decrease from reported control OHC membrane viscosities.

  18. SurA Is Involved in the Targeting to the Outer Membrane of a Tat Signal Sequence-Anchored Protein

    PubMed Central

    Rondelet, Arnaud

    2012-01-01

    The twin arginine translocation (Tat) pathway exports folded proteins from the cytoplasm to the periplasm of bacteria. The targeting of the exported proteins to the Tat pathway relies on a specific amino-terminal signal sequence, which is cleaved after exportation. In the phytopathogen Dickeya dadantii, the pectin lyase homologue PnlH is exported by the Tat pathway without cleavage of its signal sequence, which anchors PnlH into the outer membrane. In proteobacteria, the vast majority of outer membrane proteins consists of β-barrel proteins and lipoproteins. Thus, PnlH represents a new kind of outer membrane protein. In Escherichia coli, periplasmic chaperones SurA, Skp, and DegP work together with the β-barrel assembly machinery (Bam) to target and insert β-barrel proteins into the outer membrane. In this work, we showed that SurA is required for an efficient targeting of PnlH to the outer membrane. Moreover, we were able to detect an in vitro interaction between SurA and the PnlH signal sequence. Since the PnlH signal sequence contains a highly hydrophobic region, we propose that SurA protects it from the hydrophobic periplasm during targeting of PnlH to the outer membrane. We also studied the nature of the information carried by the PnlH signal sequence responsible for its targeting to the outer membrane after exportation by the Tat system. PMID:22961852

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Demarcating SurA Activities Required for Outer Membrane Targeting of Yersinia pseudotuberculosis Adhesins

    PubMed Central

    Obi, Ikenna R.

    2013-01-01

    SurA is a periplasmic protein folding factor involved in chaperoning and trafficking of outer membrane proteins across the Gram-negative bacterial periplasm. In addition, SurA also possesses peptidyl-prolyl cis/trans isomerase activity. We have previously reported that in enteropathogenic Yersinia pseudotuberculosis, SurA is needed for bacterial virulence and envelope integrity. In this study, we investigated the role of SurA in the assembly of important Yersinia adhesins. Using genetic mutation, biochemical characterization, and an in vitro-based bacterial host cell association assay, we confirmed that surface localization of the invasin adhesin is dependent on SurA. As a surA deletion also has some impact on the levels of individual components of the BAM complex in the Yersinia outer membrane, abolished invasin surface assembly could reflect both a direct loss of SurA-dependent periplasmic targeting and a potentially compromised BAM complex assembly platform in the outer membrane. To various degrees, the assembly of two other adhesins, Ail and the pH 6 antigen fibrillum PsaA, also depends on SurA. Consequently, loss of SurA leads to a dramatic reduction in Yersinia attachment to eukaryotic host cells. Genetic complementation of surA deletion mutants indicated a prominent role for SurA chaperone function in outer membrane protein assembly. Significantly, the N terminus of SurA contributed most of this SurA chaperone function. Despite a dominant chaperoning role, it was also evident that SurA isomerization activity did make a modest contribution to this assembly process. PMID:23589578

  1. Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State▿ †

    PubMed Central

    Zuber, Benoît; Chami, Mohamed; Houssin, Christine; Dubochet, Jacques; Griffiths, Gareth; Daffé, Mamadou

    2008-01-01

    The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function. PMID:18567661

  2. Analysis of Surface-Exposed Outer Membrane Proteins in Helicobacter pylori

    PubMed Central

    Voss, Bradley J.; Gaddy, Jennifer A.; McDonald, W. Hayes

    2014-01-01

    More than 50 Helicobacter pylori genes are predicted to encode outer membrane proteins (OMPs), but there has been relatively little experimental investigation of the H. pylori cell surface proteome. In this study, we used selective biotinylation to label proteins localized to the surface of H. pylori, along with differential detergent extraction procedures to isolate proteins localized to the outer membrane. Proteins that met multiple criteria for surface-exposed outer membrane localization included known adhesins, as well as Cag proteins required for activity of the cag type IV secretion system, putative lipoproteins, and other proteins not previously recognized as cell surface components. We identified sites of nontryptic cleavage consistent with signal sequence cleavage, as well as C-terminal motifs that may be important for protein localization. A subset of surface-exposed proteins were highly susceptible to proteolysis when intact bacteria were treated with proteinase K. Most Hop and Hom OMPs were susceptible to proteolysis, whereas Hor and Hof proteins were relatively resistant. Most of the protease-susceptible OMPs contain a large protease-susceptible extracellular domain exported beyond the outer membrane and a protease-resistant domain at the C terminus with a predicted β-barrel structure. These features suggest that, similar to the secretion of the VacA passenger domain, the N-terminal domains of protease-susceptible OMPs are exported through an autotransporter pathway. Collectively, these results provide new insights into the repertoire of surface-exposed H. pylori proteins that may mediate bacterium-host interactions, as well as the cell surface topology of these proteins. PMID:24769695

  3. Outer Membrane Proteins of Pseudomonas aeruginosa. Their Role in Antibiotic Susceptibility.

    DTIC Science & Technology

    1980-12-01

    is the X phage receptor which has been implicated in the maltose and maltodextrin transport system by overcoming the diffusion barrier for these...accomodate small molecules other than maltodextrins . It is significant, however, that the X receptor is used for maltose transport only when substrate...and maltodextrins (23). -4- Physiological roles of outer membrane proteins of Gram-negative bacteria other than those just discussed are: peptidoglycan

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

    SciTech Connect

    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.

  5. Modeling investigation of membrane biofouling phenomena by considering the adsorption of protein, polysaccharide and humic acid.

    PubMed

    Demneh, Seyedeh Marzieh Ghasemi; Nasernejad, Bahram; Modarres, Hamid

    2011-11-01

    The importance of solute adsorption in the biofouling membrane has been clearly verified for the performance of membrane bioreactor (MBR). In order to quantify the mechanism of static adsorption in biofouling during of MBR process, we characterize membrane biofouling caused by model solutions containing a protein (bovine serum albumin, BSA), a humic substance (humic acid, HA) and a polysaccharide (alginic acid, Alg) on commercial hydrophilic polyethersulfone (PES) membrane. For static adsorption experiments, membranes were immersed in well-defined model solutions in three temperatures (298, 308 and 318 K) to obtain equilibrium data. To determine the characteristic parameters for this process, 7 isotherm models were applied to the experimental data. Three error analysis methods; the coefficient of nonlinear regression (R(2)), the sum of the squared errors (SSE) and standard deviation of residuals (S(yx)), were used to evaluate the data and determine the best fit isotherm for each model solutions. The error values demonstrated that the Sips isotherm model provided the best fit to the experimental data. AFM images were used for determination of changes in membrane surface after adsorption. These images confirmed the results obtained from adsorption isotherm study. Thermodynamic parameters such as standard free energy (Δ(r)G(θ)), enthalpy (Δ(r)H(θ)) and entropy (Δ(r)S(θ)) changes were determined; these adsorption processes were found to be feasible and endothermic but not spontaneous. The distribution of the substances adsorbed on PES surface were more chaotic than that in the aqueous solutions. Parameters obtained in this study can be used to determine the "fouling potential" of a given feed stream and a membrane. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Prediction of structural features and application to outer membrane protein identification

    PubMed Central

    Yan, Renxiang; Wang, Xiaofeng; Huang, Lanqing; Yan, Feidi; Xue, Xiaoyu; Cai, Weiwen

    2015-01-01

    Protein three-dimensional (3D) structures provide insightful information in many fields of biology. One-dimensional properties derived from 3D structures such as secondary structure, residue solvent accessibility, residue depth and backbone torsion angles are helpful to protein function prediction, fold recognition and ab initio folding. Here, we predict various structural features with the assistance of neural network learning. Based on an independent test dataset, protein secondary structure prediction generates an overall Q3 accuracy of ~80%. Meanwhile, the prediction of relative solvent accessibility obtains the highest mean absolute error of 0.164, and prediction of residue depth achieves the lowest mean absolute error of 0.062. We further improve the outer membrane protein identification by including the predicted structural features in a scoring function using a simple profile-to-profile alignment. The results demonstrate that the accuracy of outer membrane protein identification can be improved by ~3% at a 1% false positive level when structural features are incorporated. Finally, our methods are available as two convenient and easy-to-use programs. One is PSSM-2-Features for predicting secondary structure, relative solvent accessibility, residue depth and backbone torsion angles, the other is PPA-OMP for identifying outer membrane proteins from proteomes. PMID:26104144

  7. Immunogenicity of Pseudomonas aeruginosa outer membrane antigens examined by crossed immunoelectrophoresis.

    PubMed Central

    Lam, J S; Mutharia, L M; Hancock, R E; Høiby, N; Lam, K; Baek, L; Costerton, J W

    1983-01-01

    By crossed immunoelectrophoresis 36 different anode-migrating antigens were demonstrated in sonicated antigen preparations of Pseudomonas aeruginosa. We numbered these antigens to establish a reference precipitin pattern. Antigen no. 31 was identified as the lipopolysaccharide (LPS) antigen, because it was found to be responsible for the O-group specificity and because it reacted with anti-LPS monoclonal antibodies and with Limulus amoebocyte lysate. Purified outer membrane proteins F (porin), H2, and I used as antigens formed precipitins with the reference antibodies, thus establishing their antigenicity. LPS that copurified with protein F and slightly contaminated protein H2 was detectable as an extra precipitin (antigen no. 31). The use of monoclonal antibodies specific for smooth LPS and rough LPS revealed different antigenic determinants in the LPS molecule and suggested that antigen no. 5 could be the core region of the LPS which is equivalent to the rough LPS. Antibodies against these outer membrane antigens were detected in patients with chronic P. aeruginosa pneumonia and in patients with acute P. aeruginosa bacteremia. Antibodies with the same specificity were also found in rats chronically infected with P. aeruginosa 7 days postinfection. This demonstrates the surface accessibility and antigenic reactivity of outer membrane antigens. Images PMID:6194119

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

    PubMed

    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.

  9. Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.

    PubMed Central

    Kothary, Mahendra H.; Gopinath, Gopal R.; Gangiredla, Jayanthi; Rallabhandi, Prasad V.; Harrison, Lisa M.; Yan, Qiong Q.; Chase, Hannah R.; Lee, Boram; Park, Eunbi; Yoo, YeonJoo; Chung, Taejung; Finkelstein, Samantha B.; Negrete, Flavia J.; Patel, Isha R.; Carter, Laurenda; Sathyamoorthy, Venugopal; Fanning, Séamus; Tall, Ben D.

    2017-01-01

    Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport. PMID:28232819

  10. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    PubMed

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  11. Novel outer membrane protein involved in cellulose and cellooligosaccharide degradation by Cytophaga hutchinsonii.

    PubMed

    Ji, Xiaofei; Wang, Ying; Zhang, Cong; Bai, Xinfeng; Zhang, Weican; Lu, Xuemei

    2014-08-01

    Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and -glucosidase activity was distributed on the cell surface of C. hutchinsonii.Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the -glucosidase activity,it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the -glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface -glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii.

  12. Role of the mar-sox-rob Regulon in Regulating Outer Membrane Porin Expression▿†

    PubMed Central

    Chubiz, Lon M.; Rao, Christopher V.

    2011-01-01

    Multiple factors control the expression of the outer membrane porins OmpF and OmpC in Escherichia coli. In this work, we investigated the role of the mar-sox-rob regulon in regulating outer membrane porin expression in response to salicylate. We provide both genetic and physiological evidence that MarA and Rob can independently activate micF transcription in response to salicylate, leading to reduced OmpF expression. MarA was also found to repress OmpF expression through a MicF-independent pathway. In the case of OmpC, we found that its transcription was moderately increased in response to salicylate. However, this increase was independent of MarA and Rob. Finally, we found that the reduction in OmpF expression in a tolC mutant is due primarily to Rob. Collectively, this work further clarifies the coordinated role of MarA and Rob in regulating the expression of the outer membrane porins. PMID:21398557

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

    PubMed

    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.

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

    PubMed

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

    2016-06-13

    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.

  15. Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

    PubMed

    Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

    1985-04-01

    Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains.

  16. Use of gene fusions to study outer membrane protein localization in Escherichia coli.

    PubMed Central

    Silhavy, T J; Shuman, H A; Beckwith, J; Schwartz, M

    1977-01-01

    Escherichia coli strains have been isolated that produce hybrid proteins comprised of an NH2-terminal sequence from the lamB gene product (an outer membrane protein) and a major portion of the COOH-terminal sequence of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23; a cytoplasmic protein). These proteins exhibit beta-galactosidase activity. One such strain, pop 3105, produces a hybrid protein containing very little of the lamB gene protein; the protein is found in the cytoplasm. The protein found in a second strain, pop 3186, contains much more of the lamB gene protein; a substantial fraction of the beta-galactosidase activity is found in the outer membrane, probably facing outward. These results indicate that information necessary to direct the lamB gene product to its outer membrane location is located within the lamB gene itself. The properties of such fusion strains open up the prospect of a precise genetic analysis of the genetic components involved in protein transport. Images PMID:414221

  17. Novel Outer Membrane Protein Involved in Cellulose and Cellooligosaccharide Degradation by Cytophaga hutchinsonii

    PubMed Central

    Ji, Xiaofei; Wang, Ying; Zhang, Cong; Bai, Xinfeng; Zhang, Weican

    2014-01-01

    Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and β-glucosidase activity was distributed on the cell surface of C. hutchinsonii. Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the β-glucosidase activity, it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the β-glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface β-glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii. PMID:24837387

  18. Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

    PubMed Central

    Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

    1985-01-01

    Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains. Images PMID:3980081

  19. Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation

    PubMed Central

    Schafer, Blanca; Quispe, Joel; Choudhary, Vineet; Chipuk, Jerry E.; Ajero, Teddy G.; Du, Han; Schneiter, Roger

    2009-01-01

    Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the “pores” and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25–100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin. PMID:19244344

  20. Prediction of structural features and application to outer membrane protein identification

    NASA Astrophysics Data System (ADS)

    Yan, Renxiang; Wang, Xiaofeng; Huang, Lanqing; Yan, Feidi; Xue, Xiaoyu; Cai, Weiwen

    2015-06-01

    Protein three-dimensional (3D) structures provide insightful information in many fields of biology. One-dimensional properties derived from 3D structures such as secondary structure, residue solvent accessibility, residue depth and backbone torsion angles are helpful to protein function prediction, fold recognition and ab initio folding. Here, we predict various structural features with the assistance of neural network learning. Based on an independent test dataset, protein secondary structure prediction generates an overall Q3 accuracy of ~80%. Meanwhile, the prediction of relative solvent accessibility obtains the highest mean absolute error of 0.164, and prediction of residue depth achieves the lowest mean absolute error of 0.062. We further improve the outer membrane protein identification by including the predicted structural features in a scoring function using a simple profile-to-profile alignment. The results demonstrate that the accuracy of outer membrane protein identification can be improved by ~3% at a 1% false positive level when structural features are incorporated. Finally, our methods are available as two convenient and easy-to-use programs. One is PSSM-2-Features for predicting secondary structure, relative solvent accessibility, residue depth and backbone torsion angles, the other is PPA-OMP for identifying outer membrane proteins from proteomes.

  1. Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor).

    PubMed

    Sileikyte, Justina; Petronilli, Valeria; Zulian, Alessandra; Dabbeni-Sala, Federica; Tognon, Giuseppe; Nikolov, Peter; Bernardi, Paolo; Ricchelli, Fernanda

    2011-01-14

    We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

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

    PubMed

    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.

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

    PubMed Central

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

    2008-01-01

    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 stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins. PMID:18055598

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

    PubMed

    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

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. IMPORTANCE 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

  6. Solanum Nigrum polysaccharide (SNL) extract effects in transplanted tumor-bearing mice--erythrocyte membrane fluidity and blocking of functions.

    PubMed

    Yuan, Hong-Liang; Liu, Xiao-Lei; Liu, Ying-Jie

    2014-01-01

    Solanum nigrum L. has been used in traditional Chinese medicine because of its diuretic and antipyretic effects. The present research concerned effects of crude polysaccharides isolated from Solanum nigrum L. on erythrocyte membranes of tumor-bearing S180 and H22 in mice. Fluorescence- labeled red blood cell membranes were used with DPH fluorescence spectrophotometry to examine erythrocyte membrane fluidity, and colorimetry to determine degree of erythrocyte surface membrane blocking. Extent of reaction by tumor-bearing mice with the enzyme erythrocyte membrane bubble shadow detection of red cell membrane variation in the degree of closure before and after administration. Solanum nigrum polysaccharide could significantly improve the S180 and H22 tumor-bearing mice erythrocyte membrane fluidity, compared with the control group, the difference was significant (p<0.01), SNL can significantly improve the red blood cell membrane and then S180 tumor-bearing mice sealing ability, compared with the negative control group, the difference was significant(p<0.05, p<0.01). H22 tumor-bearing mice can increase red cell membrane and then sealing ability, the difference was significant (p<0.05). Solanum nigrum polysaccharide degree of fluidity and blocking two transplanted tumors in mice restored the ability to raise the red cell membrane has a significant effect. Solanum nigrum L.-type mice transplanted tumor can affect the red blood cell membrane fluidity and re-closed, through the red cell membrane of red blood cells to enhance the immune function of the possibility of erythrocyte immunity against tumor formation garland provide experimental basis.

  7. Antibody Responses to Recombinant Protein Fragments of the Major Outer Membrane Protein and Polymorphic Outer Membrane Protein POMP90 in Chlamydophila abortus-Infected Pregnant Sheep

    PubMed Central

    Livingstone, Morag; Entrican, Gary; Wattegedera, Sean; Buxton, David; McKendrick, Iain J.; Longbottom, David

    2005-01-01

    Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans. PMID:15939753

  8. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

    PubMed Central

    Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E.; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E.; Fridberger, Anders; Zuo, Jian

    2015-01-01

    Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases. PMID:26352669

  9. The motion of a single molecule, the lambda-receptor, in the bacterial outer membrane.

    PubMed Central

    Oddershede, Lene; Dreyer, Jakob Kisbye; Grego, Sonia; Brown, Stanley; Berg-Sørensen, Kirstine

    2002-01-01

    Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo. The efficiency of this in vivo biotinylation is very low, thus enabling the attachment of a streptavidin-coated bead binding specifically to a single biotinylated lambda-receptor. The bead was used as a handle for the optical tweezers and as a marker for the single particle tracking routine. We propose a model 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. PMID:12496085

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

    PubMed

    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.

  11. Mechanics of force propagation in TonB-dependent outer membrane transport.

    PubMed

    Gumbart, James; Wiener, Michael C; Tajkhorshid, Emad

    2007-07-15

    For the uptake of scarce yet essential organometallic compounds, outer membrane transporters of Gram-negative bacteria work in concert with an energy-generating inner membrane complex, thus spanning the periplasmic space to drive active transport. Here, we examine the interaction of TonB, an inner membrane protein, with an outer membrane transporter based upon a recent crystal structure of a TonB-transporter complex to characterize two largely unknown steps of the transport cycle: how energy is transmitted from TonB to the transporter and how energy transduction initiates transport. Simulations of TonB in complex with BtuB reveal that force applied to TonB is transmitted to BtuB without disruption of the very small connection between the two, supporting a mechanical mode of coupling. Based on the results of different pulling simulations, we propose that the force transduction instigates a partial unfolding of the pore-occluding luminal domain of the transporter, a potential step in the transport cycle. Furthermore, analysis of the electrostatic potentials and salt bridge interactions between the two proteins during the simulations hints at involvement of electrostatic forces in long-range interaction and binding of TonB and BtuB.

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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, DRP1K38A, 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. PMID:26813789

  13. A Novel Mitosomal β-Barrel Outer Membrane Protein in Entamoeba

    PubMed Central

    Santos, Herbert J.; Imai, Kenichiro; Makiuchi, Takashi; Tomii, Kentaro; Horton, Paul; Nozawa, Akira; Ibrahim, Mohamed; Tozawa, Yuzuru; Nozaki, Tomoyoshi

    2015-01-01

    Entamoeba possesses a highly divergent mitochondrion-related organelle known as the mitosome. Here, we report the discovery of a novel protein in Entamoeba, which we name Mitosomal β-barrel Outer Membrane Protein of 30 kDa (MBOMP30). Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a β-barrel protein. Circular dichroism analysis showed MBOMP30 has a predominant β-sheet structure. Localization to Entamoeba histolytica mitosomes was observed through Percoll-gradient fractionation and immunofluorescence assay. Mitosomal membrane integration was demonstrated by carbonate fractionation, proteinase K digestion, and immunoelectron microscopy. Interestingly, the deletion of the putative β-signal, a sequence believed to guide β-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a ~240 kDa complex. MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes. PMID:25711150

  14. Translocation of Vibrio harveyi N,N'-diacetylchitobiase to the outer membrane of Escherichia coli.

    PubMed Central

    Jannatipour, M; Soto-Gil, R W; Childers, L C; Zyskind, J W

    1987-01-01

    The gene encoding N,N'-diacetylchitobiase (chitobiase) of the chitinolytic marine bacterium Vibrio harveyi has been isolated. While expression of the chitobiase gene (chb) was inducible by N,N'-diacetylchitobiose in V. harveyi, it was expressed constitutively when cloned in Escherichia coli, suggesting that controlling elements are not closely linked to chb. Chitobiase was found in the membrane fraction of E. coli cells containing plasmids with the cloned V. harveyi chb gene. When membranes of such cells were separated on Osborn gradients, chitobiase activity was found mainly in the outer membrane band. Translocation of the enzyme to the outer membrane was accompanied by cleavage of a signal peptide. A fusion protein, in which 22 amino acids from the amino terminus of prechitobiase were replaced with 21 amino acids from the pUC19 lacZ amino terminus, was not processed, and 99% of the activity was located in the cytoplasmic fraction. A homology to six amino acids surrounding the lipoprotein processing and modification site was found near the amino terminus of prechitobiase. Images PMID:3301816

  15. The potential and electric field in the cochlear outer hair cell membrane.

    PubMed

    Harland, Ben; Lee, Wen-han; Brownell, William E; Sun, Sean X; Spector, Alexander A

    2015-05-01

    Outer hair cell electromechanics, critically important to mammalian active hearing, is driven by the cell membrane potential. The membrane protein prestin is a crucial component of the active outer hair cell's motor. The focus of the paper is the analysis of the local membrane potential and electric field resulting from the interaction of electric charges involved. Here the relevant charges are the ions inside and outside the cell, lipid bilayer charges, and prestin-associated charges (mobile-transferred by the protein under the action of the applied field, and stationary-relatively unmoved by the field). The electric potentials across and along the membrane are computed for the case of an applied DC-field. The local amplitudes and phases of the potential under different frequencies are analyzed for the case of a DC + AC-field. We found that the effect of the system of charges alters the electric potential and internal field, which deviate significantly from their traditional linear and constant distributions. Under DC + AC conditions, the strong frequency dependence of the prestin mobile charge has a relatively small effect on the amplitude and phase of the resulting potential. The obtained results can help in a better understanding and experimental verification of the mechanism of prestin performance.

  16. A growing toolbox of techniques for studying β-barrel outer membrane protein folding and biogenesis.

    PubMed

    Horne, Jim E; Radford, Sheena E

    2016-06-15

    Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed. © 2016 The Author(s).

  17. Synthetic Effect between Envelope Stress and Lack of Outer Membrane Vesicle Production in Escherichia coli

    PubMed Central

    Schwechheimer, Carmen

    2013-01-01

    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. PMID:23852867

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

    PubMed

    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.

  19. Expression, crystallization and preliminary X-ray analysis of an outer membrane protein from Thermus thermophilus HB27

    PubMed Central

    Brosig, Alexander; Nesper, Jutta; Welte, Wolfram; Diederichs, Kay

    2008-01-01

    The cell envelope of the thermophilic bacterium Thermus thermophilus is multilayered and includes an outer membrane with integral outer membrane proteins that are not well characterized. The hypothetical protein TTC0834 from T. thermophilus HB27 was identified as a 22 kDa outer membrane protein containing eight predicted β-strands. TTC0834 was expressed with an N-­terminal His tag in T. thermophilus HB8 and detected in the S-layer/outer membrane envelope fraction. His-TTC0834 was purified and crystallized under various conditions. Native data sets were collected to 3.2 Å resolution and the best diffracting crystals belonged to space group P3121 or P3221, with unit-cell parameters a = b = 166.67, c = 97.53 Å. PMID:18540069

  20. Expression, crystallization and preliminary X-ray analysis of an outer membrane protein from Thermus thermophilus HB27.

    PubMed

    Brosig, Alexander; Nesper, Jutta; Welte, Wolfram; Diederichs, Kay

    2008-06-01

    The cell envelope of the thermophilic bacterium Thermus thermophilus is multilayered and includes an outer membrane with integral outer membrane proteins that are not well characterized. The hypothetical protein TTC0834 from T. thermophilus HB27 was identified as a 22 kDa outer membrane protein containing eight predicted beta-strands. TTC0834 was expressed with an N-terminal His tag in T. thermophilus HB8 and detected in the S-layer/outer membrane envelope fraction. His-TTC0834 was purified and crystallized under various conditions. Native data sets were collected to 3.2 A resolution and the best diffracting crystals belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 166.67, c = 97.53 A.

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

    PubMed

    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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. Outer membranes of gram-negative bacteria. XV. Transmembrane diffusion rates in lipoprotein-deficient mutants of Escherichia coli.

    PubMed Central

    Nikaido, H; Bavoil, P; Hirota, Y

    1977-01-01

    Permeability of the outer membrane to 6-aminopenicillanic acid was unaltered in an lpo mutant, lacking the Braun lipoprotein, a result suggesting that the lipoproteins by themselves form no or few diffusion pores. PMID:200601

  4. Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin.

    PubMed

    Nobre, Thatyane M; Martynowycz, Michael W; Andreev, Konstantin; Kuzmenko, Ivan; Nikaido, Hiroshi; Gidalevitz, David

    2015-12-15

    Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted as monolayers at the air-water interface, and their properties, as well as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region, but was prevented from this penetration into the modified lipopolysaccharides. Results correlate with behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains.

  5. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria

    PubMed Central

    Roier, Sandro; Zingl, Franz G.; Cakar, Fatih; Durakovic, Sanel; Kohl, Paul; Eichmann, Thomas O.; Klug, Lisa; Gadermaier, Bernhard; Weinzerl, Katharina; Prassl, Ruth; Lass, Achim; Daum, Günther; Reidl, Joachim; Feldman, Mario F.; Schild, Stefan

    2016-01-01

    Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo. PMID:26806181

  6. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria.

    PubMed

    Roier, Sandro; Zingl, Franz G; Cakar, Fatih; Durakovic, Sanel; Kohl, Paul; Eichmann, Thomas O; Klug, Lisa; Gadermaier, Bernhard; Weinzerl, Katharina; Prassl, Ruth; Lass, Achim; Daum, Günther; Reidl, Joachim; Feldman, Mario F; Schild, Stefan

    2016-01-25

    Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.

  7. Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin

    PubMed Central

    Nobre, Thatyane M.; Martynowycz, Michael W.; Andreev, Konstantin; Kuzmenko, Ivan; Nikaido, Hiroshi; Gidalevitz, David

    2015-01-01

    Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted as monolayers at the air-water interface, and their properties, as well as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region, but was prevented from this penetration into the modified lipopolysaccharides. Results correlate with behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains. PMID:26682812

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

    PubMed Central

    Findlay, Heather E; McClafferty, Heather; Ashley, Richard H

    2005-01-01

    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's topology, could provide

  9. Ergosterol content specifies targeting of tail-anchored proteins to mitochondrial outer membranes

    PubMed Central

    Krumpe, Katrin; Frumkin, Idan; Herzig, Yonatan; Rimon, Nitzan; Özbalci, Cagakan; Brügger, Britta; Rapaport, Doron; Schuldiner, Maya

    2012-01-01

    Tail-anchored (TA) proteins have a single C-terminal transmembrane domain, making their biogenesis dependent on posttranslational translocation. Despite their importance, no dedicated insertion machinery has been uncovered for mitochondrial outer membrane (MOM) TA proteins. To decipher the molecular mechanisms guiding MOM TA protein insertion, we performed two independent systematic microscopic screens in which we visualized the localization of model MOM TA proteins on the background of mutants in all yeast genes. We could find no mutant in which insertion was completely blocked. However, both screens demonstrated that MOM TA proteins were partially localized to the endoplasmic reticulum (ER) in ∆spf1 cells. Spf1, an ER ATPase with unknown function, is the first protein shown to affect MOM TA protein insertion. We found that ER membranes in ∆spf1 cells become similar in their ergosterol content to mitochondrial membranes. Indeed, when we visualized MOM TA protein distribution in yeast strains with reduced ergosterol content, they phenocopied the loss of Spf1. We therefore suggest that the inherent differences in membrane composition between organelle membranes are sufficient to determine membrane integration specificity in a eukaryotic cell. PMID:22918956

  10. Allosteric signalling in the outer membrane translocation domain of PapC usher

    PubMed Central

    Farabella, Irene; Pham, Thieng; Henderson, Nadine S; Geibel, Sebastian; Phan, Gilles; Thanassi, David G; Delcour, Anne H; Waksman, Gabriel; Topf, Maya

    2014-01-01

    PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue ‘communities’) within the translocation domain (especially around β12–β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability. DOI: http://dx.doi.org/10.7554/eLife.03532.001 PMID:25271373

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

    PubMed

    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.

  12. Molecular design of the voltage-dependent, anion-selective channel in the mitochondrial outer membrane.

    PubMed

    Guo, X W; Smith, P R; Cognon, B; D'Arcangelis, D; Dolginova, E; Mannella, C A

    1995-01-01

    The mitochondrial outer membrane contains numerous copies of a channel protein, VDAC, that is thought to be the main permeability pathway through this membrane for polar molecules and ions. Low-dose electron microscopy has been used to obtain images of two-dimensional crystals of this channel (produced by treating outer membranes from fungal mitochondria with phospholipase A2) embedded in vitreous ice or aurothioglucose. The angular orientation of the channels in the unit cell of one type of array has been determined by rotational correlation analysis. The location of the amino-terminal segment of the protein (which, according to circular dichroism, forms an alpha-helix in nonpolar solvents and detergent solutions) has been determined by labeling arrays with Fab prepared from antibodies directed against residues 1-20. The three-dimensional structure of the channel has been obtained by applying Fourier reconstruction methods to projections of tilted crystals embedded in aurothioglucose, followed by averaging of the three non-symmetry-related channels in the unit cell. The results of this study indicate that the wall of VDAC's lumen has several irregular features (uneven height, grooves) and that the aminoterminal segment extends away from the lumen in this crystalline state.

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

    PubMed Central

    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 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. PMID:24618254

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

    PubMed

    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.

  15. Long circulating micelles of an amphiphilic random copolymer bearing cell outer membrane phosphorylcholine zwitterions.

    PubMed

    Zhao, Jing; Chai, Yu-Dong; Zhang, Jing; Huang, Peng-Fei; Nakashima, Kenichi; Gong, Yong-Kuan

    2015-04-01

    Polymeric micelles with cell outer membrane mimetic structure were prepared in water from amphiphilic random copolymers bearing both the hydrophilic phosphorylcholine zwitterions and hydrophobic octadecyl side chains of cell outer membrane. The polymeric micelles showed sizes ranging from 80 nm to 120 nm in hydrodynamic diameter and zeta-potentials from -6.4 mV to -2.4 mV by dynamic light scattering measurements. The micelles loaded with 6-coumarin as a fluorescence probe were stable to investigate their blood circulation and biodistribution. The in vitro phagocytosis results using murine peritoneal macrophages showed 10-fold reduction compared with a reference micelle. The in vivo blood circulation half-life of the polymeric micelles following intravenous administration in New Zealand Rabbits was increased from 0.55 h to 90.5h. More interestingly, tissue distribution results showed that the concentration of the micelles in the kidney is 4-fold higher than that in the liver and other organs 48 h after administration. The results of this work show great promise for designing more effective stealth drug carriers that can minimize reticuloendothelial system clearance and circulate for long time to reach target by using simple cell membrane mimetic random copolymer micelles.

  16. Crystal structure of a major outer membrane protein from Thermus thermophilus HB27.

    PubMed

    Brosig, Alexander; Nesper, Jutta; Boos, Winfried; Welte, Wolfram; Diederichs, Kay

    2009-02-06

    The thermophilic eubacterium Thermus thermophilus belongs to one of the oldest branches of evolution and has a multilayered cell envelope that differs from that of modern Gram-negative bacteria. Its outer membrane contains integral outer membrane proteins (OMPs), of which only a few are characterized. TtoA, a new beta-barrel OMP, was identified by searching the genome sequence of strain HB27 for the presence of a C-terminal signature sequence. The structure of TtoA was determined to a resolution of 2.8 A, representing the first crystal structure of an OMP from a thermophilic bacterium. TtoA consists of an eight-stranded beta-barrel with a large extracellular part to which a divalent cation is bound. A five-stranded extracellular beta-sheet protrudes out of the membrane-embedded transmembrane barrel and is stabilized by a disulfide bridge. The edge of this beta-sheet forms crystal contacts that could mimic interactions with other proteins. In modern Gram-negative bacteria, the C-terminal signature sequence of OMPs is required for binding to an Omp85 family protein as a prerequisite for its assembly. We present hints that a similar assembly pathway exists in T. thermophilus by an in vitro binding assay, where unfolded TtoA binds to the Thermus Omp85 family protein TtOmp85, while a mutant without the signature sequence does not.

  17. The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1

    PubMed Central

    Keller, Michael; Taskin, Asli A.; Horvath, Susanne E.; Guan, Xue Li; Prinz, Claudia; Opalińska, Magdalena; Zorzin, Carina; van der Laan, Martin; Wenk, Markus R.; Schubert, Rolf; Wiedemann, Nils; Holzer, Martin

    2015-01-01

    Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein. PMID:26347140

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

    PubMed Central

    Maldonado, R; Wei, R; Kachlany, SC; Kazi, M; Balashova, NV

    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 interleuskin 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. PMID:21443941

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  2. General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae.

    PubMed

    Sandkvist, M; Michel, L O; Hough, L P; Morales, V M; Bagdasarian, M; Koomey, M; DiRita, V J; Bagdasarian, M

    1997-11-01

    The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.

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

    PubMed

    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.

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

    PubMed Central

    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 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 MgCl2. 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. PMID:10788373

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

    PubMed Central

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

    2016-01-01

    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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25755088

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

    PubMed

    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.

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

    PubMed

    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.

  9. Inhibition of lipopolysaccharide transport to the outer membrane in Pseudomonas aeruginosa by peptidomimetic antibiotics.

    PubMed

    Werneburg, Martina; Zerbe, Katja; Juhas, Mario; Bigler, Laurent; Stalder, Urs; Kaech, Andres; Ziegler, Urs; Obrecht, Daniel; Eberl, Leo; Robinson, John A

    2012-08-13

    The asymmetric outer membrane (OM) of Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet and phospholipid in the inner leaflet. During OM biogenesis, LPS is transported from the periplasm into the outer leaflet by a complex comprising the OM proteins LptD and LptE. Recently, a new family of macrocyclic peptidomimetic antibiotics that interact with LptD of the opportunistic human pathogen Pseudomonas aeruginosa was discovered. Here we provide evidence that the peptidomimetics inhibit the LPS transport function of LptD. One approach to monitor LPS transport involved studies of lipid A modifications. Some modifications occur only in the inner membrane while others occur only in the OM, and thus provide markers for LPS transport within the bacterial envelope. We prepared a conditional lptD mutant of P. aeruginosa PAO1 that allowed control of lptD expression from the rhamnose promoter. With this mutant, the effects caused by the antibiotic on the wild-type strain were compared with those caused by depleting LptD in the mutant strain. When LptD was depleted in the mutant, electron microscopy revealed accumulation of membrane-like material within cells and OM blebbing; this mirrored similar effects in the wild-type strain caused by the antibiotic. Moreover, the bacterium responded to the antibiotic, and to depletion of LptD, by introducing the same lipid A modifications, consistent with inhibition by the antibiotic of LptD-mediated LPS transport. This conclusion was further supported by monitoring the radiolabelling of LPS from [¹⁴C]acetate, and by fractionation of IM and OM components. Overall, the results provide support for a mechanism of action for the peptidomimetic antibiotics that involves inhibition of LPS transport to the cell surface. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Antibacterial activity and in vitro evaluation of the biocompatibility of chitosan-based polysaccharide/polyester membranes.

    PubMed

    Wu, Chin-San; Hsu, Yi-Chiang; Liao, Hsin-Tzu; Cai, Yu-Xuan

    2015-12-10

    The antibacterial activity and biocompatibility of membranes of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and chitosan (CS) (PHBV)/CS) were evaluated in this study. Maleic anhydride (MA)-grafted polyhydroxyalkanoate (PHBV-g-MA) was evaluated as an alternative to PHBV. Mouse tail skin fibroblasts (FBs) were seeded on two series of these films to assess cytocompatibility. Collagen and cell proliferation analyses indicated that PHBV, PHBV-g-MA and their composite membranes were biocompatible with respect to FB proliferation. However, FB proliferation, collagen production and the percentage of normal cells growing on PHBV/CS membranes were greater than those for PHBV-g-MA/CS membranes. Cell-cycle and apoptosis assays by FBs on the PHBV-series membrane samples were not affected by DNA content related to damage; i.e. rapid apoptosis/necrosis was not observed, demonstrating the potential of PHBV/CS or PHBV-g-MA/CS membranes for biomedical material applications. Moreover, CS-based polysaccharide enhanced the Escherichia coli (BCRC 10239) antibacterial activity of the membranes. Membranes of PHBV-g-MA or PHBV containing CS-based polysaccharide had better antibacterial activity.

  11. Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli.

    PubMed Central

    Hirai, K; Aoyama, H; Irikura, T; Iyobe, S; Mitsuhashi, S

    1986-01-01

    The mechanism of penetration of quinolones through the bacterial outer membrane was studied with lipopolysaccharide-deficient and porin-deficient mutants. The data indicated that the lipopolysaccharide layer might form a permeability barrier for hydrophobic quinolones such as nalidixic acid but not for hydrophilic quinolones such as norfloxacin and ciprofloxacin. The results also showed that quinolones with a low relative hydrophobicity appeared to permeate through OmpF porin, whereas quinolones with a low relative hydrophobicity appeared to permeate through OmpF porin, whereas quinolones with a high relative hydrophobicity appeared to permeate through both OmpF porin and phospholipid bilayers. PMID:3521490

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

    PubMed

    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.

  13. Immunization with major outer membrane proteins in experimental salmonellosis of mice.

    PubMed Central

    Kuusi, N; Nurminen, M; Saxen, H; Valtonen, M; Mäkelä, P H

    1979-01-01

    Porin (outer membrane protein) preparations extracted from a rough (Rb2) mutant of Salmonella typhimurium proved to be good immunogens in mice and rabbits. The antibody response achieved was measured by using enzyme-linked immunosorbent assay techniques. High titers of both antiporin and antilipopolysaccharide were detected in both species. The rabbit antiserum raised against the porins and the porin preparations themselves had a highly significant protective capacity against intraperitoneal Salmonella infection of mice. Absorption of the rabbit antiporin serum with lipopolysaccharide immunosorbent did not change its protective capacity in a passive immunization experiment, suggesting that the antiporin antibody preparations were the active components. Images PMID:387596

  14. Emerging Roles for Anionic Non-Bilayer Phospholipids in Fortifying the Outer Membrane Permeability Barrier

    PubMed Central

    2014-01-01

    Lately, researchers have been actively investigating Escherichia coli lptD mutants, which exhibit reduced transport of lipopolysaccharide to the cell surface. In this issue of the Journal of Bacteriology, Sutterlin et al. (H. A. Sutterlin, S. Zhang, and T. J. Silhavy, J. Bacteriol. 196:3214–3220, 2014) now reveal an important functional role for phosphatidic acid in fortifying the outer membrane permeability barrier in certain lptD mutant backgrounds. These findings come on the heels of the first reports of two LptD crystal structures, which now provide a structural framework for interpreting lptD genetics. PMID:25022852

  15. Photoelectric signals generated by bovine rod outer segment disk membranes attached to a lecithin bilayer.

    PubMed Central

    Bauer, P J; Bamberg, E; Fahr, A

    1984-01-01

    Purified bovine rod outer segment disk membranes were attached to a lecithin bilayer membrane. After photoexcitation with a 500-nm flash delivered by a dye laser, a negative photovoltage was observed on the bilayer under normal ionic strengths (100 mM KCl), which had a rise phase of 1-3 ms at 20 degrees C. The photoresponse was obviously due to bleaching of rhodopsin as it decreased for successive flashes of light. It originated most probably during the metarhodopsin-I metarhodopsin-II (meta-I-II) transition of rhodopsin because it was pH dependent at 2 degrees C but not at 20 degrees C. At 10 mM KCl, i.e., under hypotonic conditions, a positive photovoltage with slower kinetics than at high salt was observed. As the disk membranes were merely attached to the bilayer membrane, the photovoltage was apparently due to a light-induced transmembrane potential change in the disk membranes. Possible electrogenic mechanisms underlying the photosignal will be discussed. PMID:6743754

  16. Analysis of the SDS-PAGE patterns of outer membrane proteins from Escherichia coli strains that have lost the ability to form K1 antigen and varied in the susceptibility to normal human serum.

    PubMed

    Cisowska, Agnieszka; Bugla-Płoskońska, Gabriela

    2014-01-01

    We used SDS-polyacrylamide gel electrophoresis to investigate the outer membrane proteins (OMPs) band composition of 19 Escherichia coli K1 strains that have spontaneously lost the ability to form K1 polysaccharide capsule (E. coli K1-) and demonstrated different degrees of susceptibility to the bactericidal action of normal human serum. Presented results showed that there were differences between E. coli K1- strains in OMPs expressing capacity. The analysis performed on OMPs has not revealed a direct association between the different OMPs band composition and the susceptibility of these strains to the serum.

  17. Electron crystallography of PhoE porin, an outer membrane, channel- forming protein from E. coli

    SciTech Connect

    Walian, P.J.

    1989-11-01

    One approach to studying the structure of membrane proteins is the use of electron crystallography. Dr. Bing Jap has crystallized PhoE pore-forming protein (porin) from the outer membrane of escherichia coli (E. coli) into monolayer crystals. The findings of this research and those of Jap (1988, 1989) have determined these crystals to be highly ordered, yielding structural information to a resolution of better than 2.8 angstroms. The task of this thesis has been to collect and process the electron diffraction patterns necessary to generate a complete three-dimensional set of high resolution structure factor amplitudes of PhoE porin. Fourier processing of these amplitudes when combined with the corresponding phase data is expected to yield the three-dimensional structure of PhoE porin at better than 3.5 angstroms resolution. 92 refs., 33 figs., 3 tabs. (CBS)

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

    PubMed

    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.

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

    PubMed

    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.

  20. Hybrid Antibiotic Overcomes Resistance in P. aeruginosa by Enhancing Outer Membrane Penetration and Reducing Efflux.

    PubMed

    Gorityala, Bala Kishan; Guchhait, Goutam; Goswami, Sudeep; Fernando, Dinesh M; Kumar, Ayush; Zhanel, George G; Schweizer, Frank

    2016-09-22

    Therapeutic interventions to treat multidrug-resistant (MDR) Pseudomonas aeruginosa infections are severely limited and often require the use of colistin as drug of last resort. The major challenges impeding the development of novel antipseudomonal agents are the lack of cell penetration and extensive efflux. We have discovered a tobramycin-moxifloxacin hybrid core structure which enhances outer membrane permeability and reduces efflux by dissipating the proton motive force of the cytoplasmic membrane in P. aeruginosa. The optimized hybrid protects Galleria mellonella larvae from the lethal effects of MDR P. aeruginosa. Attempts to select for resistance over a period of 25 days resulted in a 2-fold increase in the minimal inhibitory concentration (MIC) for the hybrid, while moxifloxacin or tobramycin resulted in a 16- and 512-fold increase in MIC. Although the hybrid possesses potent activity against MDR, P. aeruginosa isolates the activity that can be synergized when used in combination with other classes of antibiotics.

  1. Restoration of antibody binding to blotted meningococcal outer membrane proteins using various detergents.

    PubMed

    Wedege, E; Bryn, K; Frøholm, L O

    1988-10-04

    Restoration of IgG antibody binding to heat-denatured meningococcal outer membrane proteins has been studied on immunoblots with a series of 14 detergents. Nitrocellulose strips with the blotted proteins were incubated with the detergents and sera from human volunteers vaccinated with meningococcal membrane proteins. Zwitterionic and ionic detergents, containing substituted quarternary ammonium or amino groups with a minimum of 10 C atoms in the alkyl chain, restored the antigenicity of the serotype-specific class 2 porin protein. The concentrations of the Zwittergent detergents necessary for activation decreased with increasing alkyl chain length of the homologues. Only zwitterionic detergents renatured the class 1 protein. Both proteins were weakly antigenic in the presence of the nonionic detergents Triton X-100 and Tween 20. Meningococcal lipopolysaccharide restored antibody binding to the porin, but not to the class 1 protein. Similar concentrations of lipopolysaccharides from two other gram-negative bacteria had no effect.

  2. Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

    PubMed Central

    2011-01-01

    Background The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs) often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs) have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal) for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. Results We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Conclusions Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism. PMID:21272379

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

    PubMed

    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

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

    PubMed

    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.

  5. Dual orientation of the outer membrane lipoprotein P6 of nontypeable haemophilus influenzae.

    PubMed

    Michel, Lea Vacca; Snyder, Joy; Schmidt, Rachel; Milillo, Jennifer; Grimaldi, Kyle; Kalmeta, Breanna; Khan, M Nadeem; Sharma, Sharad; Wright, Leslie Kate; Pichichero, Michael E

    2013-07-01

    The majority of outer membrane (OM) lipoproteins in Gram-negative bacteria are tethered to the membrane via an attached lipid moiety and oriented facing in toward the periplasmic space; a few lipoproteins have been shown to be surface exposed. The outer membrane lipoprotein P6 from the Gram-negative pathogenic bacterium nontypeable Haemophilus influenzae (NTHi) is surface exposed and a leading vaccine candidate for prevention of NTHi infections. However, we recently found that P6 is not a transmembrane protein as previously thought (L. V. Michel, B. Kalmeta, M. McCreary, J. Snyder, P. Craig, M. E. Pichichero, Vaccine 29:1624-1627, 2011). Here we pursued studies to show that P6 has a dual orientation, existing infrequently as surface exposed and predominantly as internally oriented toward the periplasmic space. Flow cytometry using three monoclonal antibodies with specificity for P6 showed surface staining of whole NTHi cells. Confocal microscopy imaging confirmed that antibodies targeted surface-exposed P6 of intact NTHi cells and not internal P6 in membrane-compromised or dead cells. Western blots of two wild-type NTHi strains and a mutant NTHi strain that does not express P6 showed that P6 antibodies do not detect a promiscuous epitope on NTHi. Depletion of targets to nonlipidated P6 significantly decreased bactericidal activity of human serum. Protease digestion of surface-exposed P6 demonstrated that P6 is predominantly internally localized in a manner similar to its homologue Pal in Escherichia coli. We conclude that P6 of NTHi is likely inserted into the OM in two distinct orientations, with the predominant orientation facing in toward the periplasm.

  6. Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria.

    PubMed

    Andersen, C

    2003-01-01

    For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 A into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system.

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

    PubMed

    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. © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  9. Examination of AsmA and its effect on the assembly of Escherichia coli outer membrane proteins.

    PubMed

    Deng, M; Misra, R

    1996-08-01

    asmA mutations were isolated as extragenic suppressors of an OmpF assembly mutant, OmpF315. This suppressor locus produced a protein that was present in extremely low levels and could only be visualized by Western blotting in cells where AsmA expression was induced from a plasmid. Detailed fractionation analyses showed that AsmA localized with the inner membrane. Curiously, however, the mutant OmpF assembly step influenced by AsmA occurred in the outer membrane, perhaps indicating an indirect involvement of AsmA in the assembly of outer membrane proteins. Biochemical examination of the outer membrane showed that asmA null mutations reduce lipopolysaccharide (LPS) levels, thereby lowering the ratios of glycerolphospholipids to LPS and envelope proteins to LPS in the outer membrane. Despite these quantitative alterations, no apparent structural changes in LPS or major phospholipids were noted. Reduced LPS levels in asmA mutants indicate a possible role of AsmA in LPS biogenesis. Data presented in this study suggest that asmA-mediated OmpF assembly suppression may have been achieved by altering the outer membrane fluidity, thus making it more amenable for the assembly of mutant proteins.

  10. PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane.

    PubMed

    Dalebroux, Zachary D; Matamouros, Susana; Whittington, Dale; Bishop, Russell E; Miller, Samuel I

    2014-02-04

    Gram-negative bacteria have two lipid membranes separated by a periplasmic space containing peptidoglycan. The surface bilayer, or outer membrane (OM), provides a barrier to toxic molecules, including host cationic antimicrobial peptides (CAMPs). The OM comprises an outer leaflet of lipid A, the bioactive component of lipopolysaccharide (LPS), and an inner leaflet of glycerophospholipids (GPLs). The structure of lipid A is environmentally regulated in a manner that can promote bacterial infection by increasing bacterial resistance to CAMP and reducing LPS recognition by the innate immune system. The gastrointestinal pathogen, Salmonella Typhimurium, responds to acidic pH and CAMP through the PhoPQ two-component regulatory system, which stimulates lipid A remodeling, CAMP resistance, and intracellular survival within acidified phagosomes. Work here demonstrates that, in addition to regulating lipid A structure, the S. Typhimurium PhoPQ virulence regulators also regulate acidic GPL by increasing the levels of cardiolipins and palmitoylated acylphosphatidylglycerols within the OM. Triacylated palmitoyl-PG species were diminished in strains deleted for the PhoPQ-regulated OM lipid A palmitoyltransferase enzyme, PagP. Purified PagP transferred palmitate to PG consistent with PagP acylation of both lipid A and PG within the OM. Therefore, PhoPQ coordinately regulates OM acidic GPL with lipid A structure, suggesting that GPLs cooperate with lipid A to form an OM barrier critical for CAMP resistance and intracellular survival of S. Typhimurium.

  11. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria.

    PubMed

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-08-02

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance.

  12. OMPcontact: An Outer Membrane Protein Inter-Barrel Residue Contact Prediction Method.

    PubMed

    Zhang, Li; Wang, Han; Yan, Lun; Su, Lingtao; Xu, Dong

    2017-03-01

    In the two transmembrane protein types, outer membrane proteins (OMPs) perform diverse important biochemical functions, including substrate transport and passive nutrient uptake and intake. Hence their 3D structures are expected to reveal these functions. Because experimental structures are scarce, predicted 3D structures are more adapted to OMP research instead, and the inter-barrel residue contact is becoming one of the most remarkable features, improving prediction accuracy by describing the structural information of OMPs. To predict OMP structures accurately, we explored an OMP inter-barrel residue contact prediction method: OMPcontact. Multiple OMP-specific features were integrated in the method, including residue evolutionary covariation, topology-based transmembrane segment relative residue position, OMP lipid layer accessibility, and residue evolution conservation. These features describe the properties of a residue pair in different respects: sequential, structural, evolutionary, and biochemical. Within a 3-residues slide window, a Support Vector Machine (SVM) could accurately determinate the inter-barrel contact residue pair using above features. A 5-fold cross-valuation process was applied in testing the OMPcontact performance against a non-redundant OMP set with 75 samples inside. The tests compared four evolutionary covariation methods and screen analyzed the adaptive ones for inter-barrel contact prediction. The results showed our method not only efficiently realized the prediction, but also scored the possibility for residue pairs reliably. This is expected to improve OMP tertiary structure prediction. Therefore, OMPcontact will be helpful in compiling a structural census of outer membrane protein.

  13. Toward Understanding the Outer Membrane Uptake of Small Molecules by Pseudomonas aeruginosa*

    PubMed Central

    Eren, Elif; Parkin, Jamie; Adelanwa, Ayodele; Cheneke, Belete; Movileanu, Liviu; Khalid, Syma; van den Berg, Bert

    2013-01-01

    Because small molecules enter Gram-negative bacteria via outer membrane (OM) channels, understanding OM transport is essential for the rational design of improved and new antibiotics. In the human pathogen Pseudomonas aeruginosa, most small molecules are taken up by outer membrane carboxylate channel (Occ) proteins, which can be divided into two distinct subfamilies, OccD and OccK. Here we characterize substrate transport mediated by Occ proteins belonging to both subfamilies. Based on the determination of the OccK2-glucuronate co-crystal structure, we identify the channel residues that are essential for substrate transport. We further show that the pore regions of the channels are rigid in the OccK subfamily and highly dynamic in the OccD subfamily. We also demonstrate that the substrate carboxylate group interacts with central residues of the basic ladder, a row of arginine and lysine residues that leads to and away from the binding site at the channel constriction. Moreover, the importance of the basic ladder residues corresponds to their degree of conservation. Finally, we apply the generated insights by converting the archetype of the entire family, OccD1, from a basic amino acid-specific channel into a channel with a preference for negatively charged amino acids. PMID:23467408

  14. Molecular characterization of the 98-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis.

    PubMed Central

    Pettersson, A; van der Ley, P; Poolman, J T; Tommassen, J

    1993-01-01

    When grown under iron limitation, Neisseria meningitidis expresses several additional outer membrane proteins (OMPs), which were studied to assess their vaccine potential. Two monoclonal antibodies were obtained against a 98-kDa OMP of strain 2996 (B:2b:P1.2). Cross-reactivity studies revealed that the two antibodies reacted with 44 and 42 of 74 meningococcal strains, respectively. The antibodies did not block the binding of transferrin or lactoferrin to intact cells. The structural gene for the protein, tentatively designated iroA, was isolated and sequenced. Computer analysis revealed homology to the ferric siderophore receptors in the outer membrane of Escherichia coli and to gonococcal transferrin-binding protein 1 (TbpA). The high degree of cross-reactivity and the results of Southern blot analyses, which showed that the iroA gene is also present in strains that did not react with the monoclonal antibodies, suggest that the 98-kDa OMP is well conserved among meningococci and that it is a suitable vaccine candidate. However, the antibodies were not bactericidal in an in vitro assay with human complement. Images PMID:8406871

  15. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

    PubMed Central

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-01-01

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  2. Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.

    PubMed

    Abel, Ana; Sánchez, Sandra; Arenas, Jesús; Criado, María T; Ferreirós, Carlos M

    2007-03-01

    Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons.

  3. Purification, Refolding, and Crystallization of the Outer Membrane Protein OmpG from Escherichia coli.

    PubMed

    Köster, Stefan; van Pee, Katharina; Yildiz, Özkan

    2015-01-01

    OmpG is a pore-forming protein from E. coli outer membranes. Unlike the classical outer membrane porins, which are trimers, the OmpG channel is a monomeric β-barrel made of 14 antiparallel β-strands with short periplasmic turns and longer extracellular loops. The channel activity of OmpG is pH dependent and the channel is gated by the extracellular loop L6. At neutral/high pH, the channel is open and permeable for substrate molecules with a size up to 900 Da. At acidic pH, loop L6 folds across the channel and blocks the pore. The channel blockage at acidic pH appears to be triggered by the protonation of a histidine pair on neighboring β-strands, which repel one another, resulting in the rearrangement of loop L6 and channel closure. OmpG was purified by refolding from inclusion bodies and crystallized in two and three dimensions. Crystallization and analysis by electron microscopy and X-ray crystallography revealed the fundamental mechanisms essential for the channel activity.

  4. In silico studies of outer membrane of Neisseria meningitidis por a: its expression and immunogenic properties.

    PubMed

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Irani, Shiva

    2014-01-01

    Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N.meningitidis serogroup B. The Class 1 Outer Membrane Protein (OMP) has been named porA which is a cation selective transmembrane protein of 45 KDa that forms trimeric pore in the meningococcal outer membrane. PorA from serogroup B N. meningitidis was cloned into prokaryotic expression vector pBAD-gIIIA. Recombinant protein was expressed with arabinose and affinity purified by Ni-NTA agarose, SDS-PAGE and western blotting were performed for protein determination and verification. BALB/c mice were immunized subcutaneously with purified rPorA together with alum adjuvant. Serum antibody responses to serogroups B N.meningitidis were determined by ELISA. Serum IgG response significantly increased in the group immunized with rPorA together with alum adjuvant in comparison with control groups. These results suggest that rPorA can be a potential vaccine candidate for serogroup B N.meningitidis.

  5. Vaccination with outer membrane vesicles from Francisella noatunensis reduces development of francisellosis in a zebrafish model.

    PubMed

    Brudal, Espen; Lampe, Elisabeth O; Reubsaet, Léon; Roos, Norbert; Hegna, Ida K; Thrane, Ida Marie; Koppang, Erling O; Winther-Larsen, Hanne C

    2015-01-01

    Infection of fish with the facultative intracellular bacterium Francisella noatunensis remains an unresolved problem for aquaculture industry worldwide as it is difficult to vaccinate against without using live attenuated vaccines. Outer membrane vesicles (OMVs) are biological structures shed by Gram-negative bacteria in response to various environmental stimuli. OMVs have successfully been used to vaccinate against both intracellular and extracellular pathogens, due to an ability to stimulate innate, cell-mediated and humoral immune responses. We show by using atomic force and electron microscopy that the fish pathogenic bacterium F. noatunensis subspecies noatunensis (F.n.n.) shed OMVs both in vitro into culture medium and in vivo in a zebrafish infection model. The main protein constituents of the OMV are IglC, PdpD and PdpA, all known Francisella virulence factors, in addition to the outer membrane protein FopA and the chaperonin GroEL, as analyzed by mass spectrometry. The vesicles, when used as a vaccine, reduced proliferation of the bacterium and protected zebrafish when subsequently challenged with a high dose of F.n.n. without causing adverse effects for the host. Also granulomatous responses were reduced in F.n.n.-challenged zebrafish after OMV vaccination. Taken together, the data support the possible use of OMVs as vaccines against francisellosis in fish. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Leptospiral outer membrane protein LipL32 induces inflammation and kidney injury in zebrafish larvae

    PubMed Central

    Chang, Ming-Yang; Cheng, Yi-Chuan; Hsu, Shen-Hsing; Ma, Tsu-Lin; Chou, Li-Fang; Hsu, Hsiang-Hao; Tian, Ya-Chung; Chen, Yung-Chang; Sun, Yuh-Ju; Hung, Cheng-Chieh; Pan, Rong-Long; Yang, Chih-Wei

    2016-01-01

    Leptospirosis is an often overlooked cause of acute kidney injury that can lead to multiple organ failure and even death. The principle protein that conserved in many pathogenic leptospires is the outer membrane protein LipL32. However, the role of LipL32 in the pathogenesis of renal injury in leptospirosis is not entirely clear. Here we studied the effects of LipL32 on the developing kidney in zebrafish larvae. Incubation of zebrafish larvae with Leptospira santarosai serovar Shermani induced acute tubular injury predominantly in the proximal pronephric ducts. Furthermore, microinjection of lipl32 mRNA or recombinant LipL32 protein into zebrafish larvae increased macrophage accumulation and disrupted the basolateral location of NA-K-ATPase in pronephric ducts. These changes led to substantial impairment of the pronephric kidney structure. We further demonstrated that morpholino knockdown of tlr2, but not tlr4, reduced the LipL32-induced leukocyte infiltration and kidney injury. These data demonstrate that LipL32 contributes to the renal pathology in leptospirosis and gives some clues to the potential virulence of LipL32. Our results support the use of zebrafish as a model organism for studying the disease mechanism of leptospiral infection. This model might permit the future exploration of the virulence and molecular pathways of different leptospiral outer membrane proteins. PMID:27278903

  7. Association of the outer membrane protein Omp33 with fitness and virulence of Acinetobacter baumannii.

    PubMed

    Smani, Younes; Dominguez-Herrera, Juan; Pachón, Jerónimo

    2013-11-15

    Outer membrane protein 33 (Omp33) is an outer membrane porin of Acinetobacter baumannii associated with carbapenem resistance. However, the role of Omp33 in the fitness and virulence of A. baumannii remains unknown. In the present study, we investigated the role of Omp33 in fitness and virulence of A. baumannii by using an isogenic knockout strain deficient in the omp33 gene (JPAB02), derived from the ATCC 17978 wild-type (wt). Both in vitro and in vivo defect in the growth rate was found in the JPAB02 strain in competition with the ATCC 17978 wt, highlighting the effect of Omp33 on the metabolic fitness. A significant reduction was observed both in adherence and invasion of human lung epithelial cells and in cytotoxicity of these cells and macrophages with JPAB02. In a murine peritoneal sepsis model, the JPAB02 strain exhibited lower lethal dose 0 (LD0), LD50, and LD100, and dissemination in mice, with reduced bacterial concentration in spleen and lungs. From these data, we concluded that Omp33 plays an important role for fitness and virulence of A. baumannii.

  8. The Roles of Outer Membrane Cytochromes of Shewanella and Geobacter in Extracellular Electron Transfer

    SciTech Connect

    Shi, Liang; Richardson, David; Wang, Zheming; Kerisit, Sebastien N.; Rosso, Kevin M.; Zachara, John M.; Fredrickson, Jim K.

    2009-08-01

    As key components of the electron transfer (ET) pathways used for dissimilatory reduction of solid iron [Fe(III)] and manganese [Mn(IV)] (hydr)oxides, outer membrane cytochromes MtrC and OmcA of Shewanella oneidensis MR-1 and OmcE and OmcS of Geobacter sulfurreducens mediate ET reactions extracellularly. Cell surface-exposed MtrC and OmcA can transfer electrons directly to the metal oxides. S. oneidensis MR-1 cells also secrete flavins that can facilitate ET to the oxides. The secreted flavins are thought to serve either as chelators that form soluble Fe(III)/Mn(IV)-flavin complexes or as electron shuttles that ferry the electrons from cell-associated ET proteins to the metal oxides. Cell-surface localization may also permit MtrC and OmcA to transfer electrons extracellularly to either flavin-chelated Fe(III)/Mn(IV) or oxidized flavins. OmcE and OmcS are proposed to be located on the Geobacter cell surface where they are believed to function as the intermediates to relay electrons to type IV pili, which are then hypothesized to transfer electrons directly to the metal oxides. Thus, cell surface-localization positions these outer membrane cytochromes to transfer electrons to Fe(III)/Mn(IV) oxides external to the bacterial cells either directly, indirectly, or both, demonstrating a common strategy shared by Shewanella and Geobacter for extracellular reduction of the oxides.

  9. In Silico Studies of Outer Membrane of Neisseria Meningitidis Por A: Its Expression and Immunogenic Properties

    PubMed Central

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Irani, Shiva

    2014-01-01

    Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N.meningitidis serogroup B. The Class 1 Outer Membrane Protein (OMP) has been named porA which is a cation selective transmembrane protein of 45 KDa that forms trimeric pore in the meningococcal outer membrane. PorA from serogroup B N. meningitidis was cloned into prokaryotic expression vector pBAD-gIIIA. Recombinant protein was expressed with arabinose and affinity purified by Ni-NTA agarose, SDS-PAGE and western blotting were performed for protein determination and verification. BALB/c mice were immunized subcutaneously with purified rPorA together with alum adjuvant. Serum antibody responses to serogroups B N.meningitidis were determined by ELISA. Serum IgG response significantly increased in the group immunized with rPorA together with alum adjuvant in comparison with control groups. These results suggest that rPorA can be a potential vaccine candidate for serogroup B N.meningitidis. PMID:25317403

  10. Conservation of peptide structure of outer membrane protein-macromolecular complex from Neisseria gonorrhoeae.

    PubMed Central

    Hansen, M V; Wilde, C E

    1984-01-01

    The structural conservation of an outer membrane protein of Neisseria gonorrhoeae called OMP-MC (outer membrane protein-macromolecular complex) was investigated by determining the isoelectric point and amino-terminal amino acid sequence of the protein and by using high-performance liquid chromatography for comparative tryptic peptide mapping. The 76,000-dalton subunits generated by reduction and alkylation of the native 800,000-dalton complex from six test strains focused in ultrathin gels as bands of restricted heterogeneity at an approximate pI of 7.6. Dansyl chloride labeling indicated that all strains shared glycine as the amino-terminal amino acid. Sequence analysis of OMP-MC from two strains revealed no amino acid differences within the first 11 residues. Dual-label peptide maps revealed an extremely high degree of conservation of peptide structure. The results indicate that (i) OMP-MCs isolated from various strains of N. gonorrhoeae share structural homology and (ii) the 800,000-dalton complex is a homopolymer composed of 10 to 12 apparently identical 76,000-dalton subunits. Images PMID:6421738

  11. Solution structure and dynamics of the outer membrane cytochrome OmcF from Geobacter sulfurreducens.

    PubMed

    Dantas, Joana M; Silva, Marta A; Pantoja-Uceda, David; Turner, David L; Bruix, Marta; Salgueiro, Carlos A

    2017-09-01

    Gene knock-out studies on Geobacter sulfurreducens cells showed that the outer membrane-associated monoheme cytochrome OmcF is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI). In addition, microarray analysis of an OmcF-deficient mutant revealed that many of the genes with decreased transcript level were those whose expression is up-regulated in cells grown with a graphite electrode as electron acceptor, suggesting that OmcF also regulates the electron transfer to electrode surfaces and the concomitant electricity production by G. sulfurreducens in microbial fuel cells. (15)N,(13)C-labeled OmcF was produced and NMR spectroscopy was used to determine the solution structure of the protein in the fully reduced state and the pH-dependent conformational changes. In addition, (15)N relaxation NMR experiments were used to characterize the overall and internal backbone dynamics of OmcF. The structure obtained is well-defined, with an average pairwise root mean square deviation of 0.37Å for the backbone atoms and 0.98Å for all heavy atoms. For the first time a solution structure and the protein motions were determined for an outer membrane cytochrome from G. sulfurreducens, which constitutes an important step to understand the extracellular electron transfer mechanism in Geobacter cells. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

    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.

  13. Iron- and molybdenum-repressible outer membrane proteins in competent Azotobacter vinelandii.

    PubMed

    Page, W J; von Tigerstrom, M

    1982-07-01

    Azotobacter vinelandii produced three major proteins of 93,000, 85,000, and 81,000 daltons and a minor 77,000-dalton protein in the outer membrane of Fe-limited cells, and these cells were competent for transformation by DNA. The synthesis of these proteins was repressed in Fe-sufficient medium. Mo limitation of nitrogen-fixing cells resulted in the hyperproduction of a 44,000-dalton protein and the production of a minor 77,000-dalton protein in the outer membrane. Mo limitation enhanced competence in Fe-limited medium and induced competence in Fe-sufficient medium. The 44,000-dalton protein was replaced by a 45,000-dalton protein when Fe-sufficient medium also contained NH4+, but the cells were noncompetent. The synthesis of these proteins was repressed in Mo-sufficient medium and by NH4+ in Fe-limited medium. All of the culture supernatants contained a blue-white fluorescent material (absorbance maximum, 214 nm) which appeared to coordinate Fe3+, Fe2+, MoO4(2-), WO3(2-), and VO3(-).

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

    PubMed

    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.

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

    PubMed

    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.

  16. Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

    PubMed

    Jäger, Jens; Keese, Susanne; Roessle, Manfred; Steinert, Michael; Schromm, Andra B

    2015-05-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells. © 2014 John Wiley & Sons Ltd.

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

    PubMed

    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.

  18. Bacterial Social Networks: Structure and composition of Myxococcus xanthus outer membrane vesicle chains

    PubMed Central

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

    2014-01-01

    Summary The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviors, 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 vesicles and vesicle chains that interconnect cells. We observed peritrichous display of vesicles and vesicle chains and increased abundance in biofilms compared to planktonic cultures. By applying a range of imaging techniques, including 3D Focused Ion Beam Scanning Electron Microscopy (FIB/SEM), we determined these structures to range between 30-60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine (GlcNAc) and N-acetylgalactoseamine (GalNAc) carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl membrane proteins transferred 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 likely provides a mechanism for the coordination of social activities. PMID:23848955

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

    PubMed

    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.

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

    PubMed

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    Day, Philip M.; Potter, Daniel; Inoue, Kentaro

    2014-01-01

    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. PMID:25352854

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

    PubMed

    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

  3. Identification of a Novel Bacterial Outer Membrane Interleukin-1Β-Binding Protein from Aggregatibacter actinomycetemcomitans

    PubMed Central

    Paino, Annamari; Ahlstrand, Tuuli; Nuutila, Jari; Navickaite, Indre; Lahti, Maria; Tuominen, Heidi; Välimaa, Hannamari; Lamminmäki, Urpo; Pöllänen, Marja T.; Ihalin, Riikka

    2013-01-01

    Aggregatibacteractinomycetemcomitans 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 protein for IL-1

  4. Proteolytic Cleavage of the Immunodominant Outer Membrane Protein rOmpA in Rickettsia rickettsii.

    PubMed

    Noriea, Nicholas F; Clark, Tina R; Mead, David; Hackstadt, Ted

    2017-03-15

    Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, contains two immunodominant proteins, rOmpA and rOmpB, in the outer membrane. Both rOmpA and rOmpB are conserved throughout spotted fever group rickettsiae as members of a family of autotransporter proteins. Previously, it was demonstrated that rOmpB is proteolytically processed, with the cleavage site residing near the autotransporter domain at the carboxy-terminal end of the protein, cleaving the 168-kDa precursor into apparent 120-kDa and 32-kDa fragments. The 120- and 32-kDa fragments remain noncovalently associated on the surface of the bacterium, with implications that the 32-kDa fragment functions as the membrane anchor domain. Here we present evidence for a similar posttranslational processing of rOmpA. rOmpA is expressed as a predicted 224-kDa precursor yet is observed on SDS-PAGE as a 190-kDa protein. A small rOmpA fragment of ∼32 kDa was discovered during surface proteome analysis and identified as the carboxy-terminal end of the protein. A rabbit polyclonal antibody was generated to the autotransporter region of rOmpA and confirmed a 32-kDa fragment corresponding to the calculated mass of a proteolytically cleaved rOmpA autotransporter region. N-terminal amino acid sequencing revealed a cleavage site on the carboxy-terminal side of Ser-1958 in rOmpA. An avirulent strain of R. rickettsii Iowa deficient in rOmpB processing was also defective in the processing of rOmpA. The similarities of the cleavage sites and the failure of R. rickettsii Iowa to process either rOmpA or rOmpB suggest that a single enzyme may be responsible for both processing events.IMPORTANCE Members of the spotted fever group of rickettsiae, including R. rickettsii, the etiologic agent of Rocky Mountain spotted fever, express at least four autotransporter proteins that are protective antigens or putative virulence determinants. One member of this class of proteins, rOmpB, is proteolytically processed to a

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

    PubMed

    Paino, Annamari; Ahlstrand, Tuuli; Nuutila, Jari; Navickaite, Indre; Lahti, Maria; Tuominen, Heidi; Välimaa, Hannamari; Lamminmäki, Urpo; Pöllänen, Marja T; Ihalin, Riikka

    2013-01-01

    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 protein for IL-1

  6. Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis.

    PubMed

    Ding, Yi; Fujimoto, L Miya; Yao, Yong; Plano, Gregory V; Marassi, Francesca M

    2015-02-01

    The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis

    PubMed Central

    Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Plano, Gregory V.; Marassi, Francesca M.

    2014-01-01

    The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways. PMID:25433311

  8. Layer-by-layer structured polysaccharides-based multilayers on cellulose acetate membrane: Towards better hemocompatibility, antibacterial and antioxidant activities

    NASA Astrophysics Data System (ADS)

    Peng, Lincai; Li, Hui; Meng, Yahong

    2017-04-01

    The development of multifunctional cellulose acetate (CA) membranes with enhanced hemocompatibility and antibacterial and antioxidant activities is extremely important for biomedical applications. In this work, significant improvements in hemocompatibility and antibacterial and antioxidant activities of cellulose acetate (CA) membranes were achieved via layer-by-layer (LBL) deposition of chitosan (CS) and water-soluble heparin-mimicking polysaccharides (i.e., sulfated Cantharellus cibarius polysaccharides, SCP) onto their surface. The surface chemical compositions, growth manner, surface morphologies, and wetting ability of CS/SCP multilayer-modified CA membranes were characterized, respectively. The systematical evaluation of hemocompatibility revealed that CS/SCP multilayer-modified CA membranes significantly improved blood compatibility including resistance to non-specific protein adsorption, suppression of platelet adhesion and activation, prolongation of coagulation times, inhibition of complement activation, as well as reduction in blood hemolysis. Meanwhile, CS/SCP multilayer-modified CA membranes exhibited strong growth inhibition against Escherichia coli and Staphylococcus aureus, as well as high scavenging abilities against superoxide and hydroxyl radicals. In summary, the CS/SCP multilayers could confer CA membranes with integrated hemocompatibility and antibacterial and antioxidant activities, which might have great potential application in the biomedical field.

  9. Passive immunization to outer membrane proteins MLP and PAL does not protect mice from sepsis.

    PubMed

    Valentine, Catherine H; Hellman, Judith; Beasley-Topliffe, Laura K; Bagchi, Aranya; Warren, H Shaw

    2006-01-01

    Multiple older studies report that immunoglobulin directed to rough mutant bacteria, such as E. coli J5, provides broad protection against challenge with heterologous strains of Gram-negative bacteria. This protection was initially believed to occur through binding of immunoglobulin to bacterial lipopolysaccharide (LPS). However, hundreds of millions of dollars have been invested in attempting to develop clinically-effective anti-LPS monoclonal antibodies without success, and no study has shown that IgG from this antiserum binds LPS. Identification of the protective mechanism would facilitate development of broadly protective human monoclonal antibodies for treating sepsis. IgG from this antiserum binds 2 bacterial outer membrane proteins: murein lipoprotein (MLP) and peptidoglycan-associated lipoprotein (PAL). Both of these outer membrane proteins are highly conserved, have lipid domains that are anchored in the bacterial membrane, are shed from bacteria in blebs together with LPS, and activate cells through Toll-like receptor 2. Our goal in the current work was to determine if passive immunization directed to MLP and PAL protects mice from Gram-negative sepsis. Neither monoclonal nor polyclonal IgG directed to MLP or PAL conferred survival protection in 3 different models of sepsis: cecal ligation and puncture, an infected burn model, and an infected fibrin clot model mimicking peritonitis. Our results are not supportive of the hypothesis that either anti-MLP or anti-PAL IgG are the protective antibodies in the previously described anti-rough mutant bacterial antisera. These studies suggest that a different mechanism of protection is involved.

  10. Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110.

    PubMed

    Manabe, Takayuki; Kato, Mayu; Ueno, Takayuki; Kawasaki, Kiyoshi

    2013-11-08

    Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell-cell communications and host-microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs.

  11. The construction and characterization of Neisseria gonorrhoeae lacking protein III in its outer membrane

    PubMed Central

    1989-01-01

    Protein III (PIII) is a highly conserved, antigenically stable gonococcal outer membrane protein that is closely associated with the major outer membrane protein, protein I (PI). We have previously reported the cloning of the PIII gene. This gene was inserted into the Eco RI site of the runaway plasmid pMOB45. The beta-lactamase (beta la) Bam HI restriction fragment from the gonococcal plasmid pFA3 was inserted at the Xba I site in the PIII gene. The plasmid construct was Hae III methylated and the PIII/beta la insert was excised with Eco RI and used to transform gonococcal strain F62. One beta la+, ampicillin- resistant transformant was isolated and designated 2D. A Western blot of 2D whole cell lysate was probed with affinity-purified polyclonal PIII antisera. No PIII reactivity was detected. Southern blot analysis was performed on F62 and 2D chromosomal DNA that were cut with Eco RI or Cla I. A PIII DNA probe hybridized with fragments 2.2 kb larger in strain 2D than strain F62. This corresponds to the size of the beta la insert. A beta la-specific probe hybridized with the same 2D restriction fragments as above, but did not react with any F62 fragments, confirming that homologous recombination had occurred. There were minimal phenotypic changes between 2D and its parent strain, F62. Chromosomal DNA from 2D was able to transform gonococcal strains F62, UU1, and Pgh 3-2, rendering these PIII-. 2D and other PIII- transformants can now be used to study the role of PIII in gonococcal physiology, metabolism, membrane structure, and pathogenesis. Moreover, we now have organisms from which we can purify gonococcal proteins without PIII contamination. PMID:2499656

  12. Activation of the Complement Classical Pathway (C1q Binding) by Mesophilic Aeromonas hydrophila Outer Membrane Protein

    PubMed Central

    Merino, Susana; Nogueras, Maria Mercedes; Aguilar, Alicia; Rubires, Xavier; Albertí, Sebastian; Benedí, Vicente Javier; Tomás, Juan M.

    1998-01-01

    The mechanism of killing of Aeromonas hydrophila serum-sensitive strains in nonimmune serum by the complement classical pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355–3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic Aeromonas O serogroups. PMID:9673268

  13. Antibiotic Resistance and Regulation of the Gram-Negative Bacterial Outer Membrane Barrier by Host Innate Immune Molecules

    PubMed Central

    2016-01-01

    ABSTRACT The Gram-negative outer membrane is an important barrier that provides protection against toxic compounds, which include antibiotics and host innate immune molecules such as cationic antimicrobial peptides. Recently, significant research progress has been made in understanding the biogenesis, regulation, and functioning of the outer membrane, including a recent paper from the laboratory of Dr. Brett Finlay at the University of British Columbia (J. van der Heijden et al., mBio 7:e01238-16, 2016, http://dx.doi.org/10.1128/mBio.01541-16). These investigators demonstrate that toxic oxygen radicals, such as those found in host tissues, regulate outer membrane permeability by altering the outer membrane porin protein channels to regulate the influx of oxygen radicals as well as β-lactam antibiotics. This commentary provides context about this interesting paper and discusses the prospects of utilizing increased knowledge of outer membrane biology to develop new antibiotics for antibiotic-resistant Gram-negative bacteria. PMID:27677793

  14. Deletions or duplications in the BtuB protein affect its level in the outer membrane of Escherichia coli.

    PubMed Central

    Köster, W; Gudmundsdottir, A; Lundrigan, M D; Seiffert, A; Kadner, R J

    1991-01-01

    The Escherichia coli btuB product is an outer membrane protein that mediates the TonB-coupled active transport of cobalamins and the uptake of the E colicins and bacteriophage BF23. The roles of various segments of the BtuB protein in its function or cellular localization were investigated by analysis of several genetic constructs. Hybrid proteins in which various lengths from the amino terminus of BtuB were linked to alkaline phosphatase (btuB::phoA genes) were all secreted across the cytoplasmic membrane. The BtuB-PhoA proteins that carried up to 327 amino acids of BtuB appeared to reside in the periplasmic space, whereas hybrid proteins containing at least 399 amino acids of BtuB were associated with the outer membrane. Eleven in-frame internal deletion mutations that spanned more than half of the mature sequence were prepared by combining appropriate restriction fragments from btuB variants with 6-bp linker insertions. None of the deleted proteins was able to complement any BtuB functions, and only three of them were detectable in the outer membrane, suggesting that most of the deletions affected sequences needed for stable association with the outer membrane. Duplications covering the same portions of BtuB were prepared in the same manner. All of these partial duplication variants complemented all BtuB functions, although some gave substantially reduced levels of activity. These proteins were found in the outer membrane, although some were subject to proteolytic cleavage within or near the duplicated segment. These results indicate that the insertion of BtuB into the outer membrane requires the presence of several regions of teh BtuB protein and that the presence of extra or redundant segments of the protein can be tolerated during its insertion and function. Images PMID:1885541

  15. Overcoming hysteresis to attain reversible equilibrium folding for outer membrane phospholipase A in phospholipid bilayers

    PubMed Central

    Moon, C. Preston; Kwon, Sarah; Fleming, Karen G.

    2011-01-01

    The free energy of unfolding of a membrane protein from lipids into water (ΔGw,lo) describes its equilibrium thermodynamic stability. Knowing this parameter gives insight into a membrane protein’s sequence-structure-energy relationships. However, there are few measures of membrane protein stability because of the technical difficulties associated with unfolded and partially folded states. Here, we describe experimental process that allowed us to measure the ΔGw,lo of the outer membrane phospholipase A (OmpLA) into large unilamellar vesicles (LUVs) of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). To arrive at this reversible folding condition, we screened a large number of experimental variables: temperature, incubation time, salt concentration, pH, lipid composition as well as liposome morphology. The principal challenge we encountered under most conditions was hysteresis between folding and unfolding titrations. A second factor that compromised reversible folding was the observation that a fraction of the protein population tended to aggregate. We found that hysteresis could be completely eliminated on a feasible timescale by conducting experiments at acidic pH, by the slow dilution of the protein in the initial titration setup and by utilizing a low concentration of a detergent as a temporary “holdase” to solubilize the protein upon its initial dilution into folding conditions. We confirmed that the detergent did not disrupt the LUVs using fluorescence emission of lipid-sensitive dyes and light scattering. The results of our parameter search should be generally useful for efforts to measure of ΔGw,lo for other membrane proteins. PMID:21888919

  16. Isolation and characterization of the outer membrane and lipopolysaccharide from Eikenella corrodens.

    PubMed Central

    Progulske, A; Holt, S C

    1984-01-01

    The chemical composition of the outer membrane fractions (OMFs) of Eikenella corrodens strains 23834 and 470 as well as the strain 23834 lipopolysaccharide (LPS) was determined. The OMFs were obtained by Triton X-100 treatment of the heavier membrane fraction from sucrose density centrifugation of the total membrane fraction. The resulting OMFs of strains 23834 and 470, free of cytoplasmic membrane components, were found to contain 69.6 and 75.0% (wt/wt) protein, 4.8 and 9.2% lipid, 4.6 and 4.7% carbohydrate, and 2.0 and 4.6% muramic acid, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis both OMFs contained one major peptide determined to be 33,500 daltons for the strain 23834 OMF, and 37,500 daltons for the strain 470 OMF. Analysis of the OMF fatty acids revealed hexadecanoic, hexadecenoic, octadecenoic, and lesser amounts of octadecanoic acids. Transmission electron microscopic examination of the OMFs revealed typical large sheets of membrane. Structures (10 nm in diameter) resembling pores were also evident. The E. corrodens LPS was found to be composed of 34.5% (wt/wt) carbohydrate and 25.0% lipid A. Only minute amounts of 2-keto-3-deoxyoctonate and heptose could be detected. Fatty acid analysis revealed primarily octadecanoic and hexadecanoic acids, with lesser amounts of octadecenoic acid. No hydroxy fatty acids were detected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed the E. corrodens LPS to resemble other smooth-type LPSs. Transmission electron microscopic examination revealed a vesicle-like morphology. The E. corrodens LPS appears not to be a "classical," i.e., enteric, type of LPS. Images PMID:6360892

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

    PubMed

    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.

  18. An inner membrane protein N-terminal signal sequence is able to promote efficient localisation of an outer membrane protein in Escherichia coli.

    PubMed Central

    Jackson, M E; Pratt, J M; Stoker, N G; Holland, I B

    1985-01-01

    To test the importance of N-terminal pre-sequences in translocation of different classes of membrane proteins, we exchanged the normal signal sequence of an Escherichia coli outer membrane protein, OmpF, for the pre-sequence of the inner membrane protein, DacA. The DacA-OmpF hybrid was efficiently assembled into the outer membrane in a functionally active form. Thus the pre-sequence of DacA, despite its relatively low hydrophobicity compared with that of OmpF, contains all the essential information necessary to initiate the translocation of OmpF to the outer membrane. Since processing of DacA was also shown to be dependent upon SecA we conclude that the initiation of translocation of this inner membrane polypeptide across the envelope occurs by the same mechanism as outer membrane and periplasmic proteins. The N-terminal 11 amino acids of mature OmpF, which in the hybrid are replaced by the N-terminal nine amino acids of DacA, carry no essential assembly signals since the hybrid protein is apparently assembled with equal efficiency to OmpF. Images Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3908094

  19. Enhancement of macrophage-mediated tumor cell killing by bacterial outer membrane proteins (porins).

    PubMed Central

    Weinberg, J B; Ribi, E; Wheat, R W

    1983-01-01

    Various microbial products are known to influence the function of mouse peritoneal macrophages. Lipopolysaccharide (LPS) and certain lipid A-associated proteins are known to enhance the tumoricidal effects of macrophages. The purpose of this study was to determine whether porins (outer membrane proteins) of Salmonella typhimurium G30/C21 would influence the activity of macrophages from lipid A-responsive and -unresponsive mice. Porins, extracted by a combined sodium dodecyl sulfate-EDTA method from cell walls, were free of LPS as determined by Limulus amebocyte lysate assay and appeared as a band at approximately 36,000 molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In tumor cell killing assays done under LPS-free conditions, the porins in doses of 1 to 10 ng/ml enhanced the tumoricidal effect of macrophages from bacillus Calmette-Guérin-infected C3H/HeN or C3H/HeJ mice. Protein-free LPS enhanced the tumoricidal activity of macrophages from bacillus Calmette-Guérin-infected C3H/HeN but not C3H/HeJ mice. The tumoricidal-enhancing activity of protein-free LPS was blocked by the lipid A-binding antibiotic polymyxin B sulfate, but the effects of porins were not altered by the polymyxin B sulfate. These results suggest that porins, proteins known to alter membrane function, may alter macrophage function by interaction with macrophage membranes. Images PMID:6311745

  20. Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis.

    PubMed

    Roussel, G; Matagne, A; De Bolle, X; Perpète, E A; Michaux, C

    2012-06-01

    Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella β-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-β-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination.

  1. Topographic labelling of pore-forming proteins from the outer membrane of Escherichia coli.

    PubMed Central

    Page, M G; Rosenbusch, J P

    1986-01-01

    The topography of three pore-forming proteins from the outer membrane of Escherichia coli has been explored by using two labelling techniques. Firstly, the distribution of nucleophilic residues has been investigated by selective chemical modification using arylglyoxals (for arginine residues), isothiocyanates (for lysine residues), carbodi-imides (for carboxy residues) and diazonium salts. Secondly, the membrane-embedded domains have been investigated by labelling with photoactivatable phospholipid analogues and a reagent that partitions into the membrane. Few nucleophilic groups are found to be freely accessible to pore-impermeant probes reacting in the aqueous medium. More groups are accessible to small, pore-permeant probes, suggesting that several groups of each sort are contained within the pore. In addition, there appear to be a number of arginine, lysine, carboxyl and many tyrosine residues that are rather inaccessible and that react only with small, hydrophobic probes, if at all. Amongst these more deeply buried residues there are four arginine residues and an as-yet-undetermined number of carboxy residues that appear to be essential to the structural integrity of the oligomeric molecule. Images Fig. 4. PMID:2428354

  2. Phylogenomic analysis supports the ancestral presence of LPS-outer membranes in the Firmicutes

    PubMed Central

    Antunes, Luisa CS; Poppleton, Daniel; Klingl, Andreas; Criscuolo, Alexis; Dupuy, Bruno; Brochier-Armanet, Céline; Beloin, Christophe; Gribaldo, Simonetta

    2016-01-01

    One of the major unanswered questions in evolutionary biology is when and how the transition between diderm (two membranes) and monoderm (one membrane) cell envelopes occurred in Bacteria. The Negativicutes and the Halanaerobiales belong to the classically monoderm Firmicutes, but possess outer membranes with lipopolysaccharide (LPS-OM). Here, we show that they form two phylogenetically distinct lineages, each close to different monoderm relatives. In contrast, their core LPS biosynthesis enzymes were inherited vertically, as in the majority of bacterial phyla. Finally, annotation of key OM systems in the Halanaerobiales and the Negativicutes shows a puzzling combination of monoderm and diderm features. Together, these results support the hypothesis that the LPS-OMs of Negativicutes and Halanaerobiales are remnants of an ancient diderm cell envelope that was present in the ancestor of the Firmicutes, and that the monoderm phenotype in this phylum is a derived character that arose multiple times independently through OM loss. DOI: http://dx.doi.org/10.7554/eLife.14589.001 PMID:27580370

  3. Adaptations in rod outer segment disc membranes in response to environmental lighting conditions.

    PubMed

    Rakshit, Tatini; Senapati, Subhadip; Parmar, Vipul M; Sahu, Bhubanananda; Maeda, Akiko; Park, Paul S-H

    2017-10-01

    The light-sensing rod photoreceptor cell exhibits several adaptations in response to the lighting environment. While adaptations to short-term changes in lighting conditions have been examined in depth, adaptations to long-term changes in lighting conditions are less understood. Atomic force microscopy was used to characterize the structure of rod outer segment disc membranes, the site of photon absorption by the pigment rhodopsin, to better understand how photoreceptor cells respond to long-term lighting changes. Structural properties of the disc membrane changed in response to housing mice in constant dark or light conditions and these adaptive changes required output from the phototransduction cascade initiated by rhodopsin. Among these were changes in the packing density of rhodopsin in the membrane, which was independent of rhodopsin synthesis and specifically affected scotopic visual function as assessed by electroretinography. Studies here support the concept of photostasis, which maintains optimal photoreceptor cell function with implications in retinal degenerations. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia.

    PubMed

    Wang, Wei; Yang, Lianwei; Huang, Xiumin; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Xia, Ningshao; Cheng, Tong; Zhu, Hua

    2017-09-11

    Syncytia formation has been considered important for cell-to-cell spread and pathogenesis of many viruses. As a syncytium forms, individual nuclei often congregate together, allowing close contact of nuclear membranes and possibly fusion to occur. However, there is currently no reported evidence of nuclear membrane fusion between adjacent nuclei in wild-type virus-induced syncytia. Varicella-zoster virus (VZV) is one typical syncytia-inducing virus that causes chickenpox and shingles in humans. Here, we report, for the first time, an interesting observation of apparent fusion of the outer nuclear membranes from juxtaposed nuclei that comprise VZV syncytia both in ARPE-19 human epithelial cells in vitro and in human skin xenografts in the SCID-hu mouse model in vivo. This work reveals a novel aspect of VZV-related cytopathic effect in the context of multinucleated syncytia. Additionally, the information provided by this study could be helpful for future studies on interactions of viruses with host cell nuclei. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed

    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. © 2011 Blackwell Publishing Ltd.

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

    PubMed

    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.

  7. Key diffusion mechanisms involved in regulating bidirectional water permeation across E. coli outer membrane lectin

    PubMed Central

    Sachdeva, Shivangi; Kolimi, Narendar; Nair, Sanjana Anilkumar; Rathinavelan, Thenmalarchelvi

    2016-01-01

    Capsular polysaccharides (CPSs) are major bacterial virulent determinants that facilitate host immune evasion. E. coli group1 K30CPS is noncovalently attached to bacterial surface by Wzi, a lectin. Intriguingly, structure based phylogenetic analysis indicates that Wzi falls into porin superfamily. Molecular dynamics (MD) simulations further shed light on dual role of Wzi as it also functions as a bidirectional passive water specific porin. Such a functional role of Wzi was not realized earlier, due to the occluded pore. While five water specific entry points distributed across extracellular & periplasmic faces regulate the water diffusion involving different mechanisms, a luminal hydrophobic plug governs water permeation across the channel. Coincidently, MD observed open state structure of “YQF” triad is seen in sugar-binding site of sodium-galactose cotransporters, implicating its involvement in K30CPS surface anchorage. Importance of Loop 5 (L5) in membrane insertion is yet another highlight. Change in water diffusion pattern of periplasmic substitution mutants suggests Wzi’s role in osmoregulation by aiding in K30CPS hydration, corroborating earlier functional studies. Water molecules located inside β-barrel of Wzi crystal structure further strengthens the role of Wzi in osmoregulation. Thus, interrupting water diffusion or L5 insertion may reduce bacterial virulence. PMID:27320406

  8. Membrane Frizzled Related Protein is necessary for the normal development and maintenance of photoreceptor outer segments

    PubMed Central

    Won, Jungyeon; Smith, Richard S.; Peachey, Neal S.; Wu, Jiang; Hicks, Wanda L.; Naggert, Jürgen K.; Nishina, Patsy M.

    2009-01-01

    A 4 base pair deletion in a splice donor site of the Mfrp (membrane-type frizzled-related protein) gene, herein referred to as Mfrprd6/rd6, is predicted to lead to the skipping of exon 4 and photoreceptor degeneration in retinal degeneration 6 (rd6) mutant mice. Little, however, is known about the function of the protein or how the mutation causes the degenerative retinal phenotype. Here we examine ultrastructural changes in the retina of Mfrprd6/rd6 mice to determine the earliest effects of the mutation. We also extend the reported observations of the expression pattern of the dicistronic Mfrp/C1qtnf5 message and the localization of these and other retinal pigment epithelium (RPE) and retinal proteins during development and assess the ability of RPE cells to phagocytize outer segments in mutant and WT mice. At the ultrastructural level, outer segments do not develop normally in Mfrprd6/rd6 mutants. They are disorganized and become progressively shorter as mutant mice age. Additionally, there are focal areas in which there is a reduction of apical RPE microvilli. At P25, the rod ERG a-wave of Mfrprd6/rd6 mice is reduced in amplitude by ~50% as are ERG components generated by the RPE. Examination of β-catenin localization and Fos and Tcf-1 expression, intermediates of the canonical Wnt-pathway, showed they were not different between mutant and WT mice, suggesting that MFRP may operate through an alternative pathway. Finally, impaired outer segment phagocytosis was observed in Mfrprd6/rd6 mice both in standard ambient lighting conditions and with bright light exposure when compared to WT controls. PMID:18764959

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

    PubMed

    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.

  10. Pore-forming ability of major outer membrane proteins from Wolinella recta ATCC 33238.

    PubMed Central

    Kennell, W L; Egli, C; Hancock, R E; Holt, S C

    1992-01-01

    Three major outer membrane proteins with apparent molecular masses of 43, 45, and 51 kDa were purified from Wolinella recta ATCC 33238, and their pore-forming abilities were determined by the black lipid bilayer method. The non-heat-modifiable 45-kDa protein (Omp 45) showed no pore-forming activity even at high KCl concentrations. The single-channel conductances in 1 M KCl of the heat-modifiable proteins with apparent molecular masses of 43 kDa (Omp 43) and 51 kDa (Omp 51) were 0.49 and 0.60 nS, respectively. The proteins formed nonselective channels and, as determined by experiments of ion selectivity and zero-current potential, were weakly anion selective. Images PMID:1370429

  11. Identification of an Iron-Regulated, Hemin-Binding Outer Membrane Protein in Sinorhizobium meliloti

    PubMed Central

    Battistoni, Federico; Platero, Raúl; Duran, Rosario; Cerveñansky, Carlos; Battistoni, Julio; Arias, Alicia; Fabiano, Elena

    2002-01-01

    Rhizobia are soil bacteria that are able to establish symbiotic associations with leguminous hosts. In iron-limited environments these bacteria can use iron present in heme or heme compounds (hemoglobin, leghemoglobin). Here we report the presence in Sinorhizobium meliloti of an iron-regulated outer membrane protein that is able to bind hemin but not hemoglobin. Protein assignment was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Tryptic peptides correlated with the mass measurements obtained accounted for 54% of the translated sequence of a putative heme receptor gene present in the chromosome of S. meliloti 1021. The results which we obtained suggest that this protein (designated ShmR for Sinorhizobium heme receptor) is involved in high-affinity heme-mediated iron transport. PMID:12450806

  12. Immunochemical diversity of the major outer membrane protein of avian and mammalian Chlamydia psittaci.

    PubMed Central

    Fukushi, H; Hirai, K

    1988-01-01

    Immunochemical properties of the major outer membrane protein (MOMP) of 16 strains of Chlamydia psittaci isolated from psittacine birds, budgerigars, a pigeon, turkeys, humans, cats, a muskrat, sheep, and cattle and a strain of C. trachomatis, L2/434/Bu, were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblotting analysis with hyperimmunized rabbit antisera to strains of parrot, turkey, feline, and bovine origin. The MOMPs of the strains showed variation in molecular weights and immunological specificities. Fifteen of the C. psittaci strains were classified into two avian and two mammalian types based on immunological specificity of the MOMP, whereas the other strain was not classified in this study. Immunological classification based on specificity of the MOMP by immunoblotting proved to be a valuable method to classify various strains of C. psittaci. Images PMID:3366861

  13. Proteomic analysis of Neisseria lactamica and N eisseria meningitidis outer membrane vesicle vaccine antigens.

    PubMed

    Vaughan, Thomas E; Skipp, Paul J; O'Connor, C David; Hudson, Michael J; Vipond, Richard; Elmore, Michael J; Gorringe, Andrew R

    2006-06-19

    Vaccines to prevent meningococcal disease have been developed from the outer membrane vesicles (OMVs) of Neisseria meningitidis and the related commensal organism Neisseria lactamica. In addition to lipopolysaccharide and the major porins, these vaccines contain a large number of proteins that are incompletely characterised. Here we describe comparative proteomic analyses of the N. lactamica OMV vaccine and OMVs from a serogroup B strain of N. meningitidis. Tandem mass-spectrometry data for trypsinised N. lactamica OMV vaccine were matched to an incompletely assembled genome sequence from the same strain to give 65 robust protein identifications and a further 122 single- or two-peptide matches. Fifty-seven N. meningitidis K454 proteins were identified robustly (and a further 68 from single- or two-peptide matches) by inference from the N. meningitidis MC58 genome. The results suggest that OMVs have a hitherto unappreciated complexity and pinpoint novel candidate antigens for further characterisation.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    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 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. PMID:22289177

  17. Germ-line specific variants of components of the mitochondrial outer membrane import machinery in Drosophila.

    PubMed

    Hwa, Jennifer J; Zhu, Alan J; Hiller, Mark A; Kon, Charlene Y; Fuller, Margaret T; Santel, Ansgar

    2004-08-13

    A search of the Drosophila genome for genes encoding components of the mitochondrial translocase of outer membrane (TOM) complex revealed duplication of genes encoding homologues of Tom20 and Tom40. Tom20 and Tom40 were represented by two differentially expressed homologues in the Drosophila genome. While dtom20 and dtom40 appeared to be expressed ubiquitously, the second variants, called tomboy20 and tomboy40, were expressed only in the male germ-line. Transcripts for tomboy20 and tomboy40 were detected in primary spermatocytes as well as post-meiotic stages. Transcription of tomboy20 and tomboy40 in spermatocytes was not dependent on the transcription factor Cannonball, which is responsible for controlling expression of gene products exclusively required for post-meiotic germ cell differentiation. Epitope-tagging and transient expression of dTom20 and Tomboy40 in mammalian cell culture showed proper targeting to mitochondria.

  18. Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

    PubMed

    Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

    To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.

  19. Antigenic determinants of the chlamydial major outer membrane protein resolved at a single amino acid level.

    PubMed Central

    Zhong, G M; Brunham, R C

    1991-01-01

    Antigenic determinants were identified from seven chlamydial major outer membrane proteins by using overlapping hexapeptides and polyclonal antisera. Sixty-one determinants were detected, and 30 were surface exposed on the native organisms. The two negatively charged residues, aspartic acid and glutamic acid, were found most often in determinants. Thirteen antigenic sites were further characterized by alanine substitution. Differences in fine specificities of these linear determinants were observed in alanine substitution profiles. Five determinants had adjacent critical residues, while eight had critical residues alternated with noncritical residues. Complete replacement analysis of two antigenic determinants provided more detailed information for elucidating the structural basis of the specificity of antigen-antibody interaction and suggested a correlation between sequence conservation and tolerance to amino acid substitution for antigenic sites subject to intense immune selection pressure. PMID:1705241

  20. Studies on the expression of outer membrane protein 2 in escherichia coli.

    PubMed

    Fralick, J A; Diedrich, D L

    1982-01-01

    The relative level of protein 2 expressed in the outer membrane of strains of Escherichia coli K-12 lysogenized with bacteriophage PA-2 was found to be influenced by both the growth temperature and lc+ gene dosage. An increase in either of these parameters was accompanied by an increase in the level of protein 2 up to an apparent saturation level. Any increase in the amount of protein 2 was accompanied by a concomittant decrease in the amount of OmpF and OmpC porins. This inverse relationship led to the maintenance of an approximately constant protein mass per unit of peptidoglycan. Our results are discussed in light of recent genetic studies on the regulation of the OmpF and OmpC porins and can be explained through the competition of these three matrix proteins for a common export or insertion site.

  1. Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture.

    PubMed

    Ding, Zhao-Ying; Wang, Ying-Hsuan; Huang, Yu-Cheng; Lee, Myong-Chol; Tseng, Min-Jen; Chi, Ya-Hui; Huang, Min-Lang

    2017-09-04

    Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. In this study, we identify one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand our understanding of the regulation of LINC complexes and NE architecture. © 2017 Ding et al.

  2. Antigenic Structure of Outer Membrane Protein E of Moraxella catarrhalis and Construction and Characterization of Mutants

    PubMed Central

    Murphy, Timothy F.; Brauer, Aimee L.; Yuskiw, Norine; Hiltke, Thomas J.

    2000-01-01

    Outer membrane protein E (OMP E) is a 50-kDa protein of Moraxella catarrhalis which possesses several characteristics indicating that the protein will be an effective vaccine antigen. To study the antigenic structure of OMP E, eight monoclonal antibodies were developed and characterized. Three of the antibodies recognized epitopes which are present on the bacterial surface. Fusion peptides corresponding to overlapping regions of OMP E were constructed, and immunoblot assays were performed to localize the areas of the molecule bound by the monoclonal antibodies. These studies identified a surface-exposed epitope in the region of amino acids 80 through 180. To further study the protein, two mutants which lack OMP E were constructed. In bactericidal assays, the mutants were more readily killed by normal human serum compared to the isogenic parent strains. These results indicate that OMP E is involved in the expression of serum resistance of M. catarrhalis. PMID:11035732

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

    SciTech Connect

    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.

  4. Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.

    PubMed

    Valentine, Jenny L; Chen, Linxiao; Perregaux, Emily C; Weyant, Kevin B; Rosenthal, Joseph A; Heiss, Christian; Azadi, Parastoo; Fisher, Adam C; Putnam, David; Moe, Gregory R; Merritt, Judith H; DeLisa, Matthew P

    2016-06-23

    The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Immunization with outer membrane vesicles displaying designer glycotopes yields class-switched, glycan-specific antibodies

    PubMed Central

    Valentine, Jenny L.; Chen, Linxiao; Perregaux, Emily C.; Weyant, Kevin B.; Rosenthal, Joseph A.; Heiss, Christian; Azadi, Parastoo; Fisher, Adam C.; Putnam, David; Moe, Gregory R.; Merritt, Judith H.; DeLisa, Matthew P.

    2016-01-01

    Summary The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens. PMID:27341433

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

    PubMed

    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.

  7. Outer membrane biogenesis in Escherichia coli, Neisseria meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori

    PubMed Central

    Liechti, George; Goldberg, Joanna B.

    2012-01-01

    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 OM 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 OM profile limits the effectiveness of vaccines or therapeutics that target 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 (OMPs) 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 OM a