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

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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.

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

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Soni Priya Valeru

2014-01-01

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

Redefining the essential trafficking pathway for outer membrane lipoproteins

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Grabowicz, Marcin; Silhavy, Thomas J.

2017-01-01

In Gram-negative bacteria, most lipoproteins synthesized in the inner membrane (IM) are trafficked to the outer membrane (OM). The Lol pathway is the trafficking paradigm: LolCDE releases lipoproteins from the IM; LolA shuttles them between membranes to LolB in the OM. Several OM lipoproteins are essential for viability. In apparent concordance, the Lol proteins are each essential in wild-type cells. However, we show that Escherichia coli grows well without LolA and LolB in the absence of one...

Structural Aspects of Bacterial Outer Membrane Protein Assembly.

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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.

Exploring bacterial outer membrane barrier to combat bad bugs.

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Ghai, Ishan; Ghai, Shashank

2017-01-01

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

A progenitor of the outer membrane LamB trimer.

OpenAIRE

Stader, J; Silhavy, T J

1988-01-01

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

THE OUTER MEMBRANE OF PATHOGENIC REPRESENTATIVES OF THE LEPTOSPIRA GENIUS

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A. N. Vaganova

2011-01-01

Full Text Available Abstract. Pathogenic leptospires can infect wide spectrum of hosts and they can survive in the environment long time. The outer membrane is the cellular component participated in interaction of microorganisms and environment. In present time several proteins located in the outer membrane of leptospires which are responsible for colonization of host organism, protection from influence of immune system of host, transport of substances in to the cell and other processes have been described. The outer membrane contains proteins and lipopolysaccharide molecules which have citotoxic effect. It was shown that regulation of protein composition of membranes depends on several factors of environment such as temperature, osmolarity, presence of certain substances in environment. Lipopolysaccharide and protein molecules of outer membranes have antigenic properties. These molecules can be used in practice as the components of vaccine against leptospiroses and diagnostic tools. Current review summarize information concerning structural organization of the outer membrane of leptospires, diversities of incoming parts of molecules and regulation of their synthesis. Moreover, perspectives of practical using of the outer membrane components in diagnostics and prevention of leptospiroses are presented.

Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

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Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

2016-10-01

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

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

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

2016-09-23

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

Importance of c-Type cytochromes for U(VI reduction by Geobacter sulfurreducens

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Leang Ching

2007-03-01

Full Text Available Abstract Background In order to study the mechanism of U(VI reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI with acetate serving as the electron donor was investigated. Results The ability of several c-type cytochrome deficient mutants to reduce U(VI was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60% the ability of G. sulfurreducens to reduce U(VI. Involvement in U(VI reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI reduction. A subpopulation of both wild type and U(VI reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III revealed no correlation between the impact of cytochrome deletion on U(VI reduction and reduction of Fe(III hydroxide and chelated Fe(III. Conclusion This study indicates that c-type cytochromes are involved in U(VI reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium

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

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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.

Exploring bacterial outer membrane barrier to combat bad bugs

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Ghai I

2017-08-01

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

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

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Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

2009-12-08

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

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

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Miranda Lo

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

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

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Schiffer Marianne

2005-07-01

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

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

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Kalyan C Nallaparaju

2011-03-01

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

Analysis of long-chain fatty acid binding activity in vesicles of the outer membrane generated from Escherchia coli

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Black, P.N.

1987-01-01

Escherichia coli transports long-chain fatty acids across the dual membrane by a high affinity, saturable, energy-dependent process. The fadL gene codes for an outer membrane protein which appears to act specifically as a long-chain fatty acid binding protein when fatty acid utilization is blocked by mutation. In an effort to understand the function of the fadL gene product, FLP, membranes have been isolated from fadL + and fadL - strains following osmotic lysis. Following isolation, total membranes were separated into inner and outer membrane fractions and assayed for long-chain fatty acid binding activity. Outer membrane vesicles were incubated 2-5 min at 37 0 C with 3 H oleate (C/sub 18:1/), cooled to 0 0 C, and centrifuged through a Lipidex 100 column for 3 min to remove the unbound fatty acid. The level of fatty acid binding was quantitated by scintillation counting of the eluate. Outer membrane vesicles generated from a fadL + strain bind 325 pmol fatty acid/mg protein whereas vesicles generated for a mutant strain bind 175 pmol fatty acid/mg protein. These data suggest that FLP acts at least as a long-chain fatty acid binding protein on the surface of the cell

Structural basis for alginate secretion across the bacterial outer membrane

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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.

Structural Basis for Alginate Secretion Across the Bacterial Outer Membrane

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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.

An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

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Marcin Michalik

Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

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Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S.

2005-01-01

Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V M ) of 3.3 Å 3 Da −1 , corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin

Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coli

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Gu, Jiang; Ji, Xiaowei; Qi, Jianxun; Ma, Ying; Mao, Xuhu; Zou, Quanming

2010-01-01

In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a β-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. The crystals belonged to space group I4 1 32, with unit-cell parameter a = 158.99 Å. The Matthews coefficient and solvent content were calculated to be 2.55 Å 3 Da −1 and 51.77%, respectively, for two molecules in the asymmetric unit

Periplasmic quality control in biogenesis of outer membrane proteins.

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Lyu, Zhi Xin; Zhao, Xin Sheng

2015-04-01

The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

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

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McClafferty Heather

2005-01-01

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

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

International Nuclear Information System (INIS)

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

1987-01-01

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

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

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Paramasivam Nagarajan

2012-09-01

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

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

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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.

Topological analysis of Chlamydia trachomatis L2 outer membrane protein 2

DEFF Research Database (Denmark)

Mygind, P; Christiansen, Gunna; Birkelund, Svend

1998-01-01

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

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

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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.

Substrate specificity within a family of outer membrane carboxylate channels.

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Elif Eren

2012-01-01

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

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

KAUST Repository

Gao, Jie

2015-10-26

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

Membrane fusion proteins of type I secretion system and tripartite efflux pumps share a binding motif for TolC in gram-negative bacteria.

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Minho Lee

Full Text Available The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA. In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed.

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

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George W. Liechti

2012-04-01

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

Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia.

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Wang, Wei; Yang, Lianwei; Huang, Xiumin; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Xia, Ningshao; Cheng, Tong; Zhu, Hua

2017-12-01

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.

Selective imipenem resistance in Pseudomonas aeruginosa associated with diminished outer membrane permeability.

OpenAIRE

Studemeister, A E; Quinn, J P

1988-01-01

The permeability of the outer membranes of imipenem-susceptible and imipenem-resistant clinical isolates of Pseudomonas aeruginosa was investigated by the liposome swelling assay. Sugars and cephaloridine penetrated rapidly, whereas imipenem penetrated poorly into liposomes constructed from porin-rich outer membrane fractions of the resistant isolates.

The outer membrane protein assembly machinery of Neisseria meningitidis

NARCIS (Netherlands)

Volokhina, E.B.|info:eu-repo/dai/nl/304837202

2009-01-01

Gram-negative bacteria are characterized by a cell envelope consisting of an inner membrane (IM) and an outer membrane (OM), which are separated by the peptidoglycan-containing periplasm. While the integral IM proteins are alpha-helical, all but one known integral OM proteins (OMPs) are

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

Science.gov (United States)

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

1997-09-01

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

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

International Nuclear Information System (INIS)

Zhao Haiyan; Sequeira, Reuben D.; Galeva, Nadezhda A.; Tang Liang

2011-01-01

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.

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

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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.

Porphyromonas gingivalis Outer Membrane Vesicles Mediate Coaggregation and Piggybacking of Treponema denticola and Lachnoanaerobaculum saburreum

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Daniel Grenier

2013-01-01

Full Text Available Porphyromonas gingivalis sheds outer membrane vesicles that contain several virulence factors, including adhesins. In this study, we investigated the ability of P. gingivalis outer membrane vesicles to mediate the coaggregation and piggybacking of Treponema denticola and Lachnoanaerobaculum saburreum. Marked coaggregation between T. denticola and L. saburreum occurred in the presence of P. gingivalis outer membrane vesicles. Sucrose was an effective chemoattractant for the motile species T. denticola. The addition of outer membrane vesicles to a mixture of T. denticola and L. saburreum significantly increased the number of nonmotile bacteria that migrated into a sucrose-filled capillary tube immersed in the bacterial mixture. Under optimal conditions, the number of nonmotile L. saburreum in the capillary tube increased approximately 5-fold, whereas no increase occurred when boiled vesicles were used. This study showed that P. gingivalis outer membrane vesicles mediate coaggregation between T. denticola and L. saburreum and that nonmotile bacteria can be translocated by piggybacking on spirochetes.

Expression and distribution of leptospiral outer membrane components during renal infection of hamsters

NARCIS (Netherlands)

Barnett, J. K.; Barnett, D.; Bolin, C. A.; Summers, T. A.; Wagar, E. A.; Cheville, N. F.; Hartskeerl, R. A.; Haake, D. A.

1999-01-01

The outer membrane of pathogenic Leptospira species grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41. The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during

NMR structure of temporin-1 ta in lipopolysaccharide micelles: mechanistic insight into inactivation by outer membrane.

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Rathi Saravanan

Full Text Available BACKGROUND: Antimicrobial peptides (AMPs play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS. The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane. PRINCIPAL FINDINGS: Using NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles. SIGNIFICANCE: We propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a

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

DEFF Research Database (Denmark)

Knudsen, K; Madsen, AS; Mygind, P

1999-01-01

Two genes encoding 97- to 99-kDa Chlamydia pneumoniae VR1310 outer membrane proteins (Omp4 and Omp5) with mutual similarity were cloned and sequenced. The proteins were shown to be constituents of the C. pneumoniae outer membrane complex, and the deduced amino acid sequences were similar to those...

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

Science.gov (United States)

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

2016-12-06

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

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

Science.gov (United States)

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.

In-vivo identification of direct electron transfer from Shewanella oneidensis MR-1 to electrodes via outer-membrane OmcA-MtrCAB protein complexes

Energy Technology Data Exchange (ETDEWEB)

Okamoto, Akihiro [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Nakamura, Ryuhei, E-mail: nakamura@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Hashimoto, Kazuhito, E-mail: hashimoto@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); ERATO/JST, HASHIMOTO Light Energy Conversion Project (Japan)

2011-06-30

Graphical abstract: . Display Omitted Highlights: > Monolayer biofilm of Shewanella cells was prepared on an ITO electrode. > Extracellular electron transfer (EET) process was examined with series of mutants. > Direct ET was confirmed with outer-membrane-bound OmcA-MtrCAB complex. > The EET process was not prominently influenced by capsular polysaccharide. - Abstract: The direct electron-transfer (DET) property of Shewanella bacteria has not been resolved in detail due to the complexity of in vivo electrochemistry in whole-cell systems. Here, we report the in vivo assignment of the redox signal indicative of the DET property in biofilms of Shewanella oneidensis MR-1 by cyclic voltammetry (CV) with a series of mutants and a chemical marking technique. The CV measurements of monolayer biofilms formed by deletion mutants of c-type cytochromes ({Delta}mtrA, {Delta}mtrB, {Delta}mtrC/{Delta}omcA, and {Delta}cymA), and pilin ({Delta}pilD), capsular polysaccharide ({Delta}SO3177) and menaquinone ({Delta}menD) biosynthetic proteins demonstrated that the electrochemical redox signal with a midpoint potential at 50 mV (vs. SHE) was due to an outer-membrane-bound OmcA-MtrCAB protein complex of decaheme cytochromes, and did not involve either inner-membrane-bound CymA protein or secreted menaquinone. Using the specific binding affinity of nitric monoxide for the heme groups of c-type cytochromes, we further confirmed this conclusion. The heterogeneous standard rate constant for the DET process was estimated to be 300 {+-} 10 s{sup -1}, which was two orders of magnitude higher than that previously reported for the electron shuttling process via riboflavin. Experiments using a mutant unable to produce capsular polysaccharide ({Delta}SO3177) revealed that the DET property of the OmcA-MtrCAB complex was not influenced by insulating and hydrophilic extracellular polysaccharide. Accordingly, under physiological conditions, S. oneidensis MR-1 utilizes a high density of outer-membrane

Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism.

Science.gov (United States)

Wu, Si; Ge, Xi; Lv, Zhixin; Zhi, Zeyong; Chang, Zengyi; Zhao, Xin Sheng

2011-09-15

The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.

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

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Deirdre R Ducken

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

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

DEFF Research Database (Denmark)

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

2009-01-01

important physiological role of regulatory RNA molecules in Gram-negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP-sRNA network by showing that the expression of the outer membrane protein YbfM is silenced by a conserved sRNA......In the past few years an increasing number of small non-coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the post-transcriptional level. These RNAs act under various growth and stress conditions, suggesting that one......, designated MicM (also known as RybC/SroB). The regulation is strictly dependent on the RNA chaperone Hfq, and mutational analysis indicates that MicM sequesters the ribosome binding site of ybfM mRNA by an antisense mechanism. Furthermore, we provide evidence that Hfq strongly enhances the on-rate of duplex...

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

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Daan R Speth

2012-08-01

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

[Study on immunogenicity of group A and group C meningococcal conjugate vaccine with coupling group B meningococcal outer membrane protein].

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Ma, Fu-Bao; Tao, Hong; Wang, Hong-Jun

2009-10-01

To evaluate the Immunogenicity of Group A and Group C Meningococcal conjugate Vaccine with coupling Group B Meningococcal Outer Membrane Protein (Men B-OMP). 458 healthy children aged 3-5 months, 6-23 months, 2-6 years and 7-24 years were given the Groups A and C conjugate Vaccine with MenB-OMP or other vaccine as control group to measure the pre-and post-vaccination Men A and C and B by Serum Bactericidal Assay (SBA) in the double-blind randomized controlled trial. 97.65%-100% were 4 times or greater increase in SBA titer for the healthy children given the Groups A and C conjugate Vaccine with MenB-OMP, The geometric mean titer of SBA were 1:194-1:420, which significantly higber than controls. The Group A and C conjugate Vaccine with MenB-OMP was safe and well immunogenic.

c-Type cytochrome-dependent formation of U(IV nanoparticles by Shewanella oneidensis.

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Matthew J Marshall

2006-09-01

Full Text Available Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI complexes in situ, the biomolecular mechanisms of U(VI reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI and formation of extracellular UO(2 nanoparticles. In particular, the outer membrane (OM decaheme cytochrome MtrC (metal reduction, previously implicated in Mn(IV and Fe(III reduction, directly transferred electrons to U(VI. Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO(2 nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS. In wild-type cells, this UO(2-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO(2 nanoparticles with MtrC and OmcA (outer membrane cytochrome. This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO(2 nanoparticles. In the environment, such association of UO(2 nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O(2 or transport in soils and sediments.

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

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Fujita Naoya

2011-01-01

Full Text Available Abstract 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.

The voltage-dependent anion selective channel 1 (VDAC1 topography in the mitochondrial outer membrane as detected in intact cell.

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Marianna F Tomasello

Full Text Available Voltage-Dependent Anion selective Channel maintains the permeability of the outer mitochondrial membrane and is relevant in bioenergetic metabolism and apoptosis. The structure of the protein was shown to be a β-barrel formed by 19 strands. The topology or sideness of the pore has been predicted with various approaches but a general consensus was never reached. This is an important issue since VDAC is considered receptor of Hexokinase and Bcl-2. We fused at VDAC1 C-terminus two tags separated by a caspase cleavage site. Activation in cellulo of caspases was used to eventually separate the two reporters. This experiment did not require the isolation of mitochondria and limited the possibility of outer membrane rupture due to similar procedures. Our results show that the C-terminus end of VDAC faces the mitochondrial inter-membrane space.

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

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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

Adaptation of Salmonella enterica Hadar under static magnetic field: effects on outer membrane protein pattern

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Snoussi Sarra

2012-02-01

Full Text Available Abstract Background Salmonella enterica serovar Hadar (S. Hadar is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments. Results The present study investigated the adaptation of S. Hadar under the effect of acute static magnetic field exposure (200 mT, 9 h and the impact on the outer membrane protein pattern. Via two-dimensional electrophoresis (2-DE and LC-MS/MS spectrometry, we compared the proteome of enriched-outer membrane fraction before and after exposure to a magnetic field. A total of 11 proteins, displaying more than a two-fold change, were differentially expressed in exposed cells, among which 7 were up-regulated and 4 down-regulated. These proteins were involved in the integrity of cell envelope (TolB, Pal, in the response to oxidative stress (OmpW, dihydrolipoamide dehydrogenase, UspF, in the oxidative stress status (bacterioferritin, in virulence (OmpX, Yfgl or in motility (FlgE and UspF. Complementary experiments associated the down-regulation of FlgE and UspF with an alteration of swarming, a flagella-driven motility, under SMF. Furthermore, the antibiotic disc diffusion method confirmed a decrease of gentamicin susceptibility in exposed cells. This decrease could be partly associated with the up-regulation of TolC, outer membrane component of an efflux pump. OmpA, a multifunctional protein, was up-regulated. Conclusions SMF (200 mT seems to maintain the cell envelope integrity and to submit the exposed cells to an oxidative stress. Some alterations suggest an increase of the ability of exposed cells to form biofilms.

Structure analysis of OmpC, one of the major proteins in the outer membrane of E. coli, by high resolution electron microscopy

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Chang, C.F.

1983-07-01

This dissertation is concerned with the structure analysis of a pore-forming membrane protein, OmpC, which is one of the major proteins in the outer membrane of Escherichia coli. In order to obtain structural information it was necessary to develop a suitable technique for preparing two-dimensional crystalline arrays of this membrane protein in an unfixed, unstained and hydrated condition. Electron micrographs were recorded at exposures of less than 5 electrons/A 2 in order to avoid severe radiation damage. The resulting images were crystallographically averaged, in order to overcome the statistical limitations associated with the low electron exposures. The resulting images, which extend to a resolution of approx. 13.5 A, lend themselves to a natural interpretation that is consistent with the mass density of protein, water and lipid, prior data from 2-D and 3-D structure studies of negatively stained specimens at approx. = 20 A resolution, and published spectroscopic data on the peptide chain secondary structure

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

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Goel Vikas

2001-08-01

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

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

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

2016-01-22

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin

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

2015-12-01

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 asmonolayers at the air-water interface, and their properties, aswell 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,butwas prevented fromthis penetration intothemodified lipopolysaccharides.Results correlatewith 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.

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

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Myers Garry

2009-12-01

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

Usher syndrome type 1-associated cadherins shape the photoreceptor outer segment.

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Schietroma, Cataldo; Parain, Karine; Estivalet, Amrit; Aghaie, Asadollah; Boutet de Monvel, Jacques; Picaud, Serge; Sahel, José-Alain; Perron, Muriel; El-Amraoui, Aziz; Petit, Christine

2017-06-05

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis , these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23 , encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15-containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. © 2017 Schietroma et al.

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

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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.

Entry and exit of bacterial outer membrane proteins.

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Misra, Rajeev

2015-08-01

The sites of new outer membrane protein (OMP) deposition and the fate of pre-existing OMPs are still enigmatic despite numerous concerted efforts. Rassam et al. identified mid-cell regions as the primary entry points for new OMP insertion in clusters, driving the pre-existing OMP clusters towards cell poles for long-term storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

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Yang, Chih-Wei; Wu, Mai-Szu; Pan, Ming-Jeng; Hsieh, Wang-Ju; Vandewalle, Alain; Huang, Chiu-Ching

2002-08-01

Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM.

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Bischof, Lisa Franziska; Friedrich, Carmen; Harms, Andrea; Søgaard-Andersen, Lotte; van der Does, Chris

2016-03-25

Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery ofMyxococcus xanthus PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 ofPseudomonas aeruginosaformed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1-16). PilM-N(1-16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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

1995-12-01

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

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

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Appleton Hazel

2010-02-01

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

Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

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Thomas Kieselbach

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

Identification of outer membrane proteins of Yersinia pestis through biotinylation

NARCIS (Netherlands)

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

2007-01-01

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

Differential distribution of proteins and lipids in detergent-resistant and detergent-soluble domains in rod outer segment plasma membranes and disks.

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Elliott, Michael H; Nash, Zack A; Takemori, Nobuaki; Fliesler, Steven J; McClellan, Mark E; Naash, Muna I

2008-01-01

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.

Immunization with r-Lactococcus lactis expressing outer membrane protein A of Shigella dysenteriae type-1: evaluation of oral and intranasal route of administration.

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Yagnik, B; Sharma, D; Padh, H; Desai, P

2017-02-01

To evaluate the comparative immunogenic potential of food grade Lactococcus lactis expressing outer membrane protein A (OmpA) of Shigella dysenteriae type-1 (SD-1) when administered either orally or intranasally. OmpA of SD-1 was cloned and expressed first in Escherichia coli and then in L. lactis. Presence of recombinant gene was confirmed by restriction enzyme digestion and immunoblot analysis. Using immobilized metal affinity chromatography, OmpA was purified from recombinant E. coliBL21 (DE3) and subcutaneously administered in BALB/c mice. Detection of OmpA-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) confirmed the immunogenicity of OmpA. In order to establish r-L. lactis as a mucosal delivery vehicle, it was administered orally and nasally in BALB/c mice. Serum IgG and faecal IgA were assessed through ELISA to compare the relative potential of immunization routes and immunogenic potential of r-L. lactis. Immunization via the oral route proved superior to intranasal exposure. Recombinant L. lactis expressing OmpA of SD-1 was found to be immunogenic. Oral administration of r-L. lactis elicited higher systemic and mucosal immune response when compared with the nasal route. Using food grade recombinant L. lactis has implications in the development of a prophylactic against multidrug-resistant Shigella, which can be used as a prospective vaccine candidate. Evaluating mucosal routes of immunization demonstrated that the oral route of administration elicited better immune response against OmpA of Shigella. © 2016 The Society for Applied Microbiology.

Usher syndrome type 1–associated cadherins shape the photoreceptor outer segment

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Parain, Karine; Aghaie, Asadollah; Picaud, Serge

2017-01-01

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15–containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. PMID:28495838

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

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Niehaus Karsten

2008-06-01

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

Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

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Ponnala, Shivani; Chetty, Chandramu; Veeravalli, Krishna Kumar; Dinh, Dzung H; Klopfenstein, Jeffrey D; Rao, Jasti S

2012-02-01

Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch the glycolytic metabolism of glioma cells to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated OXPHOS to glycolysis switch. The OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to changes in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from the glycolytic to the OXPHOS pathway through an inhibitory effect on Akt, ROS induction and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for the treatment of glioma.

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

Science.gov (United States)

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

2017-01-01

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

Refolding, purification and crystallization of the FrpB outer membrane iron transporter from Neisseria meningitidis

International Nuclear Information System (INIS)

Saleem, Muhammad; Prince, Stephen M.; Patel, Hema; Chan, Hannah; Feavers, Ian M.; Derrick, Jeremy P.

2012-01-01

The refolding, purification and crystallization of FrpB from the meningitis pathogen Neisseria meningitidis is described. FrpB is an integral outer membrane protein from the human pathogen Neisseria meningitidis. It is a member of the TonB-dependent transporter family and promotes the uptake of iron across the outer membrane. There is also evidence that FrpB is an antigen and hence a potential component of a vaccine against meningococcal meningitis. FrpB incorporating a polyhistidine tag was overexpressed in Escherichia coli into inclusion bodies. The protein was then solubilized in urea, refolded and purified to homogeneity. Two separate antigenic variants of FrpB were crystallized by sitting-drop vapour diffusion. Crystals of the F5-1 variant diffracted to 2.4 Å resolution and belonged to space group C2, with unit-cell parameters a = 176.5, b = 79.4, c = 75.9 Å, β = 98.3°. Crystal-packing calculations suggested the presence of a monomer in the asymmetric unit. Crystals of the F3-3 variant also diffracted to 2.4 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 85.3, b = 104.6, c = 269.1 Å. Preliminary analysis suggested the presence of an FrpB trimer in the asymmetric unit

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

Energy Technology Data Exchange (ETDEWEB)

Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

2017-09-01

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

Detergent organisation in crystals of monomeric outer membrane phospholipase A

NARCIS (Netherlands)

Snijder, HJ; Timmins, PA; Kalk, KH; Dijkstra, BW

The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H2O/D2O contrast. From the neutron diffraction at five contrasts,

Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries.

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Hoang, Hanh H; Nickerson, Nicholas N; Lee, Vincent T; Kazimirova, Anastasia; Chami, Mohamed; Pugsley, Anthony P; Lory, Stephen

2011-01-01

In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect

The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM.

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Daniel O Frank

Full Text Available The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM. In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20 by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

Science.gov (United States)

Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

2015-01-01

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

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

DEFF Research Database (Denmark)

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

1997-01-01

The virulence of 18 strains of Vibrio anguillarum serogroup 01 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only str...

Getting to the Outer Leaflet: Physiology of Phosphatidylserine Exposure at the Plasma Membrane.

Science.gov (United States)

Bevers, Edouard M; Williamson, Patrick L

2016-04-01

Phosphatidylserine (PS) is a major component of membrane bilayers whose change in distribution between inner and outer leaflets is an important physiological signal. Normally, members of the type IV P-type ATPases spend metabolic energy to create an asymmetric distribution of phospholipids between the two leaflets, with PS confined to the cytoplasmic membrane leaflet. On occasion, membrane enzymes, known as scramblases, are activated to facilitate transbilayer migration of lipids, including PS. Recently, two proteins required for such randomization have been identified: TMEM16F, a scramblase regulated by elevated intracellular Ca(2+), and XKR8, a caspase-sensitive protein required for PS exposure in apoptotic cells. Once exposed at the cell surface, PS regulates biochemical reactions involved in blood coagulation, and bone mineralization, and also regulates a variety of cell-cell interactions. Exposed on the surface of apoptotic cells, PS controls their recognition and engulfment by other cells. This process is exploited by parasites to invade their host, and in specialized form is used to maintain photoreceptors in the eye and modify synaptic connections in the brain. This review discusses what is known about the mechanism of PS exposure at the surface of the plasma membrane of cells, how actors in the extracellular milieu sense surface exposed PS, and how this recognition is translated to downstream consequences of PS exposure. Copyright © 2016 the American Physiological Society.

Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles

Science.gov (United States)

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

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

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Seong-Woon Yu

2009-10-01

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

Quantitative kinetic analysis of blood vessels in the outer membranes of chronic subdural hematomas

International Nuclear Information System (INIS)

Mori, Kentaro; Adachi, Keiji; Cho, Kajin; Ishimaru, Sumio; Maeda, Minoru

1998-01-01

Dynamic biologic modeling was used to calculate the transfer rate constant for gadolinium-diethylenetriaminepenta-acetic acid (Gd-DTPA) and capillary permeability in the outer membrane of chronic subdural hematomas and effusions. Following intravenous Gd-DTPA injection, Gd concentrations in the subdural fluid and in timed arterial blood samples were measured by ion-coupled plasma emission spectrometry in 53 chronic subdural hematomas and 18 chronic subdural effusions. The capillary surface area in outer membrane was assessed morphometrically. Transfer rate constants for subdural hematomas and subdural effusions were 12.4±1.0 and 20.6±1.7 (x 10 -4 )min -1 , respectively. Capillary permeabilities for subdural hematomas and subdural effusions were 16±1.2 and 19±3.7 ml·min -1 (mm 2 /mm 3 ) -1 , respectively. The capillary surface areas for subdural hematomas and subdural effusions were 48±3 and 77±10 mm 2 /mm 3 , respectively. The high degree of infiltration of Gd into subdural effusions reflects the high capillary surface area in the outer membrane rather than greater permeability of individual capillaries. The value of transfer rate constant was correlated inversely with the duration of the chronic subdural fluid collection. Immature outer membrane has a high transfer rate constant which allows extravasation of plasma components into the subdural space, resulting in increasing volume of the subdural effusion. Delayed magnetic resonance imaging following Gd administration may be clinically useful for estimating the age of chronic subdural fluid accumulations. (author)

Location of macular xanthophylls in the most vulnerable regions of photoreceptor outer-segment membranes.

Science.gov (United States)

Subczynski, Witold K; Wisniewska, Anna; Widomska, Justyna

2010-12-01

Lutein and zeaxanthin are two dietary carotenoids that compose the macular pigment of the primate retina. Another carotenoid, meso-zeaxanthin, is formed from lutein in the retina. A membrane location is one possible site where these dipolar, terminally dihydroxylated carotenoids, named macular xanthophylls, are accumulated in the nerve fibers and photoreceptor outer segments. Macular xanthophylls are oriented perpendicular to the membrane surface, which ensures their high solubility, stability, and significant effects on membrane properties. It was recently shown that they are selectively accumulated in membrane domains that contain unsaturated phospholipids, and thus are located in the most vulnerable regions of the membrane. This location is ideal if they are to act as lipid antioxidants, which is the most accepted mechanism through which lutein and zeaxanthin protect the retina from age-related macular degeneration. In this mini-review, we examine published data on carotenoid-membrane interactions and present our hypothesis that the specific orientation and location of macular xanthophylls maximize their protective action in membranes of the eye retina. Copyright © 2010 Elsevier Inc. All rights reserved.

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

NARCIS (Netherlands)

Waterbeemd, van de B.

2013-01-01

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

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii

International Nuclear Information System (INIS)

Park, Jeong Soon; Lee, Woo Cheol; Choi, Saehae; Yeo, Kwon Joo; Song, Jung Hyun; Han, Young-Hyun; Lee, Je Chul; Kim, Seung Il; Jeon, Young Ho; Cheong, Chaejoon; Kim, Hye-Yeon

2011-01-01

The crystallization of the OmpA periplasmic domain from A. baumannii is described. Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2 1 , with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å 3 Da −1

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

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Anna Kędziora

2016-06-01

Full Text Available 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.

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

NARCIS (Netherlands)

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

2016-01-01

Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen associated molecular patterns including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an

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

DEFF Research Database (Denmark)

Mygind, P; Christiansen, Gunna; Persson, K

1998-01-01

The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated...... fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2aa23-aa93, Chlamydia psittaci-Omp2aa23-aa94, and Chlamydia trachomatis-Omp2aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547). By an enzyme-linked immunosorbent assay with serologically defined...... patient sera, Omp2 was found to be a major immunogen of both C. pneumoniae and C. trachomatis infections (P species-specific anti-Omp2 immunoglobulins were detected....

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

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Tatini Rakshit

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

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

Science.gov (United States)

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

2011-05-01

The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane. © 2011 Blackwell Publishing Ltd.

A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization

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Rhona Kayra Stuart

2014-06-01

Full Text Available Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102 and sync_2523 (strain CC9311 are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins.

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

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Rozina Tabassum

2016-03-01

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

The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

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Ofir Bahar

2014-01-01

Full Text Available Pattern recognition receptors (PRRs play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo. In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity.

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

International Nuclear Information System (INIS)

Reid, D.M.; Molday, R.S.; Friedel, U.; Cook, N.J.

1990-01-01

After neuraminidase treatment the Na + /Ca 2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of M r 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na + /Ca 2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na + /Ca 2+ exchange activation by sodium. The authors further investigated the density of the Na + /Ca 2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na + /Ca 2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as they have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na + /Ca 2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles

Distribution of macular xanthophylls between domains in a model of photoreceptor outer segment membranes.

Science.gov (United States)

Wisniewska, Anna; Subczynski, Witold K

2006-10-15

A model of photoreceptor outer segment (POS) membranes has been proposed, consisting of an equimolar ternary mixture of 1-palmitoyl-2-docosahexaenoylphosphatidylcholine/distearoylphosphatidylcholine/cholesterol. It was shown that, as in membranes made from the raft-forming mixture, in the model of POS membranes, two domains are formed: the raft domain (detergent resistant membranes, DRM), and the bulk domain (detergent soluble membranes, DSM). Saturation-recovery EPR discrimination by oxygen transport method also demonstrated the presence of two domains in this model system in situ at a wide range of temperatures (10-55 degrees C), showing additionally that neither lutein nor zeaxanthin at 1 mol% affect the formation of these domains. These membrane domains have been separated using cold Triton X-100 extraction from a model of POS membranes containing 1 mol% of either lutein or zeaxanthin. The results indicated that the macular xanthophylls lutein and zeaxanthin are substantially excluded from DRM and remain concentrated in DSM, a domain enriched in highly unsaturated docosahexaenoyl acid which is abundant in retina membranes. The concentration of xanthophylls in DRM and DSM calculated as the mol ratio of either xanthophyll to total lipid (phospholipid+cholesterol) was 0.0028 and 0.0391, respectively. Thus, xanthophylls are about 14 times more concentrated in DSM than in DRM. No significant difference in the distribution of lutein and zeaxanthin was found. The obtained results suggest that in POS membranes macular xanthophylls should also be concentrated in domains enriched in polyunsaturated chains.

Annexins as organizers of cholesterol- and sphingomyelin-enriched membrane microdomains in Niemann-Pick type C disease.

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Domon, Magdalena; Nasir, Mehmet Nail; Matar, Gladys; Pikula, Slawomir; Besson, Françoise; Bandorowicz-Pikula, Joanna

2012-06-01

Growing evidence suggests that membrane microdomains enriched in cholesterol and sphingomyelin are sites for numerous cellular processes, including signaling, vesicular transport, interaction with pathogens, and viral infection, etc. Recently some members of the annexin family of conserved calcium and membrane-binding proteins have been recognized as cholesterol-interacting molecules and suggested to play a role in the formation, stabilization, and dynamics of membrane microdomains to affect membrane lateral organization and to attract other proteins and signaling molecules onto their territory. Furthermore, annexins were implicated in the interactions between cytosolic and membrane molecules, in the turnover and storage of cholesterol and in various signaling pathways. In this review, we focus on the mechanisms of interaction of annexins with lipid microdomains and the role of annexins in membrane microdomains dynamics including possible participation of the domain-associated forms of annexins in the etiology of human lysosomal storage disease called Niemann-Pick type C disease, related to the abnormal storage of cholesterol in the lysosome-like intracellular compartment. The involvement of annexins and cholesterol/sphingomyelin-enriched membrane microdomains in other pathologies including cardiac dysfunctions, neurodegenerative diseases, obesity, diabetes mellitus, and cancer is likely, but is not supported by substantial experimental observations, and therefore awaits further clarification.

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

DEFF Research Database (Denmark)

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

2009-01-01

The protein composition and N-terminal sequences of proteins in the outer membrane of Chlamydia trachomatis L2 were analysed following isolation of N-terminal peptides using combined fractional diagonal chromatography and identification by liquid chromatography tandem MS. Acetylation of primary a...

Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.

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Hemavathy Harikrishnan

Full Text Available Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.

Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira.

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Dhandapani, Gunasekaran; Sikha, Thoduvayil; Rana, Aarti; Brahma, Rahul; Akhter, Yusuf; Gopalakrishnan Madanan, Madathiparambil

2018-04-10

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti-leptospiral strategies. © 2018 Wiley Periodicals, Inc.

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

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Lynn G.L. Richardson

2014-06-01

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

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

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

2014-01-01

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

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

DEFF Research Database (Denmark)

Koehler, JF; Birkelund, Svend; Stephens, RS

1992-01-01

The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis....... The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including...

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32.

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Eshghi, Azad; Pinne, Marija; Haake, David A; Zuerner, Richard L; Frank, Ami; Cameron, Caroline E

2012-03-01

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

Unusual Self-Assembly of the Recombinant Chlamydia trachomatis Major Outer Membrane Protein-Based Fusion Antigen CTH522 Into Protein Nanoparticles

DEFF Research Database (Denmark)

Rose, Fabrice; Karlsen, Kasper; Jensen, Pernille

2018-01-01

Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic but is a c......Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic...... but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated......-defined secondary structural elements, and no thermal transitions were measurable. Chemical unfolding resulted monomers that upon removal of the denaturant self-assembled into higher order structures, comparable to the structure of the native protein. The conformation of CTH522 in nanoparticles is thus not entirely...

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

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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

Infectious polymorphic toxins delivered by outer membrane exchange discriminate kin in myxobacteria.

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Vassallo, Christopher N; Cao, Pengbo; Conklin, Austin; Finkelstein, Hayley; Hayes, Christopher S; Wall, Daniel

2017-08-18

Myxobacteria are known for complex social behaviors including outer membrane exchange (OME), in which cells exchange large amounts of outer membrane lipids and proteins upon contact. The TraA cell surface receptor selects OME partners based on a variable domain. However, traA polymorphism alone is not sufficient to precisely discriminate kin. Here, we report a novel family of OME-delivered toxins that promote kin discrimination of OME partners. These SitA lipoprotein toxins are polymorphic and widespread in myxobacteria. Each sitA is associated with a cognate sitI immunity gene, and in some cases a sitB accessory gene. Remarkably, we show that SitA is transferred serially between target cells, allowing the toxins to move cell-to-cell like an infectious agent. Consequently, SitA toxins define strong identity barriers between strains and likely contribute to population structure, maintenance of cooperation, and strain diversification. Moreover, these results highlight the diversity of systems evolved to deliver toxins between bacteria.

OpnS, an outer membrane porin of Xenorhabdus nematophila, confers a competitive advantage for growth in the insect host.

Science.gov (United States)

van der Hoeven, Ransome; Forst, Steven

2009-09-01

The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded beta-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the DeltaopnS strain. Coinjection of the wild-type and DeltaopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or DeltaopnS strain were colonized by the wild-type strain. In addition, the DeltaopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The DeltaopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment.

Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction.

Science.gov (United States)

Beliaev, A S; Saffarini, D A

1998-12-01

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.

Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

International Nuclear Information System (INIS)

Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

2006-01-01

The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials

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

Science.gov (United States)

Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

2016-05-01

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

Comparative Outer Membrane Protein Analysis of High and Low-Invasive Strains of Cronobacter malonaticus

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Maha A. Aldubyan

2017-11-01

Full Text Available Cronobacter are an important group of foodborne pathogens that has been linked to life-threatening infections in both infants and adults. The major infections associated with Cronobacter species are neonatal meningitis, necrotizing enterocolitis, and septicaemia. There are seven species in the Cronobacter genus, of which only three are of clinical importance; Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter turicensis. To date most studies have focussed on C. sakazakii as it is the major species associated with neonatal infections. However, recently C. malonaticus, in particular sequence type 7 (ST7, has been noted as being prevalent in adult infections and therefore warranting further investigation. In this study, eight strains of C. malonaticus ST7, that had been isolated from a wide range of sources and varied in their in vitro virulence, were chosen for proteomic analysis of their outer membrane proteins (OMPs. One-dimensional gel analysis revealed a ~29 kDa size band that was only present in the highly invasive strains. Subsequent mass spectrometric analysis identified several peptides that matched the flagellin protein. The presence of flagellin protein was confirmed in 2D gel spot. Mass spectrometry analysis of total OMPs revealed that the four highly invasive C. malonaticus strains expressed the main flagellum proteins that were absent from the four low invasive strains. These were the flagellar hook protein FlgE, flagellar hook-associated protein 1, flagellar hook-associated protein, flagellin, and flagellar hook-filament junction protein FlgL. This data indicates that C. malonaticus flagellar proteins may have an important role in the organism's invasion properties.

Growth and FIB-SEM analyses of C60 microtubes vertically synthesized on porous alumina membranes

Science.gov (United States)

Miyazawa, Kun'ichi; Kuriyama, Ryota; Shimomura, Shuichi; Wakahara, Takatsugu; Tachibana, Masaru

2014-02-01

The vertical growth of C60 microtubes (C60MTs) on anodic aluminum oxide (AAO) membranes was investigated. The C60MT size dependence on isopropyl alcohol (IPA) injection rate, into C60-saturated toluene solutions through AAO membranes, was measured. A longitudinal section of the interface between a vertically grown C60MT (V-C60MT) and a membrane was prepared by focused ion beam processing, and observed with scanning electron microscopy. No cracking was observed along the interface, suggesting good bonding. V-C60MTs exhibited spiral growth. V-C60MT planar density, wall thickness and aspect ratio all decreased with increasing IPA injection rate. The relationships among length, inner and outer diameters of V-C60MTs were also investigated by varying IPA injection rate.

Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

Science.gov (United States)

Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

2007-06-12

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

Discriminating lysosomal membrane protein types using dynamic neural network.

Science.gov (United States)

Tripathi, Vijay; Gupta, Dwijendra Kumar

2014-01-01

This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

Involvement and necessity of the Cpx regulon in the event of aberrant β-barrel outer membrane protein assembly

Science.gov (United States)

Gerken, Henri; Leiser, Owen P.; Bennion, Drew; Misra, Rajeev

2010-01-01

Summary The Cpx and σE regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the σE pathway monitors the biogenesis of β-barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of β-barrel OMP mis-assembly, by utilizing mutants expressing either a defective β-barrel OMP assembly machinery (Bam) or assembly defective β-barrel OMPs. Analysis of specific mRNAs showed that ΔcpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the σE pathway. The synthetic conditional lethal phenotype of ΔcpxR in mutant Bam or β-barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant β-barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly-defective β-barrel OMP species. Together, these results showed that both the Cpx and σE regulons are required to reduce envelope stress caused by aberrant β-barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression. PMID:20487295

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

NARCIS (Netherlands)

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

2013-01-01

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

Membrane binding properties of EBV gp110 C-terminal domain; evidences for structural transition in the membrane environment

International Nuclear Information System (INIS)

Park, Sung Jean; Seo, Min-Duk; Lee, Suk Kyeong; Lee, Bong Jin

2008-01-01

Gp110 of Epstein-Barr virus (EBV) mainly localizes on nuclear/ER membranes and plays a role in the assembly of EBV nucleocapsid. The C-terminal tail domain (gp110 CTD) is essential for the function of gp110 and the nuclear/ER membranes localization of gp110 is ruled by its C-terminal unique nuclear localization signal (NLS), consecutive four arginines. In the present study, the structural properties of gp110 CTD in membrane mimics were investigated using CD, size-exclusion chromatography, and NMR, to elucidate the effect of membrane environment on the structural transition and to compare the structural feature of the protein in the solution state with that of the membrane-bound form. CD and NMR analysis showed that gp110 CTD in a buffer solution appears to adopt a stable folding intermediate which lacks compactness, and a highly helical structure is formed only in membrane environments. The helical content of gp110 CTD was significantly affected by the negative charge as well as the size of membrane mimics. Based on the elution profiles of the size-exclusion chromatography, we found that gp110 CTD intrinsically forms a trimer, revealing that a trimerization region may exist in the C-terminal domain of gp110 like the ectodomain of gp110. The mutation of NLS (RRRR) to RTTR does not affect the overall structure of gp110 CTD in membrane mimics, while the helical propensity in a buffer solution was slightly different between the wild-type and the mutant proteins. This result suggests that not only the helicity induced in membrane environment but also the local structure around NLS may be related to trafficking to the nuclear membrane. More detailed structural difference between the wild-type and the mutant in membrane environment was examined using synthetic two peptides including the wild-type NLS and the mutant NLS

Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB

Energy Technology Data Exchange (ETDEWEB)

Tanabe, Mikio; Iverson, Tina M.; (Vanderbilt)

2010-01-28

The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P6{sub 3}. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P6{sub 3} crystal forms contained one PorB monomer in the asymmetric unit. Of the three, the P6{sub 3} crystal form had the best diffraction quality, yielding data extending to 2.3 {angstrom} resolution.

Decreased outer membrane permeability in imipenem-resistant mutants of Pseudomonas aeruginosa.

OpenAIRE

Trias, J; Dufresne, J; Levesque, R C; Nikaido, H

1989-01-01

The outer membrane of imipenem-resistant mutants of Pseudomonas aeruginosa was shown to have decreased permeability to imipenem but not to cephaloridine. These experiments were performed with intact cells and liposomes containing imipenem-hydrolyzing beta-lactamase derived from Pseudomonas maltophilia, in both cases utilizing an imipenem concentration of 50 microM. In contrast, liposome swelling assays using imipenem at 8 mM detected no significant difference between the imipenem-resistant mu...

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

Science.gov (United States)

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

Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells.

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Kelly P Nevin

Full Text Available The mechanisms by which Geobacter sulfurreducens transfers electrons through relatively thick (>50 microm biofilms to electrodes acting as a sole electron acceptor were investigated. Biofilms of Geobacter sulfurreducens were grown either in flow-through systems with graphite anodes as the electron acceptor or on the same graphite surface, but with fumarate as the sole electron acceptor. Fumarate-grown biofilms were not immediately capable of significant current production, suggesting substantial physiological differences from current-producing biofilms. Microarray analysis revealed 13 genes in current-harvesting biofilms that had significantly higher transcript levels. The greatest increases were for pilA, the gene immediately downstream of pilA, and the genes for two outer c-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane c-type cytochromes, OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface c-type cytochrome, OmcE, were more abundant and OmcS was less abundant in current-harvesting cells. Strains in which pilA, the gene immediately downstream from pilA, omcB, omcS, omcE, or omcZ was deleted demonstrated that only deletion of pilA or omcZ severely inhibited current production and biofilm formation in current-harvesting mode. In contrast, these gene deletions had no impact on biofilm formation on graphite surfaces when fumarate served as the electron acceptor. These results suggest that biofilms grown harvesting current are specifically poised for electron transfer to electrodes and that, in addition to pili, OmcZ is a key component in electron transfer through differentiated G. sulfurreducens biofilms to electrodes.

The Major Outer Membrane Protein MopB Is Required for Twitching Movement and Affects Biofilm Formation and Virulence in Two Xylella fastidiosa strains.

Science.gov (United States)

Chen, Hongyu; Kandel, Prem P; Cruz, Luisa F; Cobine, Paul A; De La Fuente, Leonardo

2017-11-01

MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a β-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

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

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Louis S Ates

2015-05-01

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

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

KAUST Repository

Ates, Louis S.

2015-05-04

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

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

KAUST Repository

Ates, Louis S.; Ummels, Roy; Commandeur, Susanna; van der Weerd, Robert; Sparrius, Marion; Weerdenburg, Eveline; Alber, Marina; Kalscheuer, Rainer; Piersma, Sander R.; Abdallah, Abdallah; Abd El Ghany, Moataz; Abdel-Haleem, Alyaa M.; Pain, Arnab; Jimé nez, Connie R.; Bitter, Wilbert; Houben, Edith N.G.

2015-01-01

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

Exposure of outer membrane proteins on the surface of Pseudomonas aeruginosa PA01 revealed by labelling with [125I]lactoperoxidase

International Nuclear Information System (INIS)

Lambert, P.A.; Booth, B.R.

1982-01-01

The authors have investigated the exposure of the major outer membrane proteins on the cell surface by treating whole cells of P. aeruginosa with [ 125 I]lactoperoxidase. This reagent catalyses the iodination of tyrosine and histidine residues of proteins in the presence of hydrogen peroxide. It is too large to penetrate the outer membrane (Msub(r) 77500), therefore it is assumed to label only those proteins which have such residues exposed on the cell surface and has been applied to a number of Gram-negative organisms. It is found that F was the major labelled protein, D1 and/or D2 were less heavily labelled, and G was very faintly labelled. In addition, two proteins (Msub(r) 72500 and 38000) which did not appear to be major outer membrane proteins were labelled. (Auth.)

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

KAUST Repository

Sun, Shipeng; Chung, Neal Tai-Shung

2013-01-01

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

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

Science.gov (United States)

Sun, Shi-Peng; Chung, Tai-Shung

2013-11-19

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

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

KAUST Repository

Sun, Shipeng

2013-11-19

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

OpnS, an Outer Membrane Porin of Xenorhabdus nematophila, Confers a Competitive Advantage for Growth in the Insect Host▿ †

Science.gov (United States)

van der Hoeven, Ransome; Forst, Steven

2009-01-01

The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded β-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the ΔopnS strain. Coinjection of the wild-type and ΔopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or ΔopnS strain were colonized by the wild-type strain. In addition, the ΔopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The ΔopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment. PMID:19465651

Molecular biology of Neisseria meningitidis class 5 and H. 8 outer membrane proteins

Energy Technology Data Exchange (ETDEWEB)

Kawula, T.H.

1987-01-01

One of the surface structures responsible for inter- and intrastrain antigenic variability in meningococci is the heat-modifiable class 5 (C.5) protein. Neisseria meningitidis strain FAM18 (a meningococcal disease isolate) expressed two different C.5 proteins (C.5a and C.5b) identifiable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We generated two monoclonal antibodies (MAbs), each specific for one of the identified C.5 proteins. The MAbs, which were bactericidal for variants expressing the appropriate C.5 protein, were used to study C.5 expression changes in FAM18. The H.8 protein is an antigenically conserved outer membrane protein expressed almost exclusively by the pathogenic Neisseria. We have cloned and sequenced an H.8 gene from N. meningitidis FAM18. The predicted H.8 amino acid sequence indicated that the most probable signal peptide processing site matched the consensus prokaryotic lipoprotein processing/modification sequence. We then showed that the H.8 protein could be labeled with {sup 14}C-palmitic acid, confirming that H.8 was a lipoprotein. Processing of the H.8 protein was inhibited by globomycin in E. coli indicating that H.8 was modified by the described lipoprotein processing/modifying pathway described in both gram negative and gram positive genera.

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

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Karin Aistleitner

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

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Science.gov (United States)

Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

1992-01-01

Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197. Images PMID:1447140

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

Science.gov (United States)

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

2017-06-01

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

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

Directory of Open Access Journals (Sweden)

Jennifer M Bomberger

2009-04-01

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

Roles of the Protruding Loop of Factor B Essential for the Localization of Lipoproteins (LolB) in the Anchoring of Bacterial Triacylated Proteins to the Outer Membrane*

Science.gov (United States)

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

2014-01-01

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed. PMID:24569999

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

Science.gov (United States)

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

2014-04-11

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

Large-scale preparation of the homogeneous LolA–lipoprotein complex and efficient in vitro transfer of lipoproteins to the outer membrane in a LolB-dependent manner

OpenAIRE

Watanabe, Shoji; Oguchi, Yuki; Yokota, Naoko; Tokuda, Hajime

2007-01-01

An ATP-binding cassette transporter LolCDE complex of Escherichia coli releases lipoproteins destined to the outer membrane from the inner membrane as a complex with a periplasmic chaperone, LolA. Interaction of the LolA–lipoprotein complex with an outer membrane receptor, LolB, then causes localization of lipoproteins to the outer membrane. As far as examined, formation of the LolA–lipoprotein complex strictly depends on ATP hydrolysis by the LolCDE complex in the presence of LolA. It has be...

Dissection of β-barrel Outer Membrane Protein Assembly Pathways through Characterizing BamA POTRA 1 Mutants of Escherichia coli

Science.gov (United States)

Bennion, Drew; Charlson, Emily S.; Coon, Eric; Misra, Rajeev

2010-01-01

Summary BamA of Escherichia coli is an essential component of the hetero-oligomeric machinery that mediates β-barrel outer membrane protein (OMP) assembly. The C- and N-termini of BamA fold into trans-membrane β-barrel and five soluble POTRA domains, respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β-barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site-specific cross-linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β-barrel OMPs except for TolC, which folds into a unique soluble α-helical barrel and an OM-anchored β-barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalyzed by the trans-membrane β-barrel domain of Bam A. PMID:20598079

Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

Science.gov (United States)

Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

2006-05-01

The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

Science.gov (United States)

Pirbadian, Sahand; Barchinger, Sarah E; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A; Reed, Samantha B; Romine, Margaret F; Saffarini, Daad A; Shi, Liang; Gorby, Yuri A; Golbeck, John H; El-Naggar, Mohamed Y

2014-09-02

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC.

Science.gov (United States)

Sun, Zhan; Bao, Lang; Li, DaoKun; Huang, Bi; Wu, Bingting

2010-09-01

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)-LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx-LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Gilles Phan

2015-11-01

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

Negative Charge Neutralization in the Loops and Turns of Outer Membrane Phospholipase A Impacts Folding Hysteresis at Neutral pH.

Science.gov (United States)

McDonald, Sarah K; Fleming, Karen G

2016-11-08

Hysteresis in equilibrium protein folding titrations is an experimental barrier that must be overcome to extract meaningful thermodynamic quantities. Traditional approaches to solving this problem involve testing a spectrum of solution conditions to find ones that achieve path independence. Through this procedure, a specific pH of 3.8 was required to achieve path independence for the water-to-bilayer equilibrium folding of outer membrane protein OmpLA. We hypothesized that the neutralization of negatively charged side chains (Asp and Glu) at pH 3.8 could be the physical basis for path-independent folding at this pH. To test this idea, we engineered variants of OmpLA with Asp → Asn and Glu → Gln mutations to neutralize the negative charges within various regions of the protein and tested for reversible folding at neutral pH. Although not fully resolved, our results show that these mutations in the periplasmic turns and extracellular loops are responsible for 60% of the hysteresis in wild-type folding. Overall, our study suggests that negative charges impact the folding hysteresis in outer membrane proteins and their neutralization may aid in protein engineering applications.

FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence.

Directory of Open Access Journals (Sweden)

Xiaojun Wu

Full Text Available Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence and FTT0602c (fmvB, which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential

Outer membrane lipoprotein biogenesis: Lol is not the end.

Science.gov (United States)

Konovalova, Anna; Silhavy, Thomas J

2015-10-05

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology. © 2015 The Author(s).

Protective immunity induced by 67 K outer membrane protein of phase I Coxiella burnetii in mice and guinea pigs

International Nuclear Information System (INIS)

Zhang, Y.X.; Zhi, N.; Yu, S.R.; Li, Q.J.; Yu, G.Q.; Zhang, X.

1994-01-01

A 67 K outer membrane protein (OMP) isolated from phase I Coxiella burnetii QiYi strain was purified with monoclonal antibodies (MoAb) coupled to CNBr-Sepharose 4B. Chemical analyses of the 67 K protein showed that it contained seventeen kids of amino acids and no lipopolysaccharides. The immunogenicity and protectivity of the 67 K protein against C. burnetii was evaluated in mice and guinea pigs bi in vitro lymphocyte proliferation assay, delayed-type skin test, antibody conversion rate, and immunization and challenge tests. Intraperitoneal injection of the 67 K protein resulted in antibody production against phase I and II whole cell antigens. The anti-67 K antibody conversion rate was found to be 100% in mice and guinea pigs as well. Lymphocytes were responses in vitro to specific antigen. In addition, delayed-type hypersensitivity appeared two weeks after immunization with the 67 K protein. Moreover, 199% of mice and guinea pigs inoculated with the 67 K protein were protected against a challenge with 10 3 ID 50 virulent C. burnetii. In conclusion, these results demonstrate that the 67 K OMP elicits in vivo and in vitro both B cell-mediated and T cell-mediated immunity in mice and guinea pigs. Thus the 67 K protein is a candidate for an effective subunit vaccine against Q fever. (author)

Iodo-gen-catalysed iodination for identification of surface-exposed outer membrane proteins of Escherichia coli K12

International Nuclear Information System (INIS)

Ferreira, L.C.S.; Almeida, D.F. de

1987-01-01

Surface proteins of Escherichia coli K12 were identified by radiolabelling using 1,3,4,6 - tatrachloro, 3-alpha, 6-alpha - diphenylgycoluryl (Iodo-Gen) and 131 I. Labelled proteins were localized in the outer membrane of the cells. Using this technique it has been possible to observe technique it has been possible to observe that the eletrophoretic pattern of surface proteins changes according to the growth phases in culture. Radiolabelling of E.coli cells inculbated at 42 0 C showed that the syntheses of two surface proteins were temperature-inducible. At least one such protein may be involved in the process of cell division in E.coli K12. (author) [pt

Iodo-gen-catalysed iodination for identification of surface-exposed outer membrane proteins of Escherichia coli K12

Energy Technology Data Exchange (ETDEWEB)

Ferreira, L C.S.; Almeida, D.F. de

1987-12-01

Surface proteins of Escherichia coli K12 were identified by radiolabelling using 1,3,4,6 - tatrachloro, 3-alpha, 6-alpha - diphenylgycoluryl (Iodo-Gen) and /sup 131/I. Labelled proteins were localized in the outer membrane of the cells. Using this technique it has been possible to observe technique it has been possible to observe that the eletrophoretic pattern of surface proteins changes according to the growth phases in culture. Radiolabelling of E.coli cells inculbated at 42/sup 0/C showed that the syntheses of two surface proteins were temperature-inducible. At least one such protein may be involved in the process of cell division in E.coli K12.

Einfluss von Legionella pneumophila outer membrane vesicles auf die bakterielle Replikation in Makrophagen

OpenAIRE

Jung, Anna Lena; Schmeck, Bernd (Prof. Dr.)

2016-01-01

Gramnegative Bakterien treten über die Sekretion verschiedenster Moleküle mit ihrer Umwelt in Kontakt. Die Freisetzung von Proteinen und Nukleinsäuren kann aber nicht nur über die bakteriellen Sekretionssysteme vermittelt werden, sondern auch über outer membrane vesicles (OMVs) erfolgen. Diese kleinen, sphäroiden Membranvesikel werden von allen gramnegativen Bakterien gebildet und können über weite Entfernung wirken, da die zu tra...

Changes in outer membrane proteins of nontypable Haemophilus influenzae in patients with chronic obstructive pulmonary disease

NARCIS (Netherlands)

Groeneveld, K.; van Alphen, L.; Eijk, P. P.; Jansen, H. M.; Zanen, H. C.

1988-01-01

Five individual colonies of Haemophilus influenzae were isolated from each of one to three cultures of sputum collected from 18 patients with chronic obstructive pulmonary disease (COPD). The isolates were studied to investigate whether the major outer membrane proteins (MOMPs) changed during

Band 3 tyrosine kinase in avian erythrocyte plasma membrane is immunologically related to pp60c-src

International Nuclear Information System (INIS)

Hillsgrove, D.; Shores, C.G.; Parker, J.C.; Maness, P.F.

1987-01-01

The authors have identified in the plasma membrane of the chicken erythrocyte a 60-kDa tyrosine-specific protein kinase immunologically related to the transforming protein pp60 v-src of Rous sarcoma virus. The erythrocyte protein kinase phosphorylated heavy chains of tumor-bearing rabbit (TBR) antibodies reactive with pp60 c-src at tyrosine in immune complex protein kinase assays. The kinase was identified as a 60-kDa protein by [ 35 S]methionine labeling of erythrocytes and by autophosphorylation in immune complexes. The kinase migrated on two-dimensional gel electrophoresis with an apparent pI and molecular mass similar to pp60 c-src . A plasma membrane-enriched fraction isolated from chicken red cells contained the majority of the kinase activity. Incubation of the plasma membrane fraction with [ 32 P]ATP resulted in tyrosine phosphorylation of the anion transport protein band 3. Band 3 phosphorylation was blocked by TBR antibodies, indicting that the kinase recognized by pp60 c-src antibodies was responsible for band 3 phosphorylation. These results demonstrate that the avian erythrocyte plasma membrane contains a tightly bound tyrosine-specific protein kinase identical or closely related to pp60 c-src and that this kinase is responsible for band 3 phosphorylation in vitro

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

KAUST Repository

Cheng, Zhen Lei; Li, Xue; Liu, Ying Da; Chung, Neal Tai-Shung

2016-01-01

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

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

KAUST Repository

Cheng, Zhen Lei

2016-01-08

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

A Western-blot assay for the detection of antibodies against pathogenic Leptospira serogroups with recombinant outer membrane protein LipL32

OpenAIRE

Hong-yuan DUAN; Zhi-guo LIU; Shao-fu QIU; Bin HE; Hai ZHAO; Li-hua SONG; Hong ZHU; Qing DUAN

2011-01-01

Objective To provide a possible antigen for rapid serodiagnosis of leptospirosis,the present study focused on the activity of immune-reaction and cross-reaction between outer membrane protein LipL32 and multi-serogroup anti-pathogenic Leptospira antibodies.Methods Based on the given sequence of LipL32 gene of Leptospira icterohaemorrhagiae strain 56601,the primer pair was designed and the DNA fragment was amplified by PCR.The amplified product was inserted into vector pET-28a-(c) to construct...

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

Science.gov (United States)

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

2009-03-01

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

Safety and Immunogenicity Testing of an Intranasal Group B Meningococcal Native Outer Membrane Vesicle Vaccine in Healthy Volunteers

National Research Council Canada - National Science Library

Drabick, Joseph

1998-01-01

An intranasal vaccine composed of native outer membrane vesicles (NOMV) not exposed to detergent or denaturing agents was prepared from the group B meningococcal strain and tested in 32 healthy adult volunteers...

Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria.

Science.gov (United States)

Sperandeo, Paola; Martorana, Alessandra M; Polissi, Alessandra

2017-11-01

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule. In this review we summarize the current knowledge on the LPS transport machinery, discuss the challenges associated with this process and present the solutions that bacterial cells have evolved to address the problem of LPS transport and assembly at the cell surface. Finally, we discuss how knowledge on LPS biogenesis can be translated for the development of novel antimicrobial therapies. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016. Published by Elsevier B.V.

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

Science.gov (United States)

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

2016-11-01

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

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

Directory of Open Access Journals (Sweden)

Elza FT Belo

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

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

KAUST Repository

Le, Ngoc Lieu

2016-01-14

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

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer membrane protein OmpL32

Science.gov (United States)

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer membrane proteins has been shown to modulate the effectiveness of the host immu...

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

NARCIS (Netherlands)

Hofstra, Harmen

1981-01-01

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

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA from Shigella dysenteriae

International Nuclear Information System (INIS)

Brillet, Karl; Meksem, Ahmed; Thompson, Andrew; Cobessi, David

2009-01-01

ShuA from S. dysenteriae was crystallized in several crystallization conditions containing detergents. Adding heavy atoms during crystallization strongly improved the crystal quality and the resolution limits. Diffraction data were collected at an energy remote from the Pb M absorption edges. As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram-negative bacteria, the TonB-dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His 6 tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X-rays beyond 3.5 Å resolution were obtained by co-crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X-ray diffraction data at 2.3 Å resolution using one crystal of ShuA-Pb, and at 3.2 Å resolution at an energy remote from the Pb M absorption edges for phasing on PROXIMA-1 at SOLEIL

Identification and characterization of a novel outer membrane protein receptor required for hemin utilization in Vibrio vulnificus

Science.gov (United States)

Datta, Shreya

2011-01-01

Vibrio vulnificus, the cause of septicemia and serious wound infection in humans and fishes, require iron for its pathogenesis. Hemin uptake through the outer membrane receptor, HupA, is one of its many mechanisms by which it acquires iron. We report here the identification of an additional TonB-dependent hemin receptor HvtA, that is needed in conjunction with the HupA protein for optimal hemin utilization. The HvtA protein is significantly homologous to other outer membrane hemin receptors and its expression in trans restored the uptake of hemin and hemoglobin, the latter to a weaker extent, in a mutant strain that was defective in both receptors. Quantitative RT-PCR suggested that transcription of the hvtA gene was iron regulated. The operon containing the hvtA gene is homologous to the operon in V. cholerae containing the hemin receptor gene hutR suggesting a vertical transmission of the hvtA cluster from V. cholerae to V. vulnificus. PMID:22015545

Cross-reactivity of antibodies against PorA after vaccination with a meningococcal B outer membrane vesicle vaccine

NARCIS (Netherlands)

Vermont, C. L.; van Dijken, H. H.; Kuipers, A. J.; van Limpt, C. J. P.; Keijzers, W. C. M.; van der Ende, A.; de Groot, R.; van Alphen, L.; van den Dobbelsteen, G. P. J. M.

2003-01-01

The cross-reactivity of PorA-specific antibodies induced by a monovalent P1.7-2,4 (MonoMen) and/or a hexavalent (HexaMen) meningococcal B outer membrane vesicle vaccine (OMV) in toddlers and school children was studied by serum bactericidal assays (SBA). First, isogenic vaccine strains and

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

Science.gov (United States)

Wall, Daniel

2014-01-01

Through cooperative interactions, bacteria can build multicellular communities. To ensure that productive interactions occur, bacteria must recognize their neighbours and respond accordingly. Molecular recognition between cells is thus a fundamental behaviour, and in bacteria important discoveries have been made. This MicroReview focuses on a recently described recognition system in myxobacteria that is governed by a polymorphic cell surface receptor called TraA. TraA regulates outer membrane exchange (OME), whereby myxobacterial cells transiently fuse their OMs to efficiently transfer proteins and lipids between cells. Unlike other transport systems, OME is rather indiscriminate in what OM goods are transferred. In contrast, the recognition of partnering cells is discriminatory and only occurs between cells that bear identical or closely related TraA proteins. Therefore TraA functions in kin recognition and, in turn, OME helps regulate social interactions between myxobacteria. Here, I discuss and speculate on the social and evolutionary implications of OME and suggest it helps to guide their transition from free-living cells into coherent and functional populations. © 2013 John Wiley & Sons Ltd.

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

Science.gov (United States)

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

2017-01-01

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

In silico local structure approach: a case study on outer membrane proteins.

Science.gov (United States)

Martin, Juliette; de Brevern, Alexandre G; Camproux, Anne-Claude

2008-04-01

The detection of Outer Membrane Proteins (OMP) in whole genomes is an actual question, their sequence characteristics have thus been intensively studied. This class of protein displays a common beta-barrel architecture, formed by adjacent antiparallel strands. However, due to the lack of available structures, few structural studies have been made on this class of proteins. Here we propose a novel OMP local structure investigation, based on a structural alphabet approach, i.e., the decomposition of 3D structures using a library of four-residue protein fragments. The optimal decomposition of structures using hidden Markov model results in a specific structural alphabet of 20 fragments, six of them dedicated to the decomposition of beta-strands. This optimal alphabet, called SA20-OMP, is analyzed in details, in terms of local structures and transitions between fragments. It highlights a particular and strong organization of beta-strands as series of regular canonical structural fragments. The comparison with alphabets learned on globular structures indicates that the internal organization of OMP structures is more constrained than in globular structures. The analysis of OMP structures using SA20-OMP reveals some recurrent structural patterns. The preferred location of fragments in the distinct regions of the membrane is investigated. The study of pairwise specificity of fragments reveals that some contacts between structural fragments in beta-sheets are clearly favored whereas others are avoided. This contact specificity is stronger in OMP than in globular structures. Moreover, SA20-OMP also captured sequential information. This can be integrated in a scoring function for structural model ranking with very promising results. (c) 2007 Wiley-Liss, Inc.

Segmentation of the Outer Contact on P-Type Coaxial Germanium Detectors

Energy Technology Data Exchange (ETDEWEB)

Hull, Ethan L.; Pehl, Richard H.; Lathrop, James R.; Martin, Gregory N.; Mashburn, R. B.; Miley, Harry S.; Aalseth, Craig E.; Hossbach, Todd W.

2006-09-21

Germanium detector arrays are needed for low-level counting facilities. The practical applications of such user facilities include characterization of low-level radioactive samples. In addition, the same detector arrays can also perform important fundamental physics measurements including the search for rare events like neutrino-less double-beta decay. Coaxial germanium detectors having segmented outer contacts will provide the next level of sensitivity improvement in low background measurements. The segmented outer detector contact allows performance of advanced pulse shape analysis measurements that provide additional background reduction. Currently, n-type (reverse electrode) germanium coaxial detectors are used whenever a segmented coaxial detector is needed because the outer boron (electron barrier) contact is thin and can be segmented. Coaxial detectors fabricated from p-type germanium cost less, have better resolution, and are larger than n-type coaxial detectors. However, it is difficult to reliably segment p-type coaxial detectors because thick (~1 mm) lithium-diffused (hole barrier) contacts are the standard outside contact for p-type coaxial detectors. During this Phase 1 Small Business Innovation Research (SBIR) we have researched the possibility of using amorphous germanium contacts as a thin outer contact of p-type coaxial detectors that can be segmented. We have developed amorphous germanium contacts that provide a very high hole barrier on small planar detectors. These easily segmented amorphous germanium contacts have been demonstrated to withstand several thousand volts/cm electric fields with no measurable leakage current (<1 pA) from charge injection over the hole barrier. We have also demonstrated that the contact can be sputter deposited around and over the curved outside surface of a small p-type coaxial detector. The amorphous contact has shown good rectification properties on the outside of a small p-type coaxial detector. These encouraging

Entry of Porphyromonas gingivalis Outer Membrane Vesicles into Epithelial Cells Causes Cellular Functional Impairment▿

Science.gov (United States)

Furuta, Nobumichi; Takeuchi, Hiroki; Amano, Atsuo

2009-01-01

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis. PMID:19737899

Helicobacter pylori Outer Membrane Protein 18 (Hp1125 Is Involved in Persistent Colonization by Evading Interferon-γ Signaling

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Yuqun Shan

2015-01-01

Full Text Available Outer membrane proteins (OMPs can induce an immune response. Omp18 (HP1125 of H. pylori is a powerful antigen that can induce significant interferon-γ (IFN-γ levels. Previous studies have suggested that IFN-γ plays an important role in H. pylori clearance. However, H. pylori has multiple mechanisms to avoid host immune surveillance for persistent colonization. We generated an omp18 mutant (H. pylori 26695 and H. pylori SS1 strain to examine whether Omp18 interacts with IFN-γ and is involved in H. pylori colonization. qRT-PCR revealed that IFN-γ induced Omp18 expression. qRT-PCR and western blot analysis revealed reduced expressions of virulence factors CagA and NapA in H. pylori 26695 with IFN-γ treatment, but they were induced in the Δomp18 strain. In C57BL/6 mice infected with H. pylori SS1 and the Δomp18 strain, the Δomp18 strain conferred defective colonization and activated a stronger inflammatory response. Signal transducer phosphorylation and transcription 1 (STAT1 activator was downregulated by the wild-type strain but not the Δomp18 strain in IFN-γ-treated macrophages. Furthermore, Δomp18 strain survival rates were poor in macrophages compared to the wild-type strain. We concluded that H. pylori Omp18 has an important function influencing IFN-γ-mediated immune response to participate in persistent colonization.

Outer membrane protein A (OmpA: a new player in shigella flexneri protrusion formation and inter-cellular spreading.

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Cecilia Ambrosi

Full Text Available Outer membrane protein A (OmpA is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92 and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the

Protein secretion and membrane insertion systems in gram-negative bacteria.

Science.gov (United States)

Saier, Milton H

2006-01-01

In contrast to other organisms, gram-negative bacteria have evolved numerous systems for protein export. Eight types are known that mediate export across or insertion into the cytoplasmic membrane, while eight specifically mediate export across or insertion into the outer membrane. Three of the former secretory pathway (SP) systems, type I SP (ISP, ABC), IIISP (Fla/Path) and IVSP (Conj/Vir), can export proteins across both membranes in a single energy-coupled step. A fourth generalized mechanism for exporting proteins across the two-membrane envelope in two distinct steps (which we here refer to as type II secretory pathways [IISP]) utilizes either the general secretory pathway (GSP or Sec) or the twin-arginine targeting translocase for translocation across the inner membrane, and either the main terminal branch or one of several protein-specific export systems for translocation across the outer membrane. We here survey the various well-characterized protein translocation systems found in living organisms and then focus on the systems present in gram-negative bacteria. Comparisons between these systems suggest specific biogenic, mechanistic and evolutionary similarities as well as major differences.

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

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Bunai Christine L

2009-02-01

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

Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector.

Science.gov (United States)

Hoelzle, L E; Hoelzle, K; Wittenbrink, M M

2003-10-01

The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography. The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full-length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.

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

Science.gov (United States)

Winter, Linda E; Barenkamp, Stephen J

2017-10-01

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

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

DEFF Research Database (Denmark)

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

1990-01-01

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

Frames and outer frames for Hilbert C^*-modules

OpenAIRE

Arambašić, Ljiljana; Bakić, Damir

2015-01-01

The goal of the present paper is to extend the theory of frames for countably generated Hilbert $C^*$-modules over arbitrary $C^*$-algebras. In investigating the non-unital case we introduce the concept of outer frame as a sequence in the multiplier module $M(X)$ that has the standard frame property when applied to elements of the ambient module $X$. Given a Hilbert $\\A$-module $X$, we prove that there is a bijective correspondence of the set of all adjointable surjections from the generalize...

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

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H.L. Santos

2002-03-01

Full Text Available SDS, C12E8, CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C12E8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K0.5 = 4.0 µM and low (K0.5 = 1.4 mM affinity sites for ATP, with negative cooperativity. Ouabain (5 mM, oligomycin (1 µg/ml and sodium vanadate (3 µM inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphaß2 association.

Outer- and middle-ear contributions to presbycusis in the Brown Norway rat.

Science.gov (United States)

Gratton, Michael Anne; Bateman, Kristin; Cannuscio, Joseph F; Saunders, James C

2008-01-01

This paper examines the contribution of the outer and middle ears to the hearing loss associated with presbycusis in Brown Norway rats. Animals were formed into two groups; young adults (2-3 months old) and aged animals (approximately 34 months old). Auditory brainstem response (ABR) thresholds were obtained with the outer ear intact or surgically removed. Tympanic membrane (TM) velocity transfer functions were measured from the umbo with the outer ear removed. The length of the auditory meatus, TM surface area, and TM thickness were quantified. The ABR thresholds were 17-26 dB less sensitive in the aged animals between 8.0 and 40.0 kHz when the outer ear was intact. A significant and reliable reduction in the aged rat velocity transfer function of 5-8 dB occurred between 10.0 and 32.0 kHz, while the low frequency velocity response was only a few decibels greater in the younger animals. The ABR threshold differences between young adult and aged ears were compensated by removing the outer/middle ear effects of aging to reveal a purely sensorineural component of presbycusis. The outer and middle ear effects were calculated directly when the ABR and TM velocity data were obtained with the outer ear removed. The outer ear intact condition was modeled in order to compare the ABR data obtained with the outer ear intact with the TM velocity data obtained with the outer removed. With either procedure, removal of the age-related contributions of the outer and middle ear to the ABR threshold resulted in similar age-related ABR threshold shifts between the two age groups. The pure sensorineural threshold shift component of the ABR response was restricted to frequencies between 5.0 and 20.0 kHz and reached a maximum of approximately 15 dB. These results support the conclusion that there is an outer- and middle-ear contribution to the threshold loss defining presbycusis. (c) 2008 S. Karger AG, Basel.

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

International Nuclear Information System (INIS)

Yao, Yong; Dutta, Samit Kumar; Park, Sang Ho; Rai, Ratan; Fujimoto, L. Miya; Bobkov, Andrey A.; Opella, Stanley J.; Marassi, Francesca M.

2017-01-01

The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13 C or 1 H detection, have very narrow line widths (0.40–0.60 ppm for 13 C, 0.11–0.15 ppm for 1 H, and 0.46–0.64 ppm for 15 N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1 H-detected solid-state NMR 1 H/ 15 N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1 H/ 15 N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

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

Science.gov (United States)

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

2014-10-01

Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Mapping of a Protective Epitope of the CopB Outer Membrane Protein of Moraxella catarrhalis

OpenAIRE

Aebi, Christoph; Cope, Leslie D.; Latimer, Jo L.; Thomas, Sharon E.; Slaughter, Clive A.; McCracken, George H.; Hansen, Eric J.

1998-01-01

A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalis in an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003–2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of the ...

Quasar 3C351: VLA maps and a deep search for optical emission in the outer lobes

International Nuclear Information System (INIS)

Kronberg, P.P.; Clarke, J.N.; van den Bergh, S.

1980-01-01

VLA radio maps of the quasar 3C351 (z=0.371) at approx.2'' and 0.''4 resolution (a) show interaction with a relatively dense intergalactic medium, (b) show that there is electron acceleration within at least one of the radio lobes, and (c) imply that the intergalactic gas density is different on one side of the source than on the other. Striking similarities are found between the northern radio lobe of 3C351 and one of the outer hotspots of Cygnus A, and possibly other similar systems, in that the outer, on-axis hotspot is resolved and cusp-shaped, and the ''secondary'' off-axis hotspot is more compact. A search for optical emission in the outer lobes shows no emission stronger than 22/sup m/ in the J band and approx.21/sup m/ in the F band. There is also no evidence at these limits for a cluster of galaxies near the radio source, as is suggested by our conclusion that it is interacting with a medium of typical intracluster density

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

DEFF Research Database (Denmark)

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

2002-01-01

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

Abundance of the multiheme c-type cytochrome OmcB increases in outer biofilm layers of electrode-grown Geobacter sulfurreducens.

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Camille S Stephen

Full Text Available When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB and cytoplasmic acetate kinase (AckA were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode. Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10-20 µm distant from the electrode surface compared to inner layers (0-10 µm. In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.

A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

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Susannah ePiek

2012-12-01

Full Text Available The Gram-negative bacterial cell envelope consists of an inner membrane (IM that surrounds the cytoplasm, and an asymmetrical outer-membrane (OM that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS, phospholipids, outer membrane proteins (OMPs and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the correct biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation and isomerisation pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, these conserved pathways have been modified to suit the lifestyle of each organism.

Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

Science.gov (United States)

Zückert, Wolfram R.

2014-01-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporterlike LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

Redefining the essential trafficking pathway for outer membrane lipoproteins

Science.gov (United States)

Grabowicz, Marcin; Silhavy, Thomas J.

2017-01-01

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins’ key function is to prevent lethal accumulation of mislocalized lipoproteins. PMID:28416660

Redefining the essential trafficking pathway for outer membrane lipoproteins.

Science.gov (United States)

Grabowicz, Marcin; Silhavy, Thomas J

2017-05-02

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins' key function is to prevent lethal accumulation of mislocalized lipoproteins.

Crystallization and preliminary crystallographic characterization of the iron-regulated outer membrane lipoprotein FrpD from Neisseria meningitidis

Czech Academy of Sciences Publication Activity Database

Sviridova, E.; Bumba, Ladislav; Řezáčová, Pavlína; Procházková, Kateřina; Kavan, Daniel; Bezouška, Karel; Kutý, Michal; Šebo, Peter; Kutá-Smatanová, Ivana

2010-01-01

Roč. 66, - (2010), s. 1119-1123 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LC06010; GA ČR GP310/06/P150 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50520514; CEZ:AV0Z60870520; CEZ:AV0Z40550506 Keywords : Fe-regulated protein D * iron-regulated proteins * outer membrane lipoproteins Subject RIV: EC - Immunology Impact factor: 0.563, year: 2010

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

DEFF Research Database (Denmark)

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

2016-01-01

Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have...... ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re...

Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

Science.gov (United States)

Kim, Nak Hyun; Hwang, Byung Kook

2015-01-01

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Open membranes in a constant C-field background and noncommutative boundary strings

International Nuclear Information System (INIS)

Kawamoto, Shoichi; Sasakura, Naoki

2000-01-01

We investigate the dynamics of open membrane boundaries in a constant C-field background. We follow the analysis for open strings in a B-field background, and take some approximations. We find that open membrane boundaries do show noncommutativity in this case by explicit calculations. Membrane boundaries are one dimensional strings, so we face a new type of noncommutativity, that is, noncommutative strings. (author)

Outer membrane biogenesis in Escherichia coli, Neisseria meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori.

Science.gov (United States)

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 associated apparatus of LPS, lipoprotein, and OMP transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to their E. coli counterparts. Eventual

Membrane Targeting of P-type ATPases in Plant Cells

International Nuclear Information System (INIS)

Harper, Jeffrey F.

2004-01-01

How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

Outer measures and weak type estimates of Hardy-Littlewood maximal operators

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Terasawa Yutaka

2006-01-01

Full Text Available We will introduce the times modified centered and uncentered Hardy-Littlewood maximal operators on nonhomogeneous spaces for . We will prove that the times modified centered Hardy-Littlewood maximal operator is weak type bounded with constant when if the Radon measure of the space has "continuity" in some sense. In the proof, we will use the outer measure associated with the Radon measure. We will also prove other results of Hardy-Littlewood maximal operators on homogeneous spaces and on the real line by using outer measures.

Molecular dynamics simulations of outer-membrane protease T from E. coli based on a hybrid coarse-grained/atomistic potential

International Nuclear Information System (INIS)

Neri, Marilisa; Anselmi, Claudio; Carnevale, Vincenzo; Vargiu, Attilio V; Carloni, Paolo

2006-01-01

Outer-membrane proteases T (OmpT) are membrane enzymes used for defense by Gram-negative bacteria. Here we use hybrid molecular mechanics/coarse-grained simulations to investigate the role of large-scale motions of OmpT from Escherichia coli for its function. In this approach, the enzyme active site is treated at the all-atom level, whilst the rest of the protein is described at the coarse-grained level. Our calculations agree well with previously reported all-atom molecular dynamics simulations, suggesting that this approach is well suitable to investigate membrane proteins. In addition, our findings suggest that OmpT large-scale conformational fluctuations might play a role for its biological function, as found for another protease class, the aspartyl proteases

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

Science.gov (United States)

Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

2017-04-28

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro , and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A: Function of the Asn-His interaction in the catalytic triad

NARCIS (Netherlands)

Snijder, H.J.; van Eerde, J.H.; Kalk, K.H.; Dekker, N.; Egmond, M.R.; Dijkstra, B.W.

2010-01-01

Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral-membrane enzyme with a unique His-Ser-Asn catalytic triad. In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate. Here the role of the uncharged

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

Science.gov (United States)

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

2000-01-01

BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum.
RESULTS—Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes.
CONCLUSIONS—AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.


Keywords: liver/kidney microsomal antibody type 1; autoimmunity; autoimmune hepatitis; hepatitis C virus infection; confocal microscopy PMID:10716687

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

Energy Technology Data Exchange (ETDEWEB)

Yao, Yong; Dutta, Samit Kumar [Sanford Burnham Prebys Medical Discovery Institute (United States); Park, Sang Ho; Rai, Ratan [University of California San Diego, Department of Chemistry and Biochemistry (United States); Fujimoto, L. Miya; Bobkov, Andrey A. [Sanford Burnham Prebys Medical Discovery Institute (United States); Opella, Stanley J. [University of California San Diego, Department of Chemistry and Biochemistry (United States); Marassi, Francesca M., E-mail: fmarassi@sbp.edu [Sanford Burnham Prebys Medical Discovery Institute (United States)

2017-03-15

The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with {sup 13}C or {sup 1}H detection, have very narrow line widths (0.40–0.60 ppm for {sup 13}C, 0.11–0.15 ppm for {sup 1}H, and 0.46–0.64 ppm for {sup 15}N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The {sup 1}H-detected solid-state NMR {sup 1}H/{sup 15}N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR {sup 1}H/{sup 15}N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

Outer membrane vesicles enhance the carcinogenic potential of Helicobacter pylori.

Science.gov (United States)

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

2008-12-01

Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.

Ail and PagC-related proteins in the entomopathogenic bacteria of Photorhabdus genus.

Directory of Open Access Journals (Sweden)

Annabelle Mouammine

Full Text Available Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.

Lateral interactions in the photoreceptor membrane: a NMR study

International Nuclear Information System (INIS)

Mollevanger, L.C.P.J.

1987-01-01

The photoreceptor membrane has an exceptionally high content of polyunsaturated fatty acyl chains combined with a high amount of phosphatidyl ethanolamine. It is situated in a cell organelle, the rod outer segment, with a high biological activity in which controlable trans-membrane currents of different ions play an important role. These characteristics make it a very interesting biological membrane to search for the existence of non-bilayer structures. Therefore in this thesis a detailed study of the polymorphic phase behaviour of the rod outer segment photoreceptor lipids was undertaken, concerning modulation of the polymorphic phase behaviour of photoreceptor membrane lipids by divalent cations and temperature, polymorphism of the individual phospholipid classes phosphatidylethanolamine and phosphatidylserine and effects of cholesterol, bilayer stabilization by (rhod)opsin. Morphologically intact rod outer segment possesses a large magnetic anisotropy. This property is used to obtain 31 P-NMR of oriented photoreceptor membranes which allows spectral analysis and identification of individual phospholipid classes, and allows to study lateral lipid diffusion in intact disk membranes. The power of high resolution solid state 13 C-NMR to study the conformation of the chromophore in rhodopsin is demonstrated. (Auth.)

Regulatory protein OmpR influences the serum resistance of Yersinia enterocolitica O:9 by modifying the structure of the outer membrane.

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Karolina Skorek

Full Text Available The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.

Double anisotropic electrically conductive flexible Janus-typed membranes.

Science.gov (United States)

Li, Xiaobing; Ma, Qianli; Tian, Jiao; Xi, Xue; Li, Dan; Dong, Xiangting; Yu, Wensheng; Wang, Xinlu; Wang, Jinxian; Liu, Guixia

2017-12-07

Novel type III anisotropic conductive films (ACFs), namely flexible Janus-typed membranes, were proposed, designed and fabricated for the first time. Flexible Janus-typed membranes composed of ordered Janus nanobelts were constructed by electrospinning, which simultaneously possess fluorescence and double electrically conductive anisotropy. For the fabrication of the Janus-typed membrane, Janus nanobelts comprising a conductive side and an insulative-fluorescent side were primarily fabricated, and then the Janus nanobelts are arranged into parallel arrays using an aluminum rotary drum as the collector to obtain a single anisotropically conductive film. Subsequently, a secondary electrospinning process was applied to the as-prepared single anisotropically conductive films to acquire the final Janus-typed membrane. For this Janus-typed membrane, namely its left-to-right structure, anisotropic electrical conduction synchronously exists on both sides, and furthermore, the two electrically conductive directions are perpendicular. By modulating the amount of Eu(BA) 3 phen complex and conducting polyaniline (PANI), the characteristics and intensity of the fluorescence-electricity dual-function in the membrane can be tuned. The high integration of this peculiar Janus-typed membrane with simultaneous double electrically conductive anisotropy-fluorescent dual-functionality is successfully realized in this study. This design philosophy and preparative technique will provide support for the design and construction of new types of special nanostructures with multi-functionality.

ClC-K chloride channels: emerging pathophysiology of Bartter syndrome type 3.

Science.gov (United States)

Andrini, Olga; Keck, Mathilde; Briones, Rodolfo; Lourdel, Stéphane; Vargas-Poussou, Rosa; Teulon, Jacques

2015-06-15

The mutations in the CLCNKB gene encoding the ClC-Kb chloride channel are responsible for Bartter syndrome type 3, one of the four variants of Bartter syndrome in the genetically based nomenclature. All forms of Bartter syndrome are characterized by hypokalemia, metabolic alkalosis, and secondary hyperaldosteronism, but Bartter syndrome type 3 has the most heterogeneous presentation, extending from severe to very mild. A relatively large number of CLCNKB mutations have been reported, including gene deletions and nonsense or missense mutations. However, only 20 CLCNKB mutations have been functionally analyzed, due to technical difficulties regarding ClC-Kb functional expression in heterologous systems. This review provides an overview of recent progress in the functional consequences of CLCNKB mutations on ClC-Kb chloride channel activity. It has been observed that 1) all ClC-Kb mutants have an impaired expression at the membrane; and 2) a minority of the mutants combines reduced membrane expression with altered pH-dependent channel gating. Although further investigation is needed to fully characterize disease pathogenesis, Bartter syndrome type 3 probably belongs to the large family of conformational diseases, in which the mutations destabilize channel structure, inducing ClC-Kb retention in the endoplasmic reticulum and accelerated channel degradation. Copyright © 2015 the American Physiological Society.

A genetic screen in Myxococcus xanthus identifies mutants that uncouple outer membrane exchange from a downstream cellular response.

Science.gov (United States)

Dey, Arup; Wall, Daniel

2014-12-01

Upon physical contact with sibling cells, myxobacteria transiently fuse their outer membranes (OMs) and exchange OM proteins and lipids. From previous work, TraA and TraB were identified to be essential factors for OM exchange (OME) in donor and recipient cells. To define the genetic complexity of OME, we carried out a comprehensive forward genetic screen. The screen was based on the observation that Myxococcus xanthus nonmotile cells, by a Tra-dependent mechanism, block swarm expansion of motile cells when mixed. Thus, mutants defective in OME or a downstream responsive pathway were readily identified as escape flares from mixed inocula seeded on agar. This screen was surprisingly powerful, as we found >50 mutants defective in OME. Importantly, all of the mutations mapped to the traAB operon, suggesting that there may be few, if any, proteins besides TraA and TraB directly required for OME. We also found a second and phenotypically different class of mutants that exhibited wild-type OME but were defective in a responsive pathway. This pathway is postulated to control inner membrane homeostasis by covalently attaching amino acids to phospholipids. The identified proteins are homologous to the Staphylococcus aureus MprF protein, which is involved in membrane adaptation and antibiotic resistance. Interestingly, we also found that a small number of nonmotile cells were sufficient to block the swarming behavior of a large gliding-proficient population. This result suggests that an OME-derived signal could be amplified from a few nonmotile producers to act on many responder cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system

International Nuclear Information System (INIS)

Rao, Vincenzo A.; Shepherd, Sharon M.; English, Grant; Coulthurst, Sarah J.; Hunter, William N.

2011-01-01

The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system. Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homotetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners

The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system

Energy Technology Data Exchange (ETDEWEB)

Rao, Vincenzo A.; Shepherd, Sharon M.; English, Grant; Coulthurst, Sarah J.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom)

2011-12-01

The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system. Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide. The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homotetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners.

Analysis of proton exchange membrane fuel cell polarization losses at elevated temperature 120 C and reduced relative humidity

Energy Technology Data Exchange (ETDEWEB)

Xu, Hui; Kunz, H. Russell [Department of Chemical Engineering, University of Connecticut, Storrs, CT (United States); Fenton, James M. [Florida Solar Energy Center, University of Central Florida, Cocoa, FL (United States)

2007-03-01

Polarization losses of proton exchange membrane (PEM) fuel cells at 120 C and reduced relative humidity (RH) were analyzed. Reduced RH affects membrane and electrode ionic resistance, catalytic activity and oxygen transport. For a cell made of Nafion {sup registered} 112 membrane and electrodes that have 35 wt.% Nafion {sup registered} and 0.3 mg/cm{sup 2} platinum supported on carbon, membrane resistance at 20%RH was 0.407 {omega} cm{sup 2} and electrode resistance 0.203 {omega} cm{sup 2}, significantly higher than 0.092 and 0.041 {omega} cm{sup 2} at 100%RH, respectively. In the kinetically controlled region, 20%RH resulted in 96 mV more cathode activation loss than 100%RH. Compared to 100%, 20%RH also produced significant oxygen transport loss across the ionomer film in the electrode, 105 mV at 600 mA/cm{sup 2}. The significant increase in polarization losses at elevated temperature and reduced RH indicates the extreme importance of designing electrodes for high temperature PEM fuel cells since membrane development has always taken most emphasis. (author)

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

Directory of Open Access Journals (Sweden)

Marlena M Wilson

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

Outer Membrane Vesicle Vaccines from Biosafe Surrogates Prevent Acute Lethal Glanders in Mice

Directory of Open Access Journals (Sweden)

Michael H. Norris

2018-01-01

Full Text Available Burkholderia mallei is a host-adapted Gram-negative mammalian pathogen that causes the severe disease glanders. Glanders can manifest as a rapid acute progression or a chronic debilitating syndrome primarily affecting solipeds and humans in close association with infected animals. In USA, B. mallei is classified as one of the most important bacterial biothreat agents. Presently, there is no licensed glanders vaccine available for humans or animals. In this work, outer membrane vesicles (OMVs were isolated from three attenuated biosafe bacterial strains, Burkholderia pseudomallei Bp82, B. thailandensis E555, and B. thailandensis TxDOH and used to vaccinate mice. B. thailandensis OMVs induced significantly higher antibody responses that were investigated. B. mallei specific serum antibody responses were of higher magnitude in mice vaccinated with B. thailandensis OMVs compared to levels in mice vaccinated with B. pseudomallei OMVs. OMVs derived from biosafe strains protected mice from acute lethal glanders with vesicles from the two B. thailandensis strains affording significant protection (>90% up to 35 days post-infection with some up to 60 days. Organ loads from 35-day survivors indicated bacteria colonization of the lungs, liver, and spleen while those from 60 days had high CFUs in the spleens. The highest antibody producing vaccine (B. thailandensis E555 OMVs also protected C57BL/6 mice from acute inhalational glanders with evidence of full protection.

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

Science.gov (United States)

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

2009-11-01

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

Localization of Rod Bipolar Cells in the Mammalian Retina Using an Antibody Against the α1c L-type Ca2+ Channel

International Nuclear Information System (INIS)

Huh, Yu-Jin; Choi, Jae-Sik; Jeon, Chang-Jin

2015-01-01

Bipolar cells transmit stimuli via graded changes in membrane potential and neurotransmitter release is modulated by Ca 2+ influx through L-type Ca 2+ channels. The purpose of this study was to determine whether the α 1 c subunit of L-type voltage-gated Ca 2+ channel (α 1 c L-type Ca 2+ channel) colocalizes with protein kinase C alpha (PKC-α), which labels rod bipolar cells. Retinal whole mounts and vertical sections from mouse, hamster, rabbit, and dog were immunolabeled with antibodies against PKC-α and α 1 c L-type Ca 2+ channel, using fluorescein isothiocyanate (FITC) and Cy5 as visualizing agents. PKC-α-immunoreactive cells were morphologically identical to rod bipolar cells as previously reported. Their cell bodies were located within the inner nuclear layer, dendritic processes branched into the outer plexiform layer, and axons extended into the inner plexiform layer. Immunostaining showed that α 1 c L-type Ca 2+ channel colocalized with PKC-α in rod bipolar cells. The identical expression of PKC-α and α 1 c L-type Ca 2+ channel indicates that the α 1 c L-type Ca 2+ channel has a specific role in rod bipolar cells, and the antibody against the α 1 c L-type Ca 2+ channel may be a useful marker for studying the distribution of rod bipolar cells in mouse, hamster, rabbit, and dog retinas

Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond.

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Zückert, Wolfram R

2014-08-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

Perforated membrane-type acoustic metamaterials

International Nuclear Information System (INIS)

Langfeldt, F.; Kemsies, H.; Gleine, W.; Estorff, O. von

2017-01-01

This letter introduces a modified design of membrane-type acoustic metamaterials (MAMs) with a ring mass and a perforation so that an airflow through the membrane is enabled. Simplified analytical investigations of the perforated MAM (PMAM) indicate that the perforation introduces a second anti-resonance, where the effective surface mass density of the PMAM is much higher than the static value. The theoretical results are validated using impedance tube measurements, indicating good agreement between the theoretical predictions and the measured data. The anti-resonances yield high low-frequency sound transmission loss values with peak values over 25 dB higher than the corresponding mass-law. - Highlights: • A new membrane-type acoustic metamaterial exhibiting negative density is presented. • The metamaterial design contains a ring mass with a perforation through the membrane. • The sound transmission loss exhibits narrow-band peaks much higher than the mass-law. • The emergence of the peaks is explained using a simple theoretical model. • Impedance tube measurements are used to validate the theoretical predictions.

Perforated membrane-type acoustic metamaterials

Energy Technology Data Exchange (ETDEWEB)

Langfeldt, F., E-mail: Felix.Langfeldt@haw-hamburg.de [Department of Automotive and Aeronautical Engineering, Hamburg University of Applied Sciences, Berliner Tor 9, D-20099 Hamburg (Germany); Kemsies, H., E-mail: Hannes.Kemsies@haw-hamburg.de [Department of Automotive and Aeronautical Engineering, Hamburg University of Applied Sciences, Berliner Tor 9, D-20099 Hamburg (Germany); Gleine, W., E-mail: Wolfgang.Gleine@haw-hamburg.de [Department of Automotive and Aeronautical Engineering, Hamburg University of Applied Sciences, Berliner Tor 9, D-20099 Hamburg (Germany); Estorff, O. von, E-mail: estorff@tu-harburg.de [Institute of Modelling and Computation, Hamburg University of Technology, Denickestr. 17, D-21073 Hamburg (Germany)

2017-04-25

This letter introduces a modified design of membrane-type acoustic metamaterials (MAMs) with a ring mass and a perforation so that an airflow through the membrane is enabled. Simplified analytical investigations of the perforated MAM (PMAM) indicate that the perforation introduces a second anti-resonance, where the effective surface mass density of the PMAM is much higher than the static value. The theoretical results are validated using impedance tube measurements, indicating good agreement between the theoretical predictions and the measured data. The anti-resonances yield high low-frequency sound transmission loss values with peak values over 25 dB higher than the corresponding mass-law. - Highlights: • A new membrane-type acoustic metamaterial exhibiting negative density is presented. • The metamaterial design contains a ring mass with a perforation through the membrane. • The sound transmission loss exhibits narrow-band peaks much higher than the mass-law. • The emergence of the peaks is explained using a simple theoretical model. • Impedance tube measurements are used to validate the theoretical predictions.

Clonality, outer-membrane proteins profile and efflux pump in KPC- producing Enterobacter sp. in Brazil.

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Rosa, Juliana Ferraz; Rizek, Camila; Marchi, Ana Paula; Guimaraes, Thais; Miranda, Lourdes; Carrilho, Claudia; Levin, Anna S; Costa, Silvia F

2017-03-17

Carbapenems resistance in Enterobacter spp. has increased in the last decade, few studies, however, described the mechanisms of resistance in this bacterium. This study evaluated clonality and mechanisms of carbapenems resistance in clinical isolates of Enterobacter spp. identified in three hospitals in Brazil (Hospital A, B and C) over 7-year. Antibiotics sensitivity, pulsed-field gel electrophoresis (PFGE), PCR for carbapenemase and efflux pump genes were performed for all carbapenems-resistant isolates. Outer-membrane protein (OMP) was evaluated based on PFGE profile. A total of 130 isolates of Enterobacter spp were analyzed, 44/105 (41, 9%) E. aerogenes and 8/25 (32,0%) E. cloacae were resistant to carbapenems. All isolates were susceptible to fosfomycin, polymyxin B and tigecycline. KPC was present in 88.6% of E. aerogenes and in all E. cloacae resistant to carbapenems. The carbapenems-resistant E. aerogenes identified in hospital A belonged to six clones, however, a predominant clone was identified in this hospital over the study period. There is a predominant clone in Hospital B and Hospital C as well. The mechanisms of resistance to carbapenems differ among subtypes. Most of the isolates co-harbored blaKPC, blaTEM and /or blaCTX associated with decreased or lost of 35-36KDa and or 39 KDa OMP. The efflux pump AcrAB-TolC gene was only identified in carbapenems-resistant E. cloacae. There was a predominant clone in each hospital suggesting that cross-transmission of carbapenems-resistant Enterobacter spp. was frequent. The isolates presented multiple mechanisms of resistance to carbapenems including OMP alteration.

The C-terminal hypervariable domain targets Aradopsis ROP9 to the invaginated pollen tube plasma membrane

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Rop9 is a small GTPase of the Type II class, whereas the often studied type I Rops play roles during pollen tube growth. In pollen, Rop9 is located at the invaginated plasma membrane that surrounds the sperm cells, whereas type I Rops are located at the apical membrane of the pollen tube. The C-ter...

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

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Michael W Stuck

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

Ferric-Pyoverdine Recognition by Fpv Outer Membrane Proteins of Pseudomonas protegens Pf-5

Science.gov (United States)

Hartney, Sierra L.; Mazurier, Sylvie; Girard, Maëva K.; Mehnaz, Samina; Davis, Edward W.; Gross, Harald; Lemanceau, Philippe

2013-01-01

The soil bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) produces two siderophores, enantio-pyochelin and a compound in the large and diverse pyoverdine family. Using high-resolution mass spectroscopy, we determined the structure of the pyoverdine produced by Pf-5. In addition to producing its own siderophores, Pf-5 also utilizes ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron. Previously, phylogenetic analysis of the 45 TonB-dependent outer membrane proteins in Pf-5 indicated that six are in a well-supported clade with ferric-pyoverdine receptors (Fpvs) from other Pseudomonas spp. We used a combination of phylogenetics, bioinformatics, mutagenesis, pyoverdine structural determinations, and cross-feeding bioassays to assign specific ferric-pyoverdine substrates to each of the six Fpvs of Pf-5. We identified at least one ferric-pyoverdine that was taken up by each of the six Fpvs of Pf-5. Functional redundancy of the Pf-5 Fpvs was also apparent, with some ferric-pyoverdines taken up by all mutants with a single Fpv deletion but not by a mutant having deletions in two of the Fpv-encoding genes. Finally, we demonstrated that phylogenetically related Fpvs take up ferric complexes of structurally related pyoverdines, thereby establishing structure-function relationships that can be employed in the future to predict the pyoverdine substrates of Fpvs in other Pseudomonas spp. PMID:23222724

Proteomic study via a non-gel based approach of meningococcal outer membrane vesicle vaccine obtained from strain CU385: a road map for discovering new antigens.

Science.gov (United States)

Gil, Jeovanis; Betancourt, L Zaro H; Sardiñas, Gretel; Yero, Daniel; Niebla, Olivia; Delgado, Maité; García, Darien; Pajón, Rolando; Sánchez, Aniel; González, Luis J; Padrón, Gabriel; Campa, Concepción; Sotolongo, Franklin; Barberó, Ramón; Guillén, Gerardo; Herrera, Luis; Besada, Vladimir

2009-05-01

This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatographymass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane.

Science.gov (United States)

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M P; Albano, E; Bianchi, F B

2000-04-01

Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack. The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum. Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes. AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.

Relative ion yields in mammalian cell components using C60 SIMS

Science.gov (United States)

Keskin, Selda; Piwowar, Alan; Hue, Jonathan; Shen, Kan; Winograd, Nicholas

2013-01-01

Time of flight secondary ion mass spectrometry has been used to better understand the influence of molecular environment on the relative ion yields of membrane lipid molecules found in high abundance in a model mammalian cell line, RAW264.7. Control lipid mixtures were prepared to simulate lipid–lipid interactions in the inner and outer leaflet of cell membranes. Compared with its pure film, the molecular ion yields of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine are suppressed when mixed with 2-dipalmitoyl-sn-glycero-3-phosphocholine. In the mixture, proton competition between 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 2-dipalmitoyl-sn-glycero-3-phosphocholine led to lower ionization efficiency. The possible mechanism for ion suppression was also investigated with 1H and 13C nuclear magnetic resonance spectroscopy. The formation of a hydroxyl bond in lipid mixtures confirms the mechanism involving proton exchange with the surrounding environment. Similar effects were observed for lipid mixtures mimicking the composition of the inner leaflet of cell membranes. The secondary molecular ion yield of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine was observed to be enhanced in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. PMID:25140069

Biogenesis and Membrane Targeting of Lipoproteins.

Science.gov (United States)

Narita, Shin-Ichiro; Tokuda, Hajime

2010-09-01

Bacterial lipoproteins represent a unique class of membrane proteins, which are anchored to membranes through triacyl chains attached to the amino-terminal cysteine. They are involved in various functions localized in cell envelope. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane, with others being in the inner membrane. All lipoproteins are synthesized in the cytoplasm with an N-terminal signal peptide, translocated across the inner membrane by the Sec translocon to the periplasmic surface of the inner membrane, and converted to mature lipoproteins through sequential reactions catalyzed by three lipoprotein-processing enzymes: Lgt, LspA, and Lnt. The sorting of lipoproteins to the outer membrane requires a system comprising five Lol proteins. An ATP-binding cassette transporter, LolCDE, initiates the sorting by mediating the detachment of lipoproteins from the inner membrane. Formation of the LolA-lipoprotein complex is coupled to this LolCDE-dependent release reaction. LolA accommodates the amino-terminal acyl chain of lipoproteins in its hydrophobic cavity, thereby generating a hydrophilic complex that can traverse the periplasmic space by diffusion. Lipoproteins are then transferred to LolB on the outer membrane and anchored to the inner leaflet of the outer membrane by the action of LolB. In contrast, since LolCDE does not recognize lipoproteins possessing Asp at position +2, these lipoproteins remain anchored to the inner membrane. Genes for Lol proteins are widely conserved among gram-negative bacteria, and Lol-mediated outer membrane targeting of lipoproteins is considered to be the general lipoprotein localization mechanism.

Lipopolysaccharide Membranes and Membrane Proteins of Pseudomonas aeruginosa Studied by Computer Simulation

Energy Technology Data Exchange (ETDEWEB)

Straatsma, TP

2006-12-01

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is also a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid

Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

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Jing Zhang

2017-12-01

Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

Origin of Outer Solar System

Science.gov (United States)

Holman, Matthew J.; Lindstrom, David (Technical Monitor)

2005-01-01

Our ongoing research program combines extensive deep and wide-field observations using a variety of observational platforms with numerical studies of the dynamics of small bodies in the outer solar system in order to advance the main scientific goals of the community studying the Kuiper belt and the outer solar system. These include: (1) determining the relative populations of the known classes of KBOs as well as other possible classes; ( 2 ) determining the size distributions or luminosity function of the individual populations or the Kuiper belt as a whole; (3) determining the inclinations distributions of these populations; (4) establishing the radial extent of the Kuiper belt; ( 5 ) measuring and relating the physical properties of different types of KBOs to those of other solar system bodies; and, (6) completing our systematic inventory of the satellites of the outer planets.

Antibiotic trapping by plasmid-encoded cmy-2-lactamase combined with reduced outer membrane permeability as a mechanism of carbapenem resistance in escherichia coli

NARCIS (Netherlands)

W.H.F. Goessens (Wil); A.K. van der Bij (Akke); R. van Boxtel (Ria); J.D.D. Pitout (J. D D); P. van Ulsen (Peter); D.C. Melles (Damian); J. Tommassen (Jan)

2013-01-01

textabstractA liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane

MOLECULAR MODELING STUDY OF THE CONTRIBUTIONS OF SIDE AMINO ACID RESIDUES OF POLYMYXIN B3 TO ITS BINDING WITH E.COLI OUTER MEMBRANE LIPOPOLYSACCHARIDE

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Lisnyak Yu. V.

2014-12-01

Full Text Available Last decades, antimicrobial peptides (AMPs are the subject of intense investigations aimed to develop effective drugs against extremely resistant nosocomial bacterial pathogens (especially Gram-negative bacteria. In particular, there has been greatly renewed interest to polymyxins, the representatives of AMPs which are specific and highly potent against Gram-negative bacteria, but have potential nephrotoxic side effect. A prerequisite of purposeful enhancement of therapeutic properties of polymyxins is a detailed knowledge of the molecular mechanisms of their interactions with cell targets. Lipopolysaccharide (LPS, the main component of the outer leaflet of outer membrane of gram-negative bacteria, is a primary cell target of polymyxins. The aim of the paper was to study the peculiarities of molecular interactions of polymyxin В3 with lipopolysaccharide of the outer membrane of gram-negative bacterium. Materials and methods The complexes of polymyxin В3 (PmВ3 and its alaninederivatives with E. coli outer membrane lipopolysaccharide were built and studied by molecular modeling methods (minimization, simulated annealing, docking. Atom coordinates of polymyxin В3 and LPS structures were taken from nuclear magnetic resonance and X-ray crystallography experiments, respectively. The AMBER03 force field was used with a 1.05 nm force cutoff. Longrange electrostatic interactions were treated by the Particle Mesh Ewald method. Results and discussion Alanine scanning of PmВ3 molecule has been carried out and the role of its side amino acid residues in the formation of complex with lipopolysaccharide has been investigated. It has been shown that substitutions of polymyxin’s Dab residues in positions 1, 3, 5, 8 and 9 for alanine markedly reduce the binding energy of PmB3-LPS complex, where as the similar substitutions of residues in positions 2, 6, 7 and 10 leave the binding energy virtually unchanged. Structural aspects of antimicrobial action of

Accelerated microevolution in an outer membrane protein (OMP of the intracellular bacteria Wolbachia

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Russell Jacob A

2010-02-01

Full Text Available Abstract Background Outer membrane proteins (OMPs of Gram-negative bacteria are key players in the biology of bacterial-host interactions. However, while considerable attention has been given to OMPs of vertebrate pathogens, relatively little is known about the role of these proteins in bacteria that primarily infect invertebrates. One such OMP is found in the intracellular bacteria Wolbachia, which are widespread symbionts of arthropods and filarial nematodes. Recent experimental studies have shown that the Wolbachia surface protein (WSP can trigger host immune responses and control cell death programming in humans, suggesting a key role of WSP for establishment and persistence of the symbiosis in arthropods. Results Here we performed an analysis of 515 unique alleles found in 831 Wolbachia isolates, to investigate WSP structure, microevolution and population genetics. WSP shows an eight-strand transmembrane β-barrel structure with four extracellular loops containing hypervariable regions (HVRs. A clustering approach based upon patterns of HVR haplotype diversity was used to group similar WSP sequences and to estimate the relative contribution of mutation and recombination during early stages of protein divergence. Results indicate that although point mutations generate most of the new protein haplotypes, recombination is a predominant force triggering diversity since the very first steps of protein evolution, causing at least 50% of the total amino acid variation observed in recently diverged proteins. Analysis of synonymous variants indicates that individual WSP protein types are subject to a very rapid turnover and that HVRs can accommodate a virtually unlimited repertoire of peptides. Overall distribution of WSP across hosts supports a non-random association of WSP with the host genus, although extensive horizontal transfer has occurred also in recent times. Conclusions In OMPs of vertebrate pathogens, large recombination impact, positive

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins

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Cristian Oliver

2017-09-01

Full Text Available Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

Science.gov (United States)

Oliver, Cristian; Hernández, Mauricio A; Tandberg, Julia I; Valenzuela, Karla N; Lagos, Leidy X; Haro, Ronie E; Sánchez, Patricio; Ruiz, Pamela A; Sanhueza-Oyarzún, Constanza; Cortés, Marcos A; Villar, María T; Artigues, Antonio; Winther-Larsen, Hanne C; Avendaño-Herrera, Ruben; Yáñez, Alejandro J

2017-01-01

Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

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

Directory of Open Access Journals (Sweden)

J. Lienard

2014-01-01

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

Aspartate tightens the anchoring of staphylococcal lipoproteins to the cytoplasmic membrane.

Science.gov (United States)

Kumari, Nimerta; Götz, Friedrich; Nguyen, Minh-Thu

2017-12-01

In gram-negative bacteria, the ABC transporter LolCDE complex translocates outer membrane-specific lipoproteins (Lpp) from the inner membrane to the outer membrane. Lpp possessing aspartate (Asp) at position +2 are not translocated because it functions as a LolCDE avoidance signal. In gram-positive bacteria, lacking an outer membrane and the Lol system, Lpp are only anchored at the outer leaflet of the cytoplasmic membrane. However, the release of Lpp particularly in pathogenic or commensal species is crucial for immune modulation. Here, we provide evidence that in Staphylococcus aureus Asp at position +2 plays a role in withholding Lpp to the cytoplasmic membrane. Screening of published exoproteomic data of S. aureus revealed that Lpp mainly with Gly or Ser at position +2 were found in exoproteome, but there was no Lpp with Asp+2. The occurrence of Lpp with Asp+2 is infrequent in gram-positive bacteria. In S. aureus USA300 only seven of the 67 Lpp possess Asp+2; among them five Lpp represented Lpl lipoproteins involved in host cell invasion. Our study demonstrated that replacing the Asp+2 present in Lpl8 with a Ser enhances its release into the supernatant. However, there is no different release of Asp+2 and Ser+2 in mprF mutant that lacks the positive charge of lysyl-phosphatidylglycerol (Lys-PG). Moreover, substitution of Ser+2 by Asp in SitC (MntC) did not lead to a decreased release indicating that in staphylococci positions +3 and +4 might also be important for a tighter anchoring of Lpp. Here, we show that Asp in position +2 and adjacent amino acids contribute in tightening the anchoring of Lpp by interaction of the negative charged Asp with the positive charged Lys-PG. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Selected Legal Challenges Relating to the Military use of Outer Space, with Specific Reference to Article IV of the Outer Space Treaty

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Anél Ferreira-Snyman

2015-12-01

Full Text Available Since the end of the Second World War the potential use of outer space for military purposes persisted to be intrinsically linked to the development of space technology and space flight. The launch of the first artificial satellite, Sputnik 1, by the USSR in 1957 made Western states realise that a surprise attack from space was a real possibility, resulting in the so-called "space-race" between the USA and the USSR. During the Cold War space activities were intrinsically linked to the political objectives, priorities and national security concerns of the USA and the Soviet Union. After the Cold War the political relevance and benefits of space continued to be recognised by states. In view of the recent emergence of new major space powers such as China, the focus has again shifted to the military use of outer space and the potential that a state with advanced space technology may use it for military purposes in order to dominate other states. Article IV of the Outer Space Treaty prohibits the installation of nuclear weapons and weapons of mass destruction in outer space and determines that the moon and other celestial bodies shall be used for peaceful purposes only. Due to the dual-use character of many space assets, the distinction between military and non-military uses of outer space is becoming increasingly blurred. This article discusses a number of legal challenges presented by article IV of the Outer Space Treaty, relating specifically to the term peaceful, the distinction between the terms militarisation and weaponisation and the nature of a space weapon. It is concluded that article IV is in many respects outdated and that it cannot address the current legal issues relating to the military use of outer space. The legal vacuum in this area may have grave consequences not only for maintaining peace and security in outer space, but also on earth. Consequently, an international dialogue on the military uses of outer space should be

Antibiotic Trapping by Plasmid-Encoded CMY-2 beta-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

NARCIS (Netherlands)

Goessens, W.H.F.; van der Bij, A.K.; van Boxtel, R.; Pitout, J.D.D.; van Ulsen, J.P.; Melles, D.C.; Tommassen, J.

2013-01-01

A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein

Novel Approaches to Immobilized Heteropoly Acid Systems for High Temperature, Low Relative Humidity Polymer-Type Membranes - Final Report

Energy Technology Data Exchange (ETDEWEB)

Herring, Andrew M; Horan, James L; Aieta, Niccolo V; Sachdeva, Sonny; Kuo, Mei-Chen; Ren, Hui; Lingutla, Anitha; Emery, Michael; Haugen, Gregory M; Yandrasits, Michael A; Sharma, Neeraj; Coggio, William D; Hamrock, Steven J; Frey, Matthew H

2012-05-20

films, Generation II films. These materials showed proton conductivities as high as 1 S/cm under high RH conditions. However, the materials suffered from compromised properties due to impure monomers and low molecular weights. Multiple lots of an HPA-immobilized fluoropolymer derived from preformed PVDF-HFP (Generation III films) were synthesized and formed into membranes at 3M and characterized at 3M to exhibit conductivity reaching approximately 75mS/cm at 120°C/40%RH using a Bekktech sample fixture and TestEquity oven (optimized membrane, at close of program). Initial fuel cell fabrication and testing for this new class of membrane yielded negative results (no measureable proton conductivity); however, the specific early membrane that was used for the two 5cm2 MEAs was later determined to have <1 mS/cm at 80°C/80%RH using the Bekktech fixture, vs. ca. 200 mS/cm at 80°C/80%RH for samples of the later-optimized type described above. Future work in this area (beyond the presently reported contract) should include additional attempts to fabricate and test fuel cells based on the later-optimized Generation II and III polymer. A manufacturing study was performed which predicted no difficulties in any future scale up of the materials.

Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

Science.gov (United States)

Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

2016-01-01

The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

The novel 2Fe–2S outer mitochondrial protein mitoNEET displays conformational flexibility in its N-terminal cytoplasmic tethering domain

International Nuclear Information System (INIS)

Conlan, Andrea R.; Paddock, Mark L.; Axelrod, Herbert L.; Cohen, Aina E.; Abresch, Edward C.; Wiley, Sandra; Roy, Melinda; Nechushtai, Rachel; Jennings, Patricia A.

2009-01-01

The crystal structure of the anti-diabetic drug target mitoNEET obtained from a GFP fusion construct (1.4 Å resolution, R factor = 20.2%) shows that the CDGSH 2Fe–2S binding domains are superimposable with previously determined non-fused constructs. However, there is considerable flexibility in the position of the outer mitochondrial tethering arms resulting in two different conformations in the crystal structure. A primary role for mitochondrial dysfunction is indicated in the pathogenesis of insulin resistance. A widely used drug for the treatment of type 2 diabetes is pioglitazone, a member of the thiazolidinedione class of molecules. MitoNEET, a 2Fe–2S outer mitochondrial membrane protein, binds pioglitazone [Colca et al. (2004 ▶), Am. J. Physiol. Endocrinol. Metab.286, E252–E260]. The soluble domain of the human mitoNEET protein has been expressed C-terminal to the superfolder green fluorescent protein and the mitoNEET protein has been isolated. Comparison of the crystal structure of mitoNEET isolated from cleavage of the fusion protein (1.4 Å resolution, R factor = 20.2%) with other solved structures shows that the CDGSH domains are superimposable, indicating proper assembly of mitoNEET. Furthermore, there is considerable flexibility in the position of the cytoplasmic tethering arms, resulting in two different conformations in the crystal structure. This flexibility affords multiple orientations on the outer mitochondrial membrane

Spontaneous complement activation on human B cells results in localized membrane depolarization and the clustering of complement receptor type 2 and C3 fragments

DEFF Research Database (Denmark)

Løbner, Morten; Leslie, Robert G Q; Prodinger, Wolfgang M

2009-01-01

While our previous studies have demonstrated that complement activation induced by complement receptors type 2 (CR2/CD21) and 1 (CR1/CD35) results in C3-fragment deposition and membrane attack complex (MAC) formation in human B cells, the consequences of these events for B-cell functions remain u...

The establishment of polarized membrane traffic in Xenopus laevis embryos.

Science.gov (United States)

Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

1992-09-01

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin

Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants.

Science.gov (United States)

Khan, Shahneaz Ali; Desclozeaux, Marion; Waugh, Courtney; Hanger, Jon; Loader, Jo; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

2016-01-01

Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A) responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses.

Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP with Two Different Adjuvants.

Directory of Open Access Journals (Sweden)

Shahneaz Ali Khan

Full Text Available Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus. In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP antigen-based vaccine, combined with immune stimulating complex (ISC adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses.

Outer hair cell piezoelectricity: frequency response enhancement and resonance behavior.

Science.gov (United States)

Weitzel, Erik K; Tasker, Ron; Brownell, William E

2003-09-01

Stretching or compressing an outer hair cell alters its membrane potential and, conversely, changing the electrical potential alters its length. This bi-directional energy conversion takes place in the cell's lateral wall and resembles the direct and converse piezoelectric effects both qualitatively and quantitatively. A piezoelectric model of the lateral wall has been developed that is based on the electrical and material parameters of the lateral wall. An equivalent circuit for the outer hair cell that includes piezoelectricity shows a greater admittance at high frequencies than one containing only membrane resistance and capacitance. The model also predicts resonance at ultrasonic frequencies that is inversely proportional to cell length. These features suggest all mammals use outer hair cell piezoelectricity to support the high-frequency receptor potentials that drive electromotility. It is also possible that members of some mammalian orders use outer hair cell piezoelectric resonance in detecting species-specific vocalizations.

Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

Science.gov (United States)

von Einem, Bjoern; Weber, Petra; Wagner, Michael; Malnar, Martina; Kosicek, Marko; Hecimovic, Silva; Arnim, Christine A F von; Schneckenburger, Herbert

2012-11-26

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

Nitazoxanide Inhibits Pilus Biogenesis by Interfering with Folding of the Usher Protein in the Outer Membrane.

Science.gov (United States)

Chahales, Peter; Hoffman, Paul S; Thanassi, David G

2016-04-01

Many bacterial pathogens assemble surface fibers termed pili or fimbriae that facilitate attachment to host cells and colonization of host tissues. The chaperone/usher (CU) pathway is a conserved secretion system that is responsible for the assembly of virulence-associated pili by many different Gram-negative bacteria. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and an integral outer membrane (OM) assembly and secretion platform termed the usher. Nitazoxanide (NTZ), an antiparasitic drug, was previously shown to inhibit the function of aggregative adherence fimbriae and type 1 pili assembled by the CU pathway in enteroaggregativeEscherichia coli, an important causative agent of diarrhea. We show here that NTZ also inhibits the function of type 1 and P pili from uropathogenicE. coli(UPEC). UPEC is the primary causative agent of urinary tract infections, and type 1 and P pili mediate colonization of the bladder and kidneys, respectively. By analysis of the different stages of the CU pilus biogenesis pathway, we show that treatment of bacteria with NTZ causes a reduction in the number of usher molecules in the OM, resulting in a loss of pilus assembly on the bacterial surface. In addition, we determine that NTZ specifically prevents proper folding of the usher β-barrel domain in the OM. Our findings demonstrate that NTZ is a pilicide with a novel mechanism of action and activity against diverse CU pathways. This suggests that further development of the NTZ scaffold may lead to new antivirulence agents that target the usher to prevent pilus assembly. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Analysis of proton exchange membrane fuel cell polarization losses at elevated temperature 120 {sup o}C and reduced relative humidity

Energy Technology Data Exchange (ETDEWEB)

Xu, Hui [Department of Chemical Engineering, University of Connecticut, Storrs, CT (United States)]. E-mail: huixu@lanl.gov; Kunz, H. Russell [Department of Chemical Engineering, University of Connecticut, Storrs, CT (United States); Fenton, James M. [Florida Solar Energy Center, University of Central Florida, Cocoa, FL (United States)

2007-03-01

Polarization losses of proton exchange membrane (PEM) fuel cells at 120 {sup o}C and reduced relative humidity (RH) were analyzed. Reduced RH affects membrane and electrode ionic resistance, catalytic activity and oxygen transport. For a cell made of Nafion (registered) 112 membrane and electrodes that have 35 wt.% Nafion (registered) and 0.3 mg/cm{sup 2} platinum supported on carbon, membrane resistance at 20%RH was 0.407 {omega} cm{sup 2} and electrode resistance 0.203 {omega} cm{sup 2}, significantly higher than 0.092 and 0.041 {omega} cm{sup 2} at 100%RH, respectively. In the kinetically controlled region, 20%RH resulted in 96 mV more cathode activation loss than 100%RH. Compared to 100%, 20%RH also produced significant oxygen transport loss across the ionomer film in the electrode, 105 mV at 600 mA/cm{sup 2}. The significant increase in polarization losses at elevated temperature and reduced RH indicates the extreme importance of designing electrodes for high temperature PEM fuel cells since membrane development has always taken most emphasis.

Dynamical evolution of V-type photometric candidates in the central and outer main belt asteroids

Science.gov (United States)

Carruba, V.; Huaman, M.

2014-07-01

V-type asteroids are associated with basaltic composition, and are supposed to be fragments of crust of differentiated objects. Most V-type asteroids in the main belt are found in the inner main belt, and are either current members of the Vesta dynamical family (Vestoids), or past members that drifted away. However, several V-type photometric candidates have been recently identified in the central and outer main belt. The origin of this large population of V-type objects is not well understood, since it seems unlikely that Vestoids crossing the 3:1 and 5:2 mean-motion resonance with Jupiter could account for the whole observed population. In this work, we investigated a possible origin of the bodies from local sources, such as the parent bodies of the Eunomia, Merxia, and Agnia asteroid families in the central main belt, and Dembowska, Eos and Magnya asteroid families in the outer main belt. Our results show that dynamical evolution from the parent bodies of the Eunomia and Merxia/Agnia families on timescales of 2 Gyr or more could be responsible for the current orbital location of most of the V-type photometric candidates in the central main belt. Studies for the outer main belt are currently in progress. by the FAPESP (grant 2011/19863-3) and CAPES (grant 15029-12-3) funding agencies.

Membrane transport of anandamide through resealed human red blood cell membranes

DEFF Research Database (Denmark)

Bojesen, I.N.; Hansen, Harald S.

2005-01-01

The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments...... at 0°C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 ± 0.023, and the rate constant...... of unidirectional flux from inside to outside is 0.361 ± 0.023 s. The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]) increases with the square root of [BSA] in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane...

Relationship between the natural history of chronic subdural hematoma and enhancement of the inner membrane on post-contrast CT scan

International Nuclear Information System (INIS)

Nakaguchi, Hiroshi; Yoshimasu, Norio; Tanishima, Takeo

2003-01-01

The purpose of this study was to determine the features and natural history of the inner membrane in CSDH (chronic subdural hematoma) patients using pre- and post-contrast CT scans. Twenty-four patients with CSDH (28 hematomas) who underwent pre- and post-contrast CT scanning prior to surgery ware studied. CSDH was classified according to the internal architecture as the homogeneous (HM) type, the laminar (LM) type, the separated (SP) type, and the trabecular (TR) type. For each type, the enhancement of the inner membrane, the outer membrane, and trabeculae was evaluated. Enhancement of the inner membrane develops as the stage of CSDH progresses. Although the inner membrane was not enhanced in the HM type, it was enhanced in almost all of the patients with the other types (p <0.0001). In 13% of patients with the LM type, 29% with the SP type, and 40% with the TR type, the inner membrane was moderately or markedly enhanced. The outer membrane was not thickened in any patient. In 88% of the patients who had contralateral hemiparesis on admission and 42% of those who did not have hemiparesis, the inner membrane was clearly enhanced (p=0.0166). This indicated that blood flow to the inner membrane from the pia matter induced hypoperfusion of the brain parenchyma beneath the hematoma in addition to the effect of direct compression. Contrast CT scanning may be useful for diagnosing the extent of enlargement of the inner membrane, predicting the risk of local brain damage, and evaluating the stage in the natural history of CSDH. (author)

Relationship between the natural history of chronic subdural hematoma and enhancement of the inner membrane on post-contrast CT scan

Energy Technology Data Exchange (ETDEWEB)

Nakaguchi, Hiroshi; Yoshimasu, Norio [Teraoka Memorial Hospital, Shinichi, Hiroshima (Japan); Tanishima, Takeo [Tokyo Kosei Nenkin Hospital (Japan)

2003-02-01

The purpose of this study was to determine the features and natural history of the inner membrane in CSDH (chronic subdural hemotoma) patients using pre- and post-contrast CT scans. Twenty-four patients with CSDH (28 hematomas) who underwent pre- and post-contrast CT scanning prior to surgery ware studied. CSDH was classified according to the internal architecture as the homogeneous (HM) type, the laminar (LM) type, the separated (SP) type, and the trabecular (TR) type. For each type, the enhancement of the inner membrane, the outer membrane, and trabeculae was evaluated. Enhancement of the inner membrane develops as the stage of CSDH progresses. Although the inner membrane was not enhanced in the HM type, it was enhanced in almost all of the patients with the other types (p <0.0001). In 13% of patients with the LM type, 29% with the SP type, and 40% with the TR type, the inner membrane was moderately or markedly enhanced. The outer membrane was not thickened in any patient. In 88% of the patients who had contralateral hemiparesis on admission and 42% of those who did not have hemiparesis, the inner membrane was clearly enhanced (p=0.0166). This indicated that blood flow to the inner membrane from the pia matter induced hypoperfusion of the brain parenchyma beneath the hematoma in addition to the effect of direct compression. Contrast CT scanning may be useful for diagnosing the extent of enlargement of the inner membrane, predicting the risk of local brain damage, and evaluating the stage in the natural history of CSDH. (author)

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

KAUST Repository

Gao, Jie; Sun, Shi-Peng; Zhu, Wen-Ping; Chung, Neal Tai-Shung

2015-01-01

. It also shows an impressive rejection to CdCl2 (94%) during long-term stability tests. The CdCl2 rejection remains higher than 90% at operating temperatures from 5 to 60 °C. This study may provide useful insights for green manufacturing of outer

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

Directory of Open Access Journals (Sweden)

Bas van de Waterbeemd

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

A comparative study on component volumes from outer to inner dental enamel in relation to enamel tufts.

Science.gov (United States)

Setally Azevedo Macena, Marcus; de Alencar e Silva Leite, Maria Luísa; de Lima Gouveia, Cíntia; de Lima, Tamires Alcoforado Sena; Athayde, Priscilla Alves Aguiar; de Sousa, Frederico Barbosa

2014-06-01

Dental enamel presents marked mechanical properties gradients from outer to inner enamel, a region lacking component volumes profiles. Tufts, structures of inner enamel, have been shown to play a role in enamel resilience. We aimed at comparing component volumes from inner to outer enamel in relation to enamel tufts. Transversal ground sections from the cervical half of unerupted human third molars (n=10) were prepared and histological points were selected along transversal lines (extending from innermost to outer enamel) traced across tufts and adjacent control areas without tufts. Component volumes were measured at each histological point. Component volumes ranges were: 70.6-98.5% (mineral), 0.02-20.78% (organic), 3.8-9.8% (total water), 3-9% (firmly bound water), and 0.02-3.3% (loosely bound water). Inner enamel presented the lowest mineral volumes and the highest non-mineral volumes. Mineral, water and organic contents differed as a function of the distance from innermost enamel but not between the tuft and control lines. Tufts presented opaqueness in polarizing microscopy (feature of fracture lines). Organic volume gradient correlated with a relatively flat profile of loosely bound water. Inner, but not outer enamel, rehydrated after air-dried enamel was heated to 50°C and re-exposed to room conditions, as predicted by the organic/water gradient profiles. Component volumes vary markedly from outer to inner enamel, but not between areas with or without tufts (that behave like fracture lines under polarizing microscopy). Copyright © 2014 Elsevier Ltd. All rights reserved.

Plasma membrane calcium ATPases and related disorders.

Science.gov (United States)

Giacomello, Marta; De Mario, Agnese; Scarlatti, Chiara; Primerano, Simona; Carafoli, Ernesto

2013-03-01

The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia. Copyright © 2012 Elsevier Ltd. All rights reserved.

Interaction mediated by the putative tip regions of MdsA and MdsC in the formation of a Salmonella-specific tripartite efflux pump.

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Saemee Song

Full Text Available To survive in the presence of a wide range of toxic compounds, gram-negative bacteria expel such compounds via tripartite efflux pumps that span both the inner and outer membranes. The Salmonella-specific MdsAB pump consists of MdsB, a resistance-nodulation-division (RND-type inner membrane transporter (IMT that requires the membrane fusion protein (MFP MdsA, and an outer membrane protein (OMP; MdsC or TolC to form a tripartite efflux complex. In this study, we investigated the role of the putative tip regions of MdsA and its OMPs, MdsC and TolC, in the formation of a functional MdsAB-mediated efflux pump. Comparative analysis indicated that although sequence homologies of MdsA and MdsC with other MFPs and OMPs, respectively, are extremely low, key residues in the putative tip regions of these proteins are well conserved. Mutagenesis studies on these conserved sites demonstrated their importance for the physical and functional interactions required to form an MdsAB-mediated pump. Our studies suggest that, despite differences in the primary amino acid sequences and functions of various OMPs and MFPs, interactions mediated by the conserved tip regions of OMP and MFP are required for the formation of functional tripartite efflux pumps in gram-negative bacteria.

Translocation mechanism of C{sub 60} and C{sub 60} derivations across a cell membrane

Energy Technology Data Exchange (ETDEWEB)

Liang, Lijun, E-mail: michael.lijunl@gmail.com [Hangzhou Dianzi University, College of Life Information Science and Instrument Engineering (China); Kang, Zhengzhong [Zhejiang University, Department of Chemistry (China); Shen, Jia-Wei, E-mail: shen.jiawei@hotmail.com [Hangzhou Normal University, School of Medicine (China)

2016-11-15

Carbon-based nanoparticles (NPs) such as fullerenes and nanotubes have been extensively studied for drug delivery in recent years. The permeation process of fullerene and its derivative molecules through membrane is essential to the utilization of fullerene-based drug delivery system, but the mechanism and the dynamics of permeation through cell membrane are still unclear. In this study, coarse-grained molecular dynamics simulations were performed to investigate the permeation process of functionalized fullerene molecules (ca. 0.72 nm) through the membrane. Our results show that single functionalized fullerene molecule in such nanoscale could permeate the lipid membrane in micro-second time scale. Pristine C{sub 60} molecules prefer to aggregate into several small clusters while C{sub 60}OH{sub 15} molecules could aggregate into one big cluster to permeate through the lipid membrane. After permeation of C{sub 60} or its derivatives into membrane, all C{sub 60} and C{sub 60}OH{sub 15} molecules disaggregated and monodispersed in the lipid membrane.

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

International Nuclear Information System (INIS)

Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M.

2015-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-04-15

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

Functional mapping of PilF and PilQ in the Pseudomonas aeruginosa type IV pilus system.

Science.gov (United States)

Koo, Jason; Tang, Tim; Harvey, Hanjeong; Tammam, Stephanie; Sampaleanu, Liliana; Burrows, Lori L; Howell, P Lynne

2013-04-30

Pseudomonas aeruginosa uses type IV pili (T4P) to interact with the environment and as key virulence factors when acting as an opportunistic pathogen. Assembly of the outer membrane PilQ secretin channel through which the pili are extruded is essential for pilus biogenesis. The P. aeruginosa T4P pilotin, PilF, is required for PilQ outer membrane localization and assembly into secretins and contains six tetratricopeptide (TPR) protein-protein interaction motifs, suggesting that the two proteins interact. In this study, we found that the first four TPR motifs of PilF are sufficient for PilQ outer membrane targeting, oligomerization, and function. Guided by our structure of PilF, site-directed mutagenesis of the protein surface revealed that a hydrophobic groove on the first TPR is required for PilF-mediated PilQ assembly. Deletion of individual domains within PilQ suggests that the N0, KH-like, or secretin domain, but not the C-terminus, interacts with PilF. Purified PilQ was found to pull down PilF from Pseudomonas cell lysates. Together, these data allow us to propose a model for PilF function in the T4P system. PilF interacts directly or indirectly with the PilQ monomer after translocation of both proteins through the inner membrane and acts as a co-chaperone with the Lol system to facilitate transit across the periplasm to the outer membrane. The mechanism of PilQ insertion and assembly, which appears to be independent of the Bam system, remains to be determined.

Development of hydrophobic clay–alumina based capillary membrane for desalination of brine by membrane distillation

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Rakhi Das

2016-09-01

Full Text Available Clay–alumina compositions of 0, 20, 40 and 55 weight percent (wt% clay and rest alumina were maintained in porous support preparation by extrusion followed by sintering at 1300 °C for 2.5 h to obtain 3 mm/2 mm (outer diameter/inner diameter capillary. 1H,1H,2H,2H-perfluorodecyltriethoxysilane (97% (C8 was used to modify the capillary surface of all compositions without any intermediate membrane layer to impart hydrophobic characteristics and compared in terms of contact angle produced by the capillaries with water and liquid entry pressure (LEPw. FTIR analysis showed that the hydrophilic surface of the capillary membranes was efficiently modified by the proposed grafting method. Capillary with 55 wt% clay produced a pore size of 1.43 micron and was considered as an ideal candidate for grafting with C8 polymer to impart surface hydrophobicity. The contact angle and LEPw value obtained for this modified membrane (C-55-M were 145° and 1 bar, respectively. The modified capillary membrane was applied for desalination of brine by air gap membrane distillation (AGMD at a feed pressure of 0.85 bar. Maximum flux obtained for C-55-M membrane was 98.66 L/m2 day at a temperature difference of 60 °C with salt rejection of 99.96%. Mass transfer coefficient of C-55-M was 16 × 10−3 mm/s at feed temperature of 70 °C.

Outer boundary as arrested history in general relativity

CERN Document Server

Lau, S

2002-01-01

We present explicit outer boundary conditions for the canonical variables of general relativity. The conditions are associated with the causal evolution of a finite Cauchy domain, a so-called quasilocal boost, and they suggest a consistent scheme for modelling such an evolution numerically. The scheme involves a continuous boost in the spacetime orthogonal complement 'orthogonal' T sub p (B) of the tangent space T sub p (B) belonging to each point p on the system boundary B. We show how the boost rate may be computed numerically via equations similar to those appearing in canonical investigations of black-hole thermodynamics (although here holding at an outer two-surface rather than the bifurcate two-surface of a Killing horizon). We demonstrate the numerical scheme on a model example, the quasilocal boost of a spherical three-ball in Minkowski spacetime. Developing our general formalism with recent hyperbolic formulations of the Einstein equations in mind, we use Anderson and York's 'Einstein-Christoffel' hy...

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function

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Wen-Jung Lu

2018-03-01

Full Text Available Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein, as well as cells lacking the outer membrane factor (OMF TolC (Tolerance to colicins. Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV, however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.

Outer membrane protein changes during bacteroid development are independent of nitrogen fixation and differ between indeterminate and determinate nodulating host plants of Rhizobium leguminosarum

NARCIS (Netherlands)

Roest, H.P.; Goosen-de Roo, L.; Wijffelman, C.A.; Maagd, de R.A.; Lugtenberg, B.J.J.

1995-01-01

The outer membrane of bacteroids contains largely decreased levels of protein antigen groups II and III in comparison with that of free-living rhizobia (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J, J. Lugtenberg, J.Bacteriol, 171:1136-1142, 1989). Since we intend to study the molecular

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

Science.gov (United States)

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Recombinant outer membrane secretin PilQ(406-770) as a vaccine candidate for serogroup B Neisseria meningitidis.

Science.gov (United States)

Haghi, Fakhri; Peerayeh, Shahin Najar; Siadat, Seyed Davar; Zeighami, Habib

2012-02-21

Secretin PilQ is an antigenically conserved outer membrane protein which is present on most meningococci. This protein naturally expressed at high levels and is essential for meningococcal pilus expression at the cell surface. A 1095 bp fragment of C-terminal of secretin pilQ from serogroup B Neisseria meningitidis was cloned into prokaryotic expression vector pET-28a. Recombinant protein was overexpressed with IPTG and affinity-purified by Ni-NTA agarose. BALB/c mice were immunized subcutaneously with purified rPilQ(406-770) mixed with Freund's adjuvant. Serum antibody responses to serogroups A and B N. meningitidis whole cells or purified rPilQ(406-770) and functional activity of antibodies were determined by ELISA and SBA, respectively. The output of rPilQ(406-770) was approximately 50% of the total bacterial proteins. Serum IgG responses were significantly increased in immunized group with PilQ(406-770) mixed with Freund's adjuvant in comparison with control groups. Antisera produced against rPilQ(406-770) demonstrated strong surface reactivity to serogroups A and B N. meningitidis tested by whole-cell ELISA. Surface reactivity to serogroup B N. meningitidis was higher than serogroup A. The sera from PilQ(406-770) immunized animals were strongly bactericidal against serogroups A and B. These results suggest that rPilQ(406-770) is a potential vaccine candidate for serogroup B N. meningitidis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants

OpenAIRE

Khan, Shahneaz Ali; Desclozeaux, Marion; Waugh, Courtney; Hanger, Jon; Loader, Jo; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

2016-01-01

Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri...

Incorporation of squalene into rod outer segments

International Nuclear Information System (INIS)

Keller, R.K.; Fliesler, S.J.

1990-01-01

We have reported previously that squalene is the major radiolabeled nonsaponifiable lipid product derived from [ 3 H]acetate in short term incubations of frog retinas. In the present study, we demonstrate that newly synthesized squalene is incorporated into rod outer segments under similar in vitro conditions. We show further that squalene is an endogenous constituent of frog rod outer segment membranes; its concentration is approximately 9.5 nmol/mumol of phospholipid or about 9% of the level of cholesterol. Pulse-chase experiments with radiolabeled precursors revealed no metabolism of outer segment squalene to sterols in up to 20 h of chase. Taken together with our previous absolute rate studies, these results suggest that most, if not all, of the squalene synthesized by the frog retina is transported to rod outer segments. Synthesis of protein is not required for squalene transport since puromycin had no effect on squalene incorporation into outer segments. Conversely, inhibition of isoprenoid synthesis with mevinolin had no effect on the incorporation of opsin into the outer segment. These latter results support the conclusion that the de novo synthesis and subsequent intracellular trafficking of opsin and isoprenoid lipids destined for the outer segment occur via independent mechanisms

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

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David J. Dowling

2016-12-01

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

DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts.

Science.gov (United States)

Kanomata, Kazuhiro; Kokabu, Shoichiro; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

2009-06-01

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

Proteomic analysis of GPI-anchored membrane proteins

DEFF Research Database (Denmark)

Jung, Hye Ryung; Jensen, Ole Nørregaard

2006-01-01

Glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) represent a subset of post-translationally modified proteins that are tethered to the outer leaflet of the plasma membrane via a C-terminal GPI anchor. GPI-APs are found in a variety of eukaryote species, from pathogenic microorganisms...... to humans. GPI-APs confer important cellular functions as receptors, enzymes and scaffolding molecules. Specific enzymes and detergent extraction methods combined with separation technologies and mass spectrometry permit proteomic analysis of GPI-APs from plasma membrane preparations to reveal cell...

Application of fire-retardant treatment to the wood in Type A unirradiated nuclear fuel outer containers

International Nuclear Information System (INIS)

Whitlow, J.D.; Luna, R.E.

1992-01-01

Packagings for transporting unirradiated nuclear fuel assemblies in the United States are commonly constructed as rectangular boxes consisting of a metal inner container, a wooden outer container, and cushioning material separating the two. The wood in the outer container is a potential source of fuel for fire. Use of a fire-retardant treatment on the wood may reduce or eliminate the damage to nuclear fuel assemblies in some types of accidents involving fire. The applicability of using fire-retardant treatments on the wood of outer containers is addressed. An approximate cost-benefit analysis to determine if fire-retardant treatments are economically justified is presented. (Author)

C-type lectins do not act as functional receptors for filovirus entry into cells

Energy Technology Data Exchange (ETDEWEB)

Matsuno, Keita; Nakayama, Eri; Noyori, Osamu [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan); Marzi, Andrea; Ebihara, Hideki [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Irimura, Tatsuro [Graduate School of Pharmaceutical Science, University of Tokyo, Tokyo (Japan); Feldmann, Heinz [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Takada, Ayato, E-mail: atakada@czc.hokudai.ac.jp [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan)

2010-12-03

Research highlights: {yields} Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. {yields} Mutant GPs mediated virus entry less efficiently than wild-type GP. {yields} Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. {yields} C-type lectins do not independently mediate filovirus entry into cells. {yields} Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

C-type lectins do not act as functional receptors for filovirus entry into cells

International Nuclear Information System (INIS)

Matsuno, Keita; Nakayama, Eri; Noyori, Osamu; Marzi, Andrea; Ebihara, Hideki; Irimura, Tatsuro; Feldmann, Heinz; Takada, Ayato

2010-01-01

Research highlights: → Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. → Mutant GPs mediated virus entry less efficiently than wild-type GP. → Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. → C-type lectins do not independently mediate filovirus entry into cells. → Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

Overexpression of an Outer Membrane Protein Associated with Decreased Susceptibility to Carbapenems in Proteus mirabilis

Science.gov (United States)

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

2015-01-01

Proteus mirabilis isolates commonly have decreased susceptibility to imipenem. Previously, we found P. mirabilis hfq mutant was more resistant to imipenem and an outer membrane protein (OMP) could be involved. Therefore, we investigated the role of this OMP in carbapenem susceptibility. By SDS-PAGE we found this OMP (named ImpR) was increased in hfq mutant and LC-MS/MS revealed it to be the homologue of Salmonella YbfM, which is a porin for chitobiose and subject to MicM (a small RNA) regulation. We demonstrated that ImpR overexpression resulted in increased carbapenem MICs in the laboratory strain and clinical isolates. Chitobiose induced expression of chb (a chitobiose utilization operon). Real-time RT-PCR and SDS-PAGE were performed to elucidate the relationship of hfq, impR, chb and MicM in P. mirabilis. We found MicM RNA was decreased in hfq mutant and chbBC-intergenic region (chbBC-IGR) overexpression strain (chbIGRov), while impR mRNA was increased in hfq mutant, micM mutant and chbIGRov strain. In addition, mutation of hfq or micM and overexpression of chbBC-IGR increased ImpR protein level. Accordingly, chitobiose made wild-type have higher levels of ImpR protein and are more resistant to carbapenems. Hfq- and MicM-complemented strains restored wild-type MICs. Mutation of both impR and hfq eliminated the increase in carbapenem MICs observed in hfq mutant and ImpR-complementation of hfq/impR double mutant resulted in MICs as hfq mutant, indicating that the ImpR-dependent decreased carbapenem susceptibility of hfq mutant. These indicate MicM was antisense to impR mRNA and was negatively-regulated by chbBC-IGR. Together, overexpression of ImpR contributed to the decreased carbapenem susceptibility in P. mirabilis. PMID:25756370

Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology Estudo comparativo de técnicas de isolamento de membrana externa de Yersinia pestis e seu uso na epidemiologia da peste

Directory of Open Access Journals (Sweden)

Frederico Guilherme Coutinho Abath

1990-04-01

Full Text Available In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.No presente estudo três técnicas para isolamento de frações enriquecidas em membrana externa de Y. pestis foram avaliadas. As técnicas utilizadas foram: centrifugação em gradiente de densidade em sacarose e solubilização diferencial com Sarkosyl ou Triton X-100. A análise por eletroforese em gel de poliacrilamida na presença de dodecil sulfato de sódio (SDS-PAGE das membranas externas extraídas pelos diferentes métodos evidenciou perfis protéicos semelhantes. A determinação das atividades de NADH-desidrogenase e succinato-desidrogenase (enzimas de membrana interna indicou que todas as preparações estudadas eram adequadas a estudos analíticos. Obteve-se evidências preliminares sobre o possível uso de perfis protéicos de membrana externa na identificação de variantes geográficos entre isolados selvagens de Y. pestis.

The Vasopressin Type-2 Receptor and Prostaglandin Receptors EP2 and EP4 can Increase Aquaporin-2 Plasma Membrane Targeting Through a cAMP Independent Pathway

DEFF Research Database (Denmark)

Olesen, Emma Tina Bisgaard; Moeller, Hanne Bjerregaard; Assentoft, Mette

2016-01-01

Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be c...

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

Science.gov (United States)

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

2015-01-01

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

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

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Kyoung Sub Choi

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

Three-dimensional structure of Wza, the protein required for translocation of group 1 capsular polysaccharide across the outer membrane of Escherichia coli.

Science.gov (United States)

Beis, Konstantinos; Collins, Richard F; Ford, Robert C; Kamis, Alhaji B; Whitfield, Chris; Naismith, James H

2004-07-02

Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-A resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein. Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two- and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-A high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 A, whereas the smaller is almost circular with a diameter of 90 A. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of approximately 40 A in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide.

Outer boundary as arrested history in general relativity

International Nuclear Information System (INIS)

Lau, Stephen R

2002-01-01

We present explicit outer boundary conditions for the canonical variables of general relativity. The conditions are associated with the causal evolution of a finite Cauchy domain, a so-called quasilocal boost, and they suggest a consistent scheme for modelling such an evolution numerically. The scheme involves a continuous boost in the spacetime orthogonal complement 'orthogonal' T p (B) of the tangent space T p (B) belonging to each point p on the system boundary B. We show how the boost rate may be computed numerically via equations similar to those appearing in canonical investigations of black-hole thermodynamics (although here holding at an outer two-surface rather than the bifurcate two-surface of a Killing horizon). We demonstrate the numerical scheme on a model example, the quasilocal boost of a spherical three-ball in Minkowski spacetime. Developing our general formalism with recent hyperbolic formulations of the Einstein equations in mind, we use Anderson and York's 'Einstein-Christoffel' hyperbolic system as the evolution equations for our numerical simulation of the model

Outer boundary as arrested history in general relativity

Science.gov (United States)

Lau, Stephen R.

2002-06-01

We present explicit outer boundary conditions for the canonical variables of general relativity. The conditions are associated with the causal evolution of a finite Cauchy domain, a so-called quasilocal boost, and they suggest a consistent scheme for modelling such an evolution numerically. The scheme involves a continuous boost in the spacetime orthogonal complement ⊥Tp(B) of the tangent space Tp(B) belonging to each point p on the system boundary B. We show how the boost rate may be computed numerically via equations similar to those appearing in canonical investigations of black-hole thermodynamics (although here holding at an outer two-surface rather than the bifurcate two-surface of a Killing horizon). We demonstrate the numerical scheme on a model example, the quasilocal boost of a spherical three-ball in Minkowski spacetime. Developing our general formalism with recent hyperbolic formulations of the Einstein equations in mind, we use Anderson and York's 'Einstein-Christoffel' hyperbolic system as the evolution equations for our numerical simulation of the model.

Role of C-type natriuretic peptide in the function of normal human sperm

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Hui Xia

2016-01-01

Full Text Available C-type natriuretic peptide (CNP is a newly discovered type of local regulatory factor that mediates its biological effects through the specific, membrane-bound natriuretic peptide receptor-B (NPR-B. Recent studies have established that CNP is closely related to male reproductive function. The aims of this study were to determine the distribution of CNP/NPR-B in human ejaculated spermatozoa through different methods (such as immunolocalization, real time polymerase chain reaction and Western Blot, and then to evaluate the influence of CNP on sperm function i n vitro, such as motility and acrosome reaction. Human semen samples were collected from consenting donors who met the criteria of the World Health Organization for normozoospermia. Our results show that the specific receptor NPR-B of CNP is localized in the acrosomal region of the head and the membrane of the front-end tail of the sperm, and there is no signal of CNP in human sperm. Compared with the control, CNP can induce a significant dose-dependent increase in spermatozoa motility and acrosome reaction. In summary, CNP/NPR-B can affect sperm motility and acrosome reaction, thus regulating the reproductive function of males. CNP may be a new key factor in regulating sperm function.

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

Energy Technology Data Exchange (ETDEWEB)

Patel, TN; Park, AHA; Bantat, S

2014-12-01

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

Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice.

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Wenping Gong

Full Text Available The obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ and tumor necrosis factor-α (TNF-α, two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii.

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

Energy Technology Data Exchange (ETDEWEB)

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

1986-04-01

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

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

Science.gov (United States)

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

2015-10-05

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2011-06-01

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

Leucine-based receptor sorting motifs are dependent on the spacing relative to the plasma membrane

DEFF Research Database (Denmark)

Geisler, C; Dietrich, J; Nielsen, B L

1998-01-01

Many integral membrane proteins contain leucine-based motifs within their cytoplasmic domains that mediate internalization and intracellular sorting. Two types of leucine-based motifs have been identified. One type is dependent on phosphorylation, whereas the other type, which includes an acidic...... amino acid, is constitutively active. In this study, we have investigated how the spacing relative to the plasma membrane affects the function of both types of leucine-based motifs. For phosphorylation-dependent leucine-based motifs, a minimal spacing of 7 residues between the plasma membrane...... and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active leucine-based motifs, a minimal spacing of 6 residues between the plasma membrane and the acidic residue was required for optimal activity of the motifs. In addition, we found that the acidic...

Membrane potential and ion transport in lung epithelial type II cells

International Nuclear Information System (INIS)

Gallo, R.L.

1986-01-01

The alveolar type II pneumocyte is critically important to the function and maintenance of pulmonary epithelium. To investigate the nature of the response of type II cells to membrane injury, and describe a possible mechanism by which these cells regulate surfactant secretion, the membrane potential of isolated rabbit type II cells was characterized. This evaluation was accomplished by measurements of the accumulation of the membrane potential probes: [ 3 H]triphenylmethylphosphonium ([ 3 H]TPMP + ), rubidium 86, and the fluorescent dye DiOC 5 . A compartmental analysis of probe uptake into mitochondrial, cytoplasmic, and non-membrane potential dependent stores was made through the use of selective membrane depolarizations with carbonycyanide M-chlorophenylhydrazone (CCCP), and lysophosphatidylcholine (LPC). These techniques and population analysis with flow cytometry, permitted the accurate evaluation of type II cell membrane potential under control conditions and under conditions which stimulated cell activity. Further analysis of ion transport by cells exposed to radiation or adrenergic stimulation revealed a common increase in Na + /K + ATPase activity, and an increase in sodium influx across the plasma membrane. This sodium influx was found to be a critical step in the initiation of surfactant secretion. It is concluded that radiation exposure as well as other pulmonary toxicants can directly affect the membrane potential and ionic regulation of type II cells. Ion transport, particularly of sodium, plays an important role in the regulation of type II cell function

Photoreceptor change and visual outcome after idiopathic epiretinal membrane removal with or without additional internal limiting membrane peeling.

Science.gov (United States)

Ahn, Seong Joon; Ahn, Jeeyun; Woo, Se Joon; Park, Kyu Hyung

2014-01-01

To compare the postoperative photoreceptor status and visual outcome after epiretinal membrane removal with or without additional internal limiting membrane (ILM) peeling. Medical records of 40 eyes from 37 patients undergoing epiretinal membrane removal with residual ILM peeling (additional ILM peeling group) and 69 eyes from 65 patients undergoing epiretinal membrane removal without additional ILM peeling (no additional peeling group) were reviewed. The length of defects in cone outer segment tips, inner segment/outer segment junction, and external limiting membrane line were measured using spectral domain optical coherence tomography images of the fovea before and at 1, 3, 6, and 12 months after the surgery. Cone outer segment tips and inner segment/outer segment junction line defects were most severe at postoperative 1 month and gradually restored at 12 months postoperatively. The cone outer segment tips line defect in the additional ILM peeling group was significantly greater than that in the no additional peeling group at postoperative 1 month (P = 0.006), and best-corrected visual acuity was significantly worse in the former group at the same month (P = 0.001). There was no significant difference in the defect size and best-corrected visual acuity at subsequent visits and recurrence rates between the two groups. Patients who received epiretinal membrane surgery without additional ILM peeling showed better visual and anatomical outcome than those with additional ILM peeling at postoperative 1 month. However, surgical outcomes were comparable between the two groups, thereafter. In terms of visual outcome and photoreceptor integrity, additional ILM peeling may not be an essential procedure.

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

Science.gov (United States)

Otsuki, Tetsuji; Ota, Toshio; Nishikawa, Tetsuo; Hayashi, Koji; Suzuki, Yutaka; Yamamoto, Jun-ichi; Wakamatsu, Ai; Kimura, Kouichi; Sakamoto, Katsuhiko; Hatano, Naoto; Kawai, Yuri; Ishii, Shizuko; Saito, Kaoru; Kojima, Shin-ichi; Sugiyama, Tomoyasu; Ono, Tetsuyoshi; Okano, Kazunori; Yoshikawa, Yoko; Aotsuka, Satoshi; Sasaki, Naokazu; Hattori, Atsushi; Okumura, Koji; Nagai, Keiichi; Sugano, Sumio; Isogai, Takao

2005-01-01

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

Antimycotic activity of fengycin C biosurfactant and its interaction with phosphatidylcholine model membranes.

Science.gov (United States)

González-Jaramillo, Lina María; Aranda, Francisco José; Teruel, José Antonio; Villegas-Escobar, Valeska; Ortiz, Antonio

2017-08-01

Lipopeptide biosurfactants constitute one of the most promising groups of compounds for the treatment and prevention of fungal diseases in plants. Bacillus subtilis strain EA-CB0015 produces iturin A, fengycin C and surfactin and it has been proven useful for the treatment of black Sigatoka disease in banana plants, an important pathology caused by the fungus Mycosphaerella fijiensis (Morelet). We have found that B. subtilis EA-CB0015 cell free supernatants and purified fractions inhibit M. fijiensis cellular growth. The effect of the purified lipopeptides mentioned above on fungal growth has been also evaluated, observing that iturin A and fengycin C inhibit mycelial growth and ascospore germination, whereas surfactin is not effective. On the hypothesis that the antifungal action of the lipopeptides is associated to their incorporation into biological membranes, ultimately leading to membrane permeabilization, a detailed biophysical study on the interaction of a new isoform of fengycin C with model dipalmitoyphosphatidylcholine (DPPC) membranes has been carried out. Differential scanning calorimetry shows that fengycin C alters the thermotropic phase transitions of DPPC, and is laterally segregated in the fluid bilayer forming domains. Fluorescent probe polarization measurements show that fengycin C does not affect the hydrophobic interior of the membrane. This latter perturbation is concomitant with a strong dehydration of the polar region of DPPC, as shown by FTIR. Fengycin-rich domains, where the surrounding DPPC molecules are highly dehydrated, may well constitute sites of membrane permeabilization leading to a leaky target membrane. These results are a solid support to explain the membrane perturbing action of fengycin, which has been related to its antifungal activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Membrane versus shell type elements in F.E. analysis of box type buildings

International Nuclear Information System (INIS)

Canetta, G.

1979-01-01

Finite element analysis of box-type buildings is discussed under typical loading conditions - gravity, seismic and temperature loads. The computation effort is recognized to be noticeably different, according to whether membrane or shell type elements are used. The relevance of membrane and bending stress components to the total stress distribution is outlined in the table below; the different role of the typical members under the various loading conditions is emphasized. (orig.)

Oblique propagating electromagnetic ion - Cyclotron instability with A.C. field in outer magnetosphere

Science.gov (United States)

Pandey, R. S.; Singh, Vikrant; Rani, Anju; Varughese, George; Singh, K. M.

2018-05-01

In the present paper Oblique propagating electromagnetic ion-cyclotron wave has been analyzed for anisotropic multi ion plasma (H+, He+, O+ ions) in earth magnetosphere for the Dione shell of L=7 i.e., the outer radiation belt of the magnetosphere for Loss-cone distribution function with a spectral index j in the presence of A.C. electric field. Detail for particle trajectories and dispersion relation has been derived by using the method of characteristic solution on the basis of wave particle interaction and transformation of energy. Results for the growth rate have been calculated numerically for various parameters and have been compared for different ions present in magnetosphere. It has been found that for studying the wave over wider spectrum, anisotropy for different values of j should be taken. The effect of frequency of A.C. electric field and angle which propagation vector make with magnetic field, on growth rate has been explained.

Type-1 cannabinoid receptors reduce membrane fluidity of capacitated boar sperm by impairing their activation by bicarbonate.

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Barbara Barboni

Full Text Available BACKGROUND: Mammalian spermatozoa acquire their full fertilizing ability (so called capacitation within the female genital tract, where they are progressively exposed to inverse gradients of inhibiting and stimulating molecules. METHODOLOGY/PRINCIPAL FINDINGS: In the present research, the effect on this process of anandamide, an endocannabinoid that can either activate or inhibit cannabinoid receptors depending on its concentration, and bicarbonate, an oviductal activatory molecule, was assessed, in order to study the role exerted by the type 1 cannabinoid receptor (CB1R in the process of lipid membrane remodeling crucial to complete capacitation. To this aim, boar sperm were incubated in vitro under capacitating conditions (stimulated by bicarbonate in the presence or in the absence of methanandamide (Met-AEA, a non-hydrolysable analogue of anandamide. The CB1R involvement was studied by using the specific inhibitor (SR141716 or mimicking its activation by adding a permeable cAMP analogue (8Br-cAMP. By an immunocytochemistry approach it was shown that the Met-AEA inhibits the bicarbonate-dependent translocation of CB1R from the post-equatorial to equatorial region of sperm head. In addition it was found that Met-AEA is able to prevent the bicarbonate-induced increase in membrane disorder and the cholesterol extraction, both preliminary to capacitation, acting through a CB1R-cAMP mediated pathway, as indicated by MC540 and filipin staining, EPR spectroscopy and biochemical analysis on whole membranes (CB1R activity and on membrane enriched fraction (C/P content and anisotropy. CONCLUSIONS/SIGNIFICANCE: Altogether, these data demonstrate that the endocannabinoid system strongly inhibits the process of sperm capacitation, acting as membrane stabilizing agent, thus increasing the basic knowledge on capacitation-related signaling and potentially opening new perspectives in diagnostics and therapeutics of male infertility.

Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival.

Science.gov (United States)

Caine, Jennifer A; Lin, Yi-Pin; Kessler, Julie R; Sato, Hiromi; Leong, John M; Coburn, Jenifer

2017-12-01

Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced. © 2017 John Wiley & Sons Ltd.

Self-assembling membranes and related methods thereof

Science.gov (United States)

Capito, Ramille M; Azevedo, Helena S; Stupp, Samuel L

2013-08-20

The present invention relates to self-assembling membranes. In particular, the present invention provides self-assembling membranes configured for securing and/or delivering bioactive agents. In some embodiments, the self-assembling membranes are used in the treatment of diseases, and related methods (e.g., diagnostic methods, research methods, drug screening).

Ion selectivity of the cation transport system of isolated intact cattle rod outer segments: evidence for a direct communication between the rod plasma membrane and the rod disk membranes.

Science.gov (United States)

Schnetkamp, P P

1980-05-08

The ion selectivity of cation transport through the plasma membrane of isolated intact cattle rod outer segments (rods) is investigated by means of 45Ca-exchange experiments and light-scattering experiments. These techniques appear to provide complementary information: the 45Ca experiments (45Ca fluxes in rods) describe electroneutral antiport, whereas the light-scattering experiments (shrinkage and swelling of rods upon hypertonic shocks with various electrolytes) reveal electrogenic uniport. Electroneutral symport of ions (salt transport) does not take place without addition of external ionophores and application of salts of weak acids. 1. Intact rods recover from a hypertonic shock in the presence of FCCP when lithium, sodium and potassium acetate are applied, but not when ammonium chloride, calcium and magnesium acetate are used. This indicates that the plasma membrane of isolated intact cattle rods is relatively permeable to net transport of Na+, Li+ and K+, and relatively impermeable to net transport of Cl-, Mg2+ and Ca2+ under conditions that do not give rise to diffusion potentials. 2. Rapid (t1/2 exchange diffusion of internal 45Ca with external Na+, Ca2+, Sr2+ and Ba2+, respectively. 3. All tested cations lower the rate of 45Ca uptake. The latter can be described by a single rate constant indicating a homogeneous rod preparation and a homogeneous endogenous Ca2+ pool. However, only those cations which stimulate 45Ca efflux from preloaded rods lower the final equilibrium of 45Ca uptake. Except for the effects of K+, Rb+ and Cs+ the reduction of the rate of 45Ca uptake by external cations appears to arise from competition for a common site on the plasms membrane. The observed affinities for this site do not correlate with actual transport (as indicated by the ability to stimulate 45Ca efflux). 4. K+ increases the affinity of the exchange diffusion system to Ca2+ from 1 microM to 0.15 microM and changes the relative affinities with respect to Ca2+ for the

The role of complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) in promoting C3 fragment deposition and membrane attack complex formation on normal peripheral human B cells

DEFF Research Database (Denmark)

Nielsen, Claus Henrik; Pedersen, Morten Løbner; Marquart, Hanne Vibeke Hansen

2002-01-01

Normal human B lymphocytes are known to activate the alternative pathway (AP) of complement, leading to C3-fragment deposition and membrane attack complex (MAC) formation. The process is mediated via complement receptor type 2 (CR2, CD21), with complement receptor type 1 (CR1, CD35) playing...... a subsidiary role. In this study, we examine the relative contributions of CR1 and CR2 to the deposition of C3 fragments and MAC on B lymphocytes under circumstances where all complement pathways are operational. C3-fragment deposition and MAC formation were assessed on human peripheral B lymphocytes...... in the presence of 30% autologous serum. Blocking the CR2 ligand-binding site with monoclonal antibody (mAb) FE8 resulted in significant reduction (37.9+/-11.9%) in C3-fragment deposition, whereas MAC formation was only marginally affected (12.1+/-22.2% reduction). Blocking the CR1 binding-site resulted...

A membrane wave equation for Q.C.D. (SU(infinity))

International Nuclear Information System (INIS)

Botelho, L.C.L.

1988-01-01

It is proposed a quantum membrane wave functional describing the interaction between a colored SU(N c ) membrane and a quantized Yang-Mills field. Additionally, its associated wave equation in the t'Hooft N c ->infinity limit is deduced. (A.C.A.S.) [pt

Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation.

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Ficarra, Florencia A; Grandellis, Carolina; Galván, Estela M; Ielpi, Luis; Feil, Regina; Lunn, John E; Gottig, Natalia; Ottado, Jorgelina

2017-06-01

Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild-type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Changes in Fundus Autofluorescence after Anti-vascular Endothelial Growth Factor According to the Type of Choroidal Neovascularization in Age-related Macular Degeneration.

Science.gov (United States)

Lee, Ji Young; Chung, Hyewon; Kim, Hyung Chan

2016-02-01

To describe the changes of fundus autofluorescence (FAF) in patients with age-related macular degeneration before and after intravitreal injection of anti-vascular endothelial growth factor according to the type of choroidal neovascularization (CNV) and to evaluate the correlation of FAF with spectral domain optical coherence tomography (SD-OCT) parameters and vision. This was a retrospective study. Twenty-one treatment-naïve patients with neovascular age-related macular degeneration were included. Study eyes were divided into two groups according to the type of CNV. Fourteen eyes were type 1 CNV and seven eyes were type 2 CNV. All eyes underwent a complete ophthalmologic examination, including an assessment of best-corrected visual acuity, SD-OCT, fluorescein angiography, and FAF imaging, before and 3 months after intravitreal anti-vascular endothelial growth factor injection. Gray scales of FAF image for CNV areas, delineated as in fluorescein angiography, were analyzed using the ImageJ program, which were adjusted by comparison with normal background areas. Correlation of changes in FAF with changes in SD-OCT parameters, including CNV thickness, photoreceptor inner and outer segment junction disruption length, external limiting membrane disruption length, central macular thickness, subretinal fluid, and intraretinal fluid were analyzed. Eyes with both type 1 and type 2 CNV showed reduced FAF before treatment. The mean gray scales (%) of type 1 and type 2 CNV were 52.20% and 42.55%, respectively. The background values were 106.72 and 96.86. After treatment, the mean gray scales (%) of type 1 CNV and type 2 CNV were changed to 57.61% (p = 0.005) and 57.93% (p = 0.008), respectively. After treatment, CNV thickness, central macular thickness, and inner and outer segment junction disruption length were decreased while FAF increased. FAF was noted to be reduced in eyes with newly diagnosed wet age-related macular degeneration, but increased after anti

Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase.

Science.gov (United States)

Moreno, S N; Mason, R P; Docampo, R

1984-12-10

At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.

Modeling electrically active viscoelastic membranes.

Directory of Open Access Journals (Sweden)

Sitikantha Roy

Full Text Available The membrane protein prestin is native to the cochlear outer hair cell that is crucial to the ear's amplification and frequency selectivity throughout the whole acoustic frequency range. The outer hair cell exhibits interrelated dimensional changes, force generation, and electric charge transfer. Cells transfected with prestin acquire unique active properties similar to those in the native cell that have also been useful in understanding the process. Here we propose a model describing the major electromechanical features of such active membranes. The model derived from thermodynamic principles is in the form of integral relationships between the history of voltage and membrane resultants as independent variables and the charge density and strains as dependent variables. The proposed model is applied to the analysis of an active force produced by the outer hair cell in response to a harmonic electric field. Our analysis reveals the mechanism of the outer hair cell active (isometric force having an almost constant amplitude and phase up to 80 kHz. We found that the frequency-invariance of the force is a result of interplay between the electrical filtering associated with prestin and power law viscoelasticity of the surrounding membrane. Paradoxically, the membrane viscoelasticity boosts the force balancing the electrical filtering effect. We also consider various modes of electromechanical coupling in membrane with prestin associated with mechanical perturbations in the cell. We consider pressure or strains applied step-wise or at a constant rate and compute the time course of the resulting electric charge. The results obtained here are important for the analysis of electromechanical properties of membranes, cells, and biological materials as well as for a better understanding of the mechanism of hearing and the role of the protein prestin in this mechanism.

Outer grid strap protruding spring repair apparatus

International Nuclear Information System (INIS)

Widener, W.H.

1987-01-01

This patent describes a nuclear fuel assembly grid spring repair apparatus for repairing a spring formed on an outer strap of a fuel assembly grid and having a portion protruding outwardly beyond the strap, the apparatus comprising: (a) a support frame defining an opening and having means defining a guide channel extending along the opening in a first direction; (b) means mounted on the frame and being adjustable for attaching the frame to the outer strap of the support grid so that the frame opening is aligned with the outwardly protruding spring on the outer strap; (c) an outer slide having a passageway defined therethrough and being mounted in the guide channel for reciprocable movement along the frame opening in the first direction for aligning the passageway with the outwardly protruding portion of the spring on the outer strap. The outer slide also has means defining a guide way extending along the passageway in a second direction generally orthogonal to the first direction; (d) a spring reset mechanism being operable for resetting the protruding spring to a nonprotruding position relative to the outer strap when the mechanism is aligned with the protruding portion of the spring; and (e) an inner slide supporting the spring reset mechanism and being mounted to the guide way for reciprocable movement along the passageway of the outer slide in the second direction for aligning the spring reset mechanism with the protruding portion of the spring on the outer strap

[Modification of retinal photoreceptor membranes and Ca ion binding].

Science.gov (United States)

Korchagin, V P; Berman, A L; Shukoliukov, S A; Rychkova, M P; Etingof, R N

1978-10-01

Calcium binding by modified photoreceptor membranes of cattle retina has been studied. Ca2+-binding the membranes significantly changes after C-phospholipase treatment, displaying the initial growth (less than 65% of lipid phosphorus removed) with subsequent decrease (more than 65% of phosphorus removed). Liposomes of the photoreceptor membranes lipids were found to bind more calcium than do the native photoreceptor membranes. Proteolytic enzymes (papaine, pronase) splitting some rhodopsin fragments do not affect the ability of the membrane to bind Ca2+. The increase of light-induced Ca-binding is observed only after the outer segments preincubation under conditions providing for rhodopsin phosphorylation. This effect was observed also after the splitting of the rhodopsin fragment by papaine. It is concluded that calcium binding in the photoreceptor membranes is mainly due to the phosphate groups of phospholipids.

Plasma membrane organization and dynamics is probe and cell line dependent.

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Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue

Role of the Cationic C-Terminal Segment of Melittin on Membrane Fragmentation.

Science.gov (United States)

Therrien, Alexandre; Fournier, Alain; Lafleur, Michel

2016-05-05

The widespread distribution of cationic antimicrobial peptides capable of membrane fragmentation in nature underlines their importance to living organisms. In the present work, we determined the impact of the electrostatic interactions associated with the cationic C-terminal segment of melittin, a 26-amino acid peptide from bee venom (net charge +6), on its binding to model membranes and on the resulting fragmentation. In order to detail the role played by the C-terminal charges, we prepared a melittin analogue for which the four cationic amino acids in positions 21-24 were substituted with the polar residue citrulline, providing a peptide with the same length and amphiphilicity but with a lower net charge (+2). We compared the peptide bilayer affinity and the membrane fragmentation for bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) mixtures. It is shown that neutralization of the C-terminal considerably increased melittin affinity for zwitterionic membranes. The unfavorable contribution associated with transferring the cationic C-terminal in a less polar environment was reduced, leaving the hydrophobic interactions, which drive the peptide insertion in bilayers, with limited counterbalancing interactions. The presence of negatively charged lipids (DPPS) in bilayers increased melittin binding by introducing attractive electrostatic interactions, the augmentation being, as expected, greater for native melittin than for its citrullinated analogue. The membrane fragmentation power of the peptide was shown to be controlled by electrostatic interactions and could be modulated by the charge carried by both the membrane and the lytic peptide. The analysis of the lipid composition of the extracted fragments from DPPC/DPPS bilayers revealed no lipid specificity. It is proposed that extended phase separations are more susceptible to lead to the extraction of a lipid species in a specific manner

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

Science.gov (United States)

Lai, Zengzu; Schreiber, John R

2011-05-01

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

Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Science.gov (United States)

Rossi, Omar; Pesce, Isabella; Giannelli, Carlo; Aprea, Susanna; Caboni, Mariaelena; Citiulo, Francesco; Valentini, Sara; Ferlenghi, Ilaria; MacLennan, Calman Alexander; D'Oro, Ugo; Saul, Allan; Gerke, Christiane

2014-09-05

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Further Characterization of the Capsule-Like Complex (CLC Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles

Directory of Open Access Journals (Sweden)

Anna E. Champion

2018-06-01

Full Text Available Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A is the most virulent subspecies. The live vaccine strain (LVS of subspecies holarctica produces a capsule-like complex (CLC that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylation mutant would be attenuated in mice. The CLCs isolated from LVS extracted with 0.5% phenol or 1 M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1 M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM. The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant

Design of outer-rotor type multipolar SR motor for electric vehicle

International Nuclear Information System (INIS)

Nakamura, Kenji; Suzuki, Yosuke; Goto, Hiroki; Ichinokura, Osamu

2005-01-01

In this paper, we design an outer-rotor type multipolar switched reluctance (SR) motor, and examine an application of the SR motor to an electric vehicle (EV). The design is based on a nonlinear magnetic circuit model proposed by the authors. Using the model, we can calculate dynamic characteristics of a SR motor accurately. Furthermore, by combining the nonlinear magnetic circuit model with a motor drive circuit and motion equation of an EV, we can predict dynamic characteristics such as the maximum speed, acceleration torque, and a battery current of the EV

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans

International Nuclear Information System (INIS)

Peschek, G.A.; Wastyn, M.; Trnka, M.; Molitor, V.; Fry, I.V.; Packer, L.

1989-01-01

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [ 35 S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min -1 (mg of protein) -1 , respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa 3 -type enzyme from the properties of its redox-active and EDTA-resistant Cu 2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa 3 -type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa 3 -type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme

The C-terminal HRET sequence of Kv1.3 regulates gating rather than targeting of Kv1.3 to the plasma membrane.

Science.gov (United States)

Voros, Orsolya; Szilagyi, Orsolya; Balajthy, András; Somodi, Sándor; Panyi, Gyorgy; Hajdu, Péter

2018-04-12

Kv1.3 channels are expressed in several cell types including immune cells, such as T lymphocytes. The targeting of Kv1.3 to the plasma membrane is essential for T cell clonal expansion and assumed to be guided by the C-terminus of the channel. Using two point mutants of Kv1.3 with remarkably different features compared to the wild-type Kv1.3 (A413V and H399K having fast inactivation kinetics and tetraethylammonium-insensitivity, respectively) we showed that both Kv1.3 channel variants target to the membrane when the C-terminus was truncated right after the conserved HRET sequence and produce currents identical to those with a full-length C-terminus. The truncation before the HRET sequence (NOHRET channels) resulted in reduced membrane-targeting but non-functional phenotypes. NOHRET channels did not display gating currents, and coexpression with wild-type Kv1.3 did not rescue the NOHRET-A413V phenotype, no heteromeric current was observed. Interestingly, mutants of wild-type Kv1.3 lacking HRET(E) (deletion) or substituted with five alanines for the HRET(E) motif expressed current indistinguishable from the wild-type. These results demonstrate that the C-terminal region of Kv1.3 immediately proximal to the S6 helix is required for the activation gating and conduction, whereas the presence of the distal region of the C-terminus is not exclusively required for trafficking of Kv1.3 to the plasma membrane.

Transfer of fatty acids from the 1-position of phosphatidyl-ethanolamine to the major outer membrane lipoprotein of E coli

International Nuclear Information System (INIS)

Jackowski, S.; Rock, C.O.

1986-01-01

The fatty acids esterified to Braun's lipoprotein are derived from the phospholipid pool in E. coli. Mutants lacking acyl-CoA synthetase activity (fadD) incorporated extracellular fatty acids specifically into the 1-position of phosphatidylethanolamine (PtdEtn). This pathway was blocked by chloramphenicol and was depressed by preventing the acylation of the amino terminus of the lipoprotein with globomycin. Transfer of fatty acids to lipoprotein was investigated in fadD mutants harboring hybrid plasmids containing either the lipoprotein gene or a lipoprotein-β-lactamase gene fusion under control of the lactose promoter. Labeling of the 1-position of the PtdEtn pool prior to induction of lipoprotein biosynthesis resulted in the transfer of fatty acids from PtdEtn to the lipoproteins. Induction of lipoprotein synthesis in the presence of exogenous [1- 14 C]palmitate increased the amount of radioactivity entering the PtdEtn pool and efficiently labeled lipoprotein acyl moieties. Lipoprotein fatty acids derived from the 1-position of PtdEtn were resistant to hydroxylamine hydrolysis, and globomycin reduced the incorporation of exogenous [1- 14 C]palmitic acid into lipoproteins by 80% suggesting that the fatty acid is attached to the amino terminus. These data illustrate the metabolic relationship between turnover of fatty acids in the 1-position of PtdEtn and the maturation of the major outer membrane lipoprotein

In Situ Spectral Kinetics of Cr(VI) Reduction by c-Type Cytochromes in A Suspension of Living Shewanella putrefaciens 200

Science.gov (United States)

Liu, Tongxu; Li, Xiaomin; Li, Fangbai; Han, Rui; Wu, Yundang; Yuan, Xiu; Wang, Ying

2016-07-01

Although c-type cytochromes (c-Cyts) mediating metal reduction have been mainly investigated with in vitro purified proteins of dissimilatory metal reducing bacteria, the in vivo behavior of c-Cyts is still unclear given the difficulty in measuring the proteins of intact cells. Here, c-Cyts in living Shewanella putrefaciens 200 (SP200) was successfully quantified using diffuse-transmission UV/Vis spectroscopy due to the strong absorbance of hemes, and the in situ spectral kinetics of Cr(VI) reduction by c-Cyts were examined over time. The reduced product Cr(III) observed on the cell surface may play a role in inhibiting the Cr(VI) reduction and reducing the cell numbers with high concentrations (>200 μM) of Cr(VI) evidenced by the 16S rRNA analysis. A brief kinetic model was established with two predominant reactions, redox transformation of c-Cyts and Cr(VI) reduction by reduced c-Cyts, but the fitting curves were not well-matched with c-Cyts data. The Cr(III)-induced inhibitory effect to the cellular function of redox transformation of c-Cyts was then added to the model, resulting in substantially improved the model fitting. This study provides a case of directly examining the reaction properties of outer-membrane enzyme during microbial metal reduction processes under physiological conditions.

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways.

Science.gov (United States)

Qu, Yan; Dubyak, George R

2009-06-01

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell-cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies.

SiC-BASED HYDROGEN SELECTIVE MEMBRANES FOR WATER-GAS-SHIFT REACTION; F

International Nuclear Information System (INIS)

Paul K.T. Liu

2001-01-01

This technical report summarizes our activities conducted in Yr II. In Yr I we successfully demonstrated the feasibility of preparing the hydrogen selective SiC membrane with a chemical vapor deposition (CVD) technique. In addition, a SiC macroporous membrane was fabricated as a substrate candidate for the proposed SiC membrane. In Yr II we have focused on the development of a microporous SiC membrane as an intermediate layer between the substrate and the final membrane layer prepared from CVD. Powders and supported thin silicon carbide films (membranes) were prepared by a sol-gel technique using silica sol precursors as the source of silicon, and phenolic resin as the source of carbon. The powders and films were prepared by the carbothermal reduction reaction between the silica and the carbon source. The XRD analysis indicates that the powders and films consist of SiC, while the surface area measurement indicates that they contain micropores. SEM and AFM studies of the same films also validate this observation. The powders and membranes were also stable under different corrosive and harsh environments. The effects of these different treatments on the internal surface area, pore size distribution, and transport properties, were studied for both the powders and the membranes using the aforementioned techniques and XPS. Finally the SiC membrane materials are shown to have satisfactory hydrothermal stability for the proposed application. In Yr III, we will focus on the demonstration of the potential benefit using the SiC membrane developed from Yr I and II for the water-gas-shift (WGS) reaction

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

International Nuclear Information System (INIS)

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

2014-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-08-01

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

Immune dysfunction in Niemann?Pick disease type C

OpenAIRE

Platt, Nick; Speak, Annelise O.; Colaco, Alexandria; Gray, James; Smith, David A.; Williams, Ian M.; Wallom, Kerri?Lee; Platt, Frances M.

2015-01-01

Abstract Lysosomal storage diseases are inherited monogenic disorders in which lysosome function is compromised. Although individually very rare, they occur at a collective frequency of approximately one in five thousand live births and usually have catastrophic consequences for health. The lysosomal storage diseases Niemann?Pick disease type C (NPC) is caused by mutations predominantly in the lysosomal integral membrane protein NPC1 and clinically presents as a progressive neurodegenerative ...

Differences in outer membrane proteins of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis

International Nuclear Information System (INIS)

Batteiger, B.E.; Jones, R.B.

1985-01-01

The lymphogranuloma venereum (LGV) and trachoma biovars of Chlamydia trachomatis exhibit differences in biological properties both in vivo and in vitro. To identify analogous biochemical differences, the authors studied the molecular charges of chlamydial outer membrane proteins (OMPs) by means of isoelectric focusing and nonequilibrium pH gradient electrophoresis. Analysis of proteins of whole elementary bodies biosynthetically labeled with L-[35S]cysteine revealed that most chlamydial proteins were neutral or acidic. The major OMPs (MOMPs) of all strains tested were acidic and had apparent isoelectric points (pIs) that varied within narrow limits despite differences in molecular mass of up to 3,000 daltons (Da). However, a low-molecular-mass cysteine-rich OMP analogous to that previously described for Chlamydia psittaci varied consistently in molecular mass (12,500 versus 12,000 Da) and pI (5.4 versus 6.9) between LGV strains and trachoma strains, respectively. OMPs with a molecular mass of 60,000 Da in the trachoma biovar strains had pIs in the 7.3 to 7.7 range. However, analogous OMPs in the LGV strains existed as a doublet with a molecular mass of about 60,000 Da. These data indicate substantial differences in biochemical characteristics of analogous OMPs in the LGV and trachoma biovars. Such differences are the first structural differences described between LGV and trachoma strains which support their distinction into separate biovars and may be related to some of their biological differences

Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

International Nuclear Information System (INIS)

Witt, P.L.; Bownds, M.D.

1987-01-01

Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger

Age-related collagen turnover of the interstitial matrix and basement membrane: Implications of age- and sex-dependent remodeling of the extracellular matrix.

Science.gov (United States)

Kehlet, Stephanie N; Willumsen, Nicholas; Armbrecht, Gabriele; Dietzel, Roswitha; Brix, Susanne; Henriksen, Kim; Karsdal, Morten A

2018-01-01

The extracellular matrix (ECM) plays a vital role in maintaining normal tissue function. Collagens are major components of the ECM and there is a tight equilibrium between degradation and formation of these proteins ensuring tissue health and homeostasis. As a consequence of tissue turnover, small collagen fragments are released into the circulation, which act as important biomarkers in the study of certain tissue-related remodeling factors in health and disease. The aim of this study was to establish an age-related collagen turnover profile of the main collagens of the interstitial matrix (type I and III collagen) and basement membrane (type IV collagen) in healthy men and women. By using well-characterized competitive ELISA-assays, we assessed specific fragments of degraded (C1M, C3M, C4M) and formed (PINP, Pro-C3, P4NP7S) type I, III and IV collagen in serum from 617 healthy men and women ranging in ages from 22 to 86. Subjects were divided into 5-year age groups according to their sex and age. Groups were compared using Kruskal-Wallis adjusted for Dunn's multiple comparisons test and Mann-Whitney t-test. Age-specific changes in collagen turnover was most profound for type I collagen. PINP levels decreased in men with advancing age, whereas in women, the level decreased in early adulthood followed by an increase around the age of menopause (age 40-60). Sex-specific changes in type I, III and IV collagen turnover was present at the age around menopause (age 40-60) with women having an increased turnover. In summary, collagen turnover is affected by age and sex with the interstitial matrix and the basement membrane being differently regulated. The observed changes needs to be accounted for when measuring ECM related biomarkers in clinical studies.

Mitochondrial outer membrane permeabilization increases reactive oxygen species production and decreases mean sperm velocity but is not associated with DNA fragmentation in human sperm.

Science.gov (United States)

Treulen, F; Uribe, P; Boguen, R; Villegas, J V

2016-02-01

Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa? Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity. MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed. The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry. MOMP was associated with ΔΨm dissipation (P < 0.05), increased ROS production (P < 0.05) and decreased mean sperm velocity (P < 0.05), but it was not associated with DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions. The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors. The results obtained in this

Characterization of retrokeratoprosthetic membranes in the Boston type 1 keratoprosthesis.

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Stacy, Rebecca C; Jakobiec, Frederick A; Michaud, Norman A; Dohlman, Claes H; Colby, Kathryn A

2011-03-01

To evaluate retroprosthetic membranes that can occur in 25% to 65% of patients with the Boston type 1 keratoprosthesis (KPro). Two patients with Peter anomaly and 2 with neurotrophic scarred corneas underwent revisions of their type 1 KPros because of visually compromising retroprosthetic membranes. The excised membranes were studied by light microscopy with hematoxylin-eosin, periodic acid-Schiff, and toluidine blue stains. Immunohistochemical and transmission electron microscopic examination were also used. Light microscopic examination revealed that the retro-KPro fibrous membranes originated from the host's corneal stroma. These mildly to moderately vascularized membranes grew through gaps in the Descemet membrane to reach behind the KPro back plate and adhere to the anterior iris surface, which had undergone partial lysis. In 2 cases, the fibrous membranes merged at the pupil with matrical portions of metaplastic lens epithelium, forming a bilayered structure that crossed the optical axis. Retro-KPro membranes stained positively for α-smooth muscle actin but negatively for pancytokeratin. Electron microscopy confirmed the presence of actin filaments within myofibroblasts and small surviving clusters of metaplastic lens epithelial cells. Stromal downgrowth, rather than epithelial downgrowth, was the major element of the retro-KPro membranes in this series. Metaplastic lens epithelium also contributed to opacification of the visual axis. Florid membranous inflammation was not a prominent finding and thus probably not a requisite stimulus for membrane development. Further advances in prosthetic design and newer antifibroproliferative agents may reduce membrane formation.

A distance measurement between specific sites on the cytoplasmic surface of bovine rhodopsin in rod outer segment disk membranes.

Science.gov (United States)

Albert, A D; Watts, A; Spooner, P; Groebner, G; Young, J; Yeagle, P L

1997-08-14

Structural information on mammalian integral membrane proteins is scarce. As part of work on an alternative approach to the structure of bovine rhodopsin, a method was devised to obtain an intramolecular distance between two specific sites on rhodopsin while in the rod outer segment disk membrane. In this report, the distance between the rhodopsin kinase phosphorylation site(s) on the carboxyl terminal and the top of the third transmembrane helix was measured on native rhodopsin. Rhodopsin was labeled with a nuclear spin label (31P) by limited phosphorylation with rhodopsin kinase. Major phosphorylation occurs at serines 343 and 338 on the carboxyl terminal. The phosphorylated rhodopsin was then specifically labeled on cysteine 140 with an electron spin label. Magic angle spinning 31P-nuclear magnetic resonance revealed the resonance arising from the phosphorylated protein. The enhancement of the transverse relaxation of this resonance by the paramagnetic spin label was observed. The strength of this perturbation was used to determine the through-space distance between the phosphorylation site(s) and the spin label position. A distance of 18 +/- 3 A was obtained.

Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS

Science.gov (United States)

Dunstan, Rhys A.; Heinz, Eva; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Purcell, Anthony W.; Evans, Timothy J.; Praszkier, Judyta; Robins-Browne, Roy M.; Strugnell, Richard A.; Korotkov, Konstantin V.; Lithgow, Trevor

2013-01-01

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS. PMID:23326233

cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

Science.gov (United States)

De Jonge, Hugo R.; Tilly, Ben C.; Hogema, Boris M.; Pfau, Daniel J.; Kelley, Catherine A.; Kelley, Megan H.; Melita, August M.; Morris, Montana T.; Viola, Ryan M.

2013-01-01

The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG. PMID:24259420

Structural adaptations of proteins to different biological membranes

Science.gov (United States)

Pogozheva, Irina D.; Tristram-Nagle, Stephanie; Mosberg, Henry I.; Lomize, Andrei L.

2013-01-01

To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30-31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5 Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20-22 Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3-4 Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5-8 Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds. PMID:23811361

Increase in average foveal thickness after internal limiting membrane peeling

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Kumagai K

2017-04-01

Full Text Available Kazuyuki Kumagai,1 Mariko Furukawa,1 Tetsuyuki Suetsugu,1 Nobuchika Ogino2 1Department of Ophthalmology, Kami-iida Daiichi General Hospital, 2Department of Ophthalmology, Nishigaki Eye Clinic, Aichi, Japan Purpose: To report the findings in three cases in which the average foveal thickness was increased after a thin epiretinal membrane (ERM was removed by vitrectomy with internal limiting membrane (ILM peeling.Methods: The foveal contour was normal preoperatively in all eyes. All cases underwent successful phacovitrectomy with ILM peeling for a thin ERM. The optical coherence tomography (OCT images were examined before and after the surgery. The changes in the average foveal (1 mm thickness and the foveal areas within 500 µm from the foveal center were measured. The postoperative changes in the inner and outer retinal areas determined from the cross-sectional OCT images were analyzed.Results: The average foveal thickness and the inner and outer foveal areas increased significantly after the surgery in each of the three cases. The percentage increase in the average foveal thickness relative to the baseline thickness was 26% in Case 1, 29% in Case 2, and 31% in Case 3. The percentage increase in the foveal inner retinal area was 71% in Case 1, 113% in Case 2, and 110% in Case 3, and the percentage increase in foveal outer retinal area was 8% in Case 1, 13% in Case 2, and 18% in Case 3.Conclusion: The increase in the average foveal thickness and the inner and outer foveal areas suggests that a centripetal movement of the inner and outer retinal layers toward the foveal center probably occurred due to the ILM peeling. Keywords: internal limiting membrane, optical coherence tomography, average foveal thickness, epiretinal membrane, vitrectomy

Transmission-Blocking Antibodies against Mosquito C-Type Lectins for Dengue Prevention

Science.gov (United States)

Liu, Yang; Zhang, Fuchun; Liu, Jianying; Xiao, Xiaoping; Zhang, Siyin; Qin, Chengfeng; Xiang, Ye; Wang, Penghua; Cheng, Gong

2014-01-01

C-type lectins are a family of proteins with carbohydrate-binding activity. Several C-type lectins in mammals or arthropods are employed as receptors or attachment factors to facilitate flavivirus invasion. We previously identified a C-type lectin in Aedes aegypti, designated as mosquito galactose specific C-type lectin-1 (mosGCTL-1), facilitating the attachment of West Nile virus (WNV) on the cell membrane. Here, we first identified that 9 A. aegypti mosGCTL genes were key susceptibility factors facilitating DENV-2 infection, of which mosGCTL-3 exhibited the most significant effect. We found that mosGCTL-3 was induced in mosquito tissues with DENV-2 infection, and that the protein interacted with DENV-2 surface envelop (E) protein and virions in vitro and in vivo. In addition, the other identified mosGCTLs interacted with the DENV-2 E protein, indicating that DENV may employ multiple mosGCTLs as ligands to promote the infection of vectors. The vectorial susceptibility factors that facilitate pathogen invasion may potentially be explored as a target to disrupt the acquisition of microbes from the vertebrate host. Indeed, membrane blood feeding of antisera against mosGCTLs dramatically reduced mosquito infective ratio. Hence, the immunization against mosGCTLs is a feasible approach for preventing dengue infection. Our study provides a future avenue for developing a transmission-blocking vaccine that interrupts the life cycle of dengue virus and reduces disease burden. PMID:24550728

Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera.

Science.gov (United States)

Hoelzle, Ludwig E; Hoelzle, Katharina; Wittenbrink, Max M

2004-10-05

Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-10-31

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

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Outer magnetosphere

International Nuclear Information System (INIS)

Schardt, A.W.; Behannon, K.W.; Lepping, R.P.; Carbary, J.F.; Eviatar, A.; Siscoe, G.L.

1984-01-01

Similarities between the Saturnian and terrestrial outer magnetosphere are examined. Saturn, like earth, has a fully developed magnetic tail, 80 to 100 RS in diameter. One major difference between the two outer magnetospheres is the hydrogen and nitrogen torus produced by Titan. This plasma is, in general, convected in the corotation direction at nearly the rigid corotation speed. Energies of magnetospheric particles extend to above 500 keV. In contrast, interplanetary protons and ions above 2 MeV have free access to the outer magnetosphere to distances well below the Stormer cutoff. This access presumably occurs through the magnetotail. In addition to the H+, H2+, and H3+ ions primarily of local origin, energetic He, C, N, and O ions are found with solar composition. Their flux can be substantially enhanced over that of interplanetary ions at energies of 0.2 to 0.4 MeV/nuc

The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

Science.gov (United States)

Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

2014-01-01

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes.

Science.gov (United States)

Agarwal, Shailesh R; Gratwohl, Jackson; Cozad, Mia; Yang, Pei-Chi; Clancy, Colleen E; Harvey, Robert D

2018-01-01

Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes. Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that β-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment. Conclusion: These results suggest that different membrane

Influence of membrane fluidity on human immunodeficiency virus type 1 entry

International Nuclear Information System (INIS)

Harada, Shinji; Yusa, Keisuke; Monde, Kazuaki; Akaike, Takaaki; Maeda, Yosuke

2005-01-01

For penetration of human immunodeficiency virus type 1 (HIV-1), formation of fusion-pores might be required for accumulating critical numbers of fusion-activated gp41, followed by multiple-site binding of gp120 with receptors, with the help of fluidization of the plasma membrane and viral envelope. Correlation between HIV-1 infectivity and fluidity was observed by treatment of fluidity-modulators, indicating that infectivity was dependent on fluidity. A 5% decrease in fluidity suppressed the HIV-1 infectivity by 56%. Contrarily, a 5% increase in fluidity augmented the infectivity by 2.4-fold. An increased temperature of 40 deg C or treatment of 0.2% xylocaine after viral adsorption at room temperature enhanced the infectivity by 2.6- and 1.5-fold, respectively. These were inhibited by anti-CXCR4 peptide, implying that multiple-site binding was accelerated at 40 deg C or by xylocaine. Thus, fluidity of both the plasma membrane and viral envelope was required to form the fusion-pore and to complete the entry of HIV-1

Superdiffusive motion of membrane-targeting C2 domains

Science.gov (United States)

Campagnola, Grace; Nepal, Kanti; Schroder, Bryce W.; Peersen, Olve B.; Krapf, Diego

2015-12-01

Membrane-targeting domains play crucial roles in the recruitment of signalling molecules to the plasma membrane. For most peripheral proteins, the protein-to-membrane interaction is transient. After proteins dissociate from the membrane they have been observed to rebind following brief excursions in the bulk solution. Such membrane hops can have broad implications for the efficiency of reactions on membranes. We study the diffusion of membrane-targeting C2 domains using single-molecule tracking in supported lipid bilayers. The ensemble-averaged mean square displacement (MSD) exhibits superdiffusive behaviour. However, traditional time-averaged MSD analysis of individual trajectories remains linear and does not reveal superdiffusion. Our observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution. These hopping events allow proteins to explore large areas in a short time. The experimental results are shown to be consistent with analytical models of bulk-mediated diffusion and numerical simulations.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors.

Science.gov (United States)

Benedicto, Ignacio; Lehmann, Guillermo L; Ginsberg, Michael; Nolan, Daniel J; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M; Prusky, Glen T; Llanos, Pierre; Rabbany, Sina Y; Maminishkis, Arvydas; Miller, Sheldon S; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-05-19

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.

Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

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Fulcher, Robert A; Cole, Leah E; Janowicz, Diane M; Toffer, Kristen L; Fortney, Kate R; Katz, Barry P; Orndorff, Paul E; Spinola, Stanley M; Kawula, Thomas H

2006-05-01

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

Niemann-Pick type C disease: molecular mechanisms and potential therapeutic approaches

OpenAIRE

Rosenbaum, Anton I.; Maxfield, Frederick R.

2011-01-01

Cholesterol is an important lipid of mammalian cells. Its unique physicochemical properties modulate membrane behavior and it serves as the precursor for steroid hormones, oxysterols and vitamin D. Cholesterol is effluxed from the late endosomes/lysosomes via the concerted action of at least two distinct proteins: Niemann-Pick C1 and Niemann-Pick C2. Mutations in these two proteins manifest as Niemann-Pick type C disease – a very rare, usually fatal, autosomal, recessive, neurovisceral, lysos...

Cell membrane disruption stimulates cAMP and Ca2+ signaling to potentiate cell membrane resealing in neighboring cells

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Tatsuru Togo

2017-12-01

Full Text Available Disruption of cellular plasma membranes is a common event in many animal tissues, and the membranes are usually rapidly resealed. Moreover, repeated membrane disruptions within a single cell reseal faster than the initial wound in a protein kinase A (PKA- and protein kinase C (PKC-dependent manner. In addition to wounded cells, recent studies have demonstrated that wounding of Madin-Darby canine kidney (MDCK cells potentiates membrane resealing in neighboring cells in the short-term by purinergic signaling, and in the long-term by nitric oxide/protein kinase G signaling. In the present study, real-time imaging showed that cell membrane disruption stimulated cAMP synthesis and Ca2+ mobilization from intracellular stores by purinergic signaling in neighboring MDCK cells. Furthermore, inhibition of PKA and PKC suppressed the ATP-mediated short-term potentiation of membrane resealing in neighboring cells. These results suggest that cell membrane disruption stimulates PKA and PKC via purinergic signaling to potentiate cell membrane resealing in neighboring MDCK cells.

[Ultrastructural organization of cytoplasmatic membrane of Anaerobacter polyendosporus studied by electron microscopic cryofractography].

Science.gov (United States)

Duda, V I; Suzina, N E; Dmitriev, V V

2001-01-01

Anaerobacter polyendosporus cells do not have typical mesosomes. However, the analysis of this anaerobic multispore bacterium by electron microscopic cryofractography showed that its cytoplasmic membrane contains specific intramembrane structures in the form of flat lamellar inverted lipid membranes tenths of nanometers to several microns in size. It was found that these structures are located in the hydrophobic interior between the outer and inner leaflets of the cytoplasmic membrane and do not contain intramembrane particles that are commonly present on freeze-fracture replicas. The flat inverted lipid membranes were revealed in bacterial cells cultivated under normal growth conditions, indicating the existence of a complex-type compartmentalization in biological membranes, which manifests itself in the formation of intramembrane compartments having the appearance of vesicles and inverted lipid membranes.

Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

Science.gov (United States)

Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

2009-05-01

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

Transcriptional responses of Leptospira interrogans to host innate immunity: significant changes in metabolism, oxygen tolerance, and outer membrane.

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Feng Xue

Full Text Available BACKGROUND: Leptospira interrogans is the major causative agent of leptospirosis. Phagocytosis plays important roles in the innate immune responses to L. interrogans infection, and L. interrogans can evade the killing of phagocytes. However, little is known about the adaptation of L. interrogans during this process. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the interaction of pathogenic Leptospira and innate immunity, we employed microarray and comparative genomics analyzing the responses of L. interrogans to macrophage-derived cells. During this process, L. interrogans altered expressions of many genes involved in carbohydrate and lipid metabolism, energy production, signal transduction, transcription and translation, oxygen tolerance, and outer membrane proteins. Among them, the catalase gene expression was significantly up-regulated, suggesting it may contribute to resisting the oxidative pressure of the macrophages. The expressions of several major outer membrane protein (OMP genes (e.g., ompL1, lipL32, lipL41, lipL48 and ompL47 were dramatically down-regulated (10-50 folds, consistent with previous observations that the major OMPs are differentially regulated in vivo. The persistent down-regulations of these major OMPs were validated by immunoblotting. Furthermore, to gain initial insight into the gene regulation mechanisms in L. interrogans, we re-defined the transcription factors (TFs in the genome and identified the major OmpR TF gene (LB333 that is concurrently regulated with the major OMP genes, suggesting a potential role of LB333 in OMPs regulation. CONCLUSIONS/SIGNIFICANCE: This is the first report on global responses of pathogenic Leptospira to innate immunity, which revealed that the down-regulation of the major OMPs may be an immune evasion strategy of L. interrogans, and a putative TF may be involved in governing these down-regulations. Alterations of the leptospiral OMPs up interaction with host antigen

Pseudomonas aeruginosa outer membrane vesicles triggered by human mucosal fluid and lysozyme can prime host tissue surfaces for bacterial adhesion

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Matteo Maria Emiliano Metruccio

2016-06-01

Full Text Available Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release Outer Membrane Vesicles (OMVs in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to PBS controls (~100 fold. TEM and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (~4-fold, P < 0.01. Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections.

Molecular analysis of interactions between dendrimers and asymmetric membranes at different transport stages.

Science.gov (United States)

He, XiaoCong; Qu, ZhiGuo; Xu, Feng; Lin, Min; Wang, JiuLing; Shi, XingHua; Lu, TianJian

2014-01-07

Studying dendrimer-biomembrane interactions is important for understanding drug and gene delivery. In this study, coarse-grained molecular dynamics simulations were performed to investigate the behaviors of polyamidoamine (PAMAM) dendrimers (G4 and G5) as they interacted with asymmetric membranes from different sides of the bilayer, thus mimicking different dendrimer transport stages. The G4 dendrimer could insert into the membrane during an equilibrated state, and the G5 dendrimer could induce pore formation in the membrane when the dendrimers interacted with the outer side (outer interactions) of an asymmetric membrane [with 10% dipalmitoyl phosphatidylserine (DPPS) in the inner leaflet of the membrane]. During the interaction with the inner side of the asymmetric membrane (inner interactions), the G4 and G5 dendrimers only adsorbed onto the membrane. As the membrane asymmetry increased (e.g., increased DPPS percentage in the inner leaflet of the membrane), the G4 and G5 dendrimers penetrated deeper into the membrane during the outer interactions and the G4 and G5 dendrimers were adsorbed more tightly onto the membrane for the inner interactions. When the DPPS content reached 50%, the G4 dendrimer could completely penetrate through the membrane from the outer side to the inner side. Our study provides molecular understanding and reference information about different dendrimer transport stages during drug and gene delivery.

The Outer Membrane Protein OmpW Forms an Eight-Stranded beta-Barrel with a Hydrophobic Channel

International Nuclear Information System (INIS)

Hong, H.; Patel, D.; Tamm, L.; van den Berg, B.

2006-01-01

Escherichia coli OmpW belongs to a family of small outer membrane (OM) proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. In order to gain insight into the function of these proteins we have determined the crystal structure of Escherichia coli OmpW to 2.7 Angstroms resolution. The structure shows that OmpW forms an eight-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound LDAO detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of LDAO. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial OM

Mitofilin complexes: conserved organizers of mitochondrial membrane architecture.

Science.gov (United States)

Zerbes, Ralf M; van der Klei, Ida J; Veenhuis, Marten; Pfanner, Nikolaus; van der Laan, Martin; Bohnert, Maria

2012-11-01

Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membrane, mitofilins are part of hetero-oligomeric protein complexes that have been termed the mitochondrial inner membrane organizing system (MINOS). MINOS integrity is required for the maintenance of the characteristic morphology of the inner mitochondrial membrane, with an inner boundary region closely apposed to the outer membrane and cristae membranes, which form large tubular invaginations that protrude into the mitochondrial matrix and harbor the enzyme complexes of the oxidative phosphorylation machinery. MINOS deficiency comes along with a loss of crista junction structures and the detachment of cristae from the inner boundary membrane. MINOS has been conserved in evolution from unicellular eukaryotes to humans, where alterations of MINOS subunits are associated with multiple pathological conditions.

Genetic Analysis of the Mode of Interplay between an ATPase Subunit and Membrane Subunits of the Lipoprotein-Releasing ATP-Binding Cassette Transporter LolCDE†

OpenAIRE

Ito, Yasuko; Matsuzawa, Hitomi; Matsuyama, Shin-ichi; Narita, Shin-ichiro; Tokuda, Hajime

2006-01-01

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the i...

Loss of elongation factor P disrupts bacterial outer membrane integrity

DEFF Research Database (Denmark)

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

2012-01-01

background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious......Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants......, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits...

Selective association of outer surface lipoproteins with the lipid rafts of Borrelia burgdorferi.

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Toledo, Alvaro; Crowley, Jameson T; Coleman, James L; LaRocca, Timothy J; Chiantia, Salvatore; London, Erwin; Benach, Jorge L

2014-03-11

Borrelia burgdorferi contains unique cholesterol-glycolipid-rich lipid rafts that are associated with lipoproteins. These complexes suggest the existence of macromolecular structures that have not been reported for prokaryotes. Outer surface lipoproteins OspA, OspB, and OspC were studied for their participation in the formation of lipid rafts. Single-gene deletion mutants with deletions of ospA, ospB, and ospC and a spontaneous gene mutant, strain B313, which does not express OspA and OspB, were used to establish their structural roles in the lipid rafts. All mutant strains used in this study produced detergent-resistant membranes, a common characteristic of lipid rafts, and had similar lipid and protein slot blot profiles. Lipoproteins OspA and OspB but not OspC were shown to be associated with lipid rafts by transmission electron microscopy. When the ability to form lipid rafts in live B. burgdorferi spirochetes was measured by fluorescence resonance energy transfer (FRET), strain B313 showed a statistically significant lower level of segregation into ordered and disordered membrane domains than did the wild-type and the other single-deletion mutants. The transformation of a B313 strain with a shuttle plasmid containing ospA restored the phenotype shared by the wild type and the single-deletion mutants, demonstrating that OspA and OspB have redundant functions. In contrast, a transformed B313 overexpressing OspC neither rescued the FRET nor colocalized with the lipid rafts. Because these lipoproteins are expressed at different stages of the life cycle of B. burgdorferi, their selective association is likely to have an important role in the structure of prokaryotic lipid rafts and in the organism's adaptation to changing environments. IMPORTANCE Lipid rafts are cholesterol-rich clusters within the membranes of cells. Lipid rafts contain proteins that have functions in sensing the cell environment and transmitting signals. Although selective proteins are present in

Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope.

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Preetinder K Dhanoa

Full Text Available BACKGROUND: Tail-anchored (TA proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34 and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9. Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. CONCLUSIONS/SIGNIFICANCE: Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie

Detection of nearest neighbors to specific fluorescently tagged ligands in rod outer segment and lymphocyte plasma membranes by photosensitization of 5-iodonaphthyl 1-azide

International Nuclear Information System (INIS)

Raviv, Y.; Bercovici, T.; Gitler, C.; Salomon, Y.

1989-01-01

Lima bean agglutinin-fluorescein 5-isothiocyanate conjugate (FluNCS-lima bean lectin) interacts with specific receptor molecules on membranes both from the rod outer segment (ROS) of the frog retina and from S49 mouse lymphoma cells. When [125I]-5-iodonaphthyl 1-azide (125I-INA), which freely and randomly partitions into the lipid bilayer, is added to membranes and the suspension is irradiated at 480 nm, the FluNCS-conjugated lectin photosensitizes the [125I]INA but only at discrete sites. This results in the selective labeling of specific proteins: an 88-kDa protein on ROS membranes and a 56-kDa protein on S49 plasma membranes. Labeling is dependent upon the interaction of the FluNCS-lectin with glycosylated receptor sites, since N-acetylgalactosamine, but not methyl alpha-mannoside, blocked labeling of the 56-kDa protein on S49 membranes. In contrast, a random labeling pattern of membrane proteins was observed upon irradiation at 480 nm using other fluorescein conjugates, such as FluNCS-bovine serum albumin (FluNCS-BSA) or FluNCS-soybean trypsin inhibitor (FluNCS-STI), which interact with cell membranes in a nonselective manner, or with N-(fluorescein-5-thiocarbamoyl)-n-undecyclamine (FluNCS-NHC11), which is freely miscible in the membrane lipid. Random labeling was also obtained by direct photoexcitation of [125I]INA at 314 nm, with no distinct labeling of the 88- and 56-kDa proteins in the respective membranes. These results suggest that protein ligands can be used to guide sensitizers to discrete receptor sites and lead to their selective labeling by photosensitized activation of [125I]INA

The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter

OpenAIRE

Modali, Sita D.; Zgurskaya, Helen I.

2011-01-01

Escherichia coli MacAB-TolC is a tri-partite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation o...

The mitochondrial outer membrane protein mitoNEET is a redox enzyme catalyzing electron transfer from FMNH2 to oxygen or ubiquinone.