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Chalton, David Allen. "Engineering of outer membrane proteins." Thesis, University of Newcastle Upon Tyne, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.430336.
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Cox, Katherine L. "Molecular dynamics of outer membrane proteins." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427881.
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Millar, Douglas G. "Assembly pathways of outer mitochondrial membrane proteins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0022/NQ29951.pdf.
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Millar, Douglas G. "Assembly pathways of outer mitochondrial membrane proteins." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=42046.
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The pathway of membrane insertion and assembly of a signal anchor sequence specific for the outer mitochondrial membrane has been investigated. Signal anchor protein insertion into the outer membrane, in vitro, was found to overlap with a general import pathway followed by the outer membrane protein, porin, as well as matrix-targeted proteins. However, signal anchor protein insertion did not require a postreceptor import step involved in porin insertion and matrix protein translocation. Also, in contrast to the membrane insertion of porin, signal anchor protein insertion did not require nucleoside triphosphates for transfer of bound precursor to the membrane assembled form. Following outer membrane integration, a hybrid protein containing the signal anchor sequence of yeast Tom70 was found to assemble into homodimers. Dimerization was mediated by the transmembrane domain. A sequence motif containing alanine residues clustered on one face of the predicted membrane-spanning $ alpha$-helix was important for dimerization, perhaps allowing favourable close packing of the transmembrane helices.
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Gagnon, Jean-Nicolas. "Molecular interactions of bacterial outer membrane proteins." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81333.
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Transport of iron-siderophores and vitamin B12 across the outer membrane (OM) of Gram-negative bacteria requires energy from the proton motive force delivered by the TonB/ExbB/ExbD complex. TonB-dependent OM receptors such as FhuA, FepA, FecA and BtuB possess a Ton box: a conserved motif located proximal to their N-terminus that has been shown to interact with TonB. However, other sites on OM receptors have been proposed to participate in interactions with TonB. To identify novel sites of interactions with TonB, we selected TonB-binding peptides from a random library of peptides displayed on phages. Fifteen peptides displayed sequence similarities to known TonB-dependent OM receptors. Of the fifteen, eight were mapped to regions of FhuA, FepA and FecA for which crystal structures are available. DNA sequences for selected peptides were fused to malE and displayed at the N-terminus of the E. coli maltose binding protein (MBP) for further characterization. Surface plasmon resonance experiments revealed that when the peptides were monovalently displayed on MBP, they retained TonB-binding activity thereby permitting assessments of their binding characteristics. In vitro and in vivo assays demonstrated that only FhuA-mapping peptides could disrupt TonB-FhuA interactions, indicating that TonB selectively binds to multiple regions distinct for each OM receptor.
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Bond, Peter J. "Simulation studies of bacterial outer membrane proteins." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.419258.
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Melillo, Amanda Adeline. "Identification of Francisella tularensis Outer Membrane Proteins." University of Toledo Health Science Campus / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=mco1121867713.
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Habib, Shukry James. "Biogenesis and function of mitochondrial outer membrane proteins." Diss., [S.l.] : [s.n.], 2006. http://edoc.ub.uni-muenchen.de/archive/00006312.
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Yue, Kevin Kin Man. "Assembly of outer membrane proteins in Escherichia coli." Thesis, University of Liverpool, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.257436.
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Crago, Aimee Marie. "Virulence-related outer membrane proteins of Salmonella typhimurium." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624399.
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Pongprayoon, Prapasiri. "Molecular modelling of β-barrel outer membrane proteins." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:0ed0c22f-027e-4be1-a64c-0819888bbebc.
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In Gram-negative bacteria, the Outer membrane (OM) acts as a first barrier to screen unwanted compounds whilst enabling ions and very small solutes to diffuse into the cell. Most of nutrients and essential ions are effectively transported across a membrane via the outer membrane proteins (OMPs). The water-filled β- barrel OMPs are called porins. These pores are classified into two groups, non- specific and substrate-specific porins. Each of them has different mechanisms to facilitate its substrate translocation. To reveal the process of substrate permeation and selectivity in microscopic detail, molecular dynamics (MD) simulations and applications were performed in this thesis. The studies in this thesis focus on a series of classical porins. These proteins share similar feature where extracellular loop(s) (generally loop 3 (L3)) is folded into the middle of the pore and act as a constriction site which is important for substrate specificity and selectivity. The studies firstly concentrate on the structural properties and dynamics of the general trimeric porins, OmpC and OmpF whose sequences share 60% identity. OmpC and OmpF are found to have similar mechanism of latching loop (L2) to maintain trimeric stability. The smaller pore size allows OmpC to be more cation-selective than OmpF. Additionally, the major driving force for cation permeation in both porins is not from electrostatic properties. This differs from the phosphate-selective porin, trimeric OprP, where a phosphate diffusion depends on electrostatic interactions with positively charged pore-lining residues. The charge brush-like behavior of interior Arg and Lys residues plays a major role in phosphate selectivity. Also, the free energy profiles (PMF) reveal two key regions that are important for differentiating phosphate from other anions. The brush-like mechanism of OprP were also implanted to the simplified model pores in order to determine the possibility of transferring phosphate-selective properties of OprP to a model which may be useful for future design of nanopores. It is found that the duplication of functional residues and pore cavity can turn a model into the highly phosphate-selective pore. Importantly, the phosphate-binding affinity is dependent on the ability of the pore to interfere and occupy the hydration shell of a translocating phosphate where such ability can be maximized by an increase in sidechain flexibility. In case of uptake of more complex substrates, OpdK also employs a constriction site to select its substrate, aromatic vanillate (VNL) with total charge of -1. Unlike ion-specific porins, the free VNL is attracted by polar and aromatic interactions and sequentially directed through the periplasmic vestibule by charged residues insides the pore. The correct orientation of VNL on arrival is crucial for OpdK to recognize and enable the permeation process.
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Sun, Eileen Soomie. "Subfamily I Treponema pallidum repeat proteins : sequence variation and immunity /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/9305.
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Moslavac, Suncana. "Outer membrane proteins of Anabaena sp. strain PCC 7120." Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-72771.
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Schesser, Bartra Sara Celinda. "Outer membrane proteins of Yersinia pestis : Ail and OmpA." Doctoral thesis, Umeå universitet, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-33956.
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A vast number of studies have been completed on the virulence determinants of Yersinia spp.; however, the focus of many of these studies has been on the virulence plasmid and the plasmid-encoded Type three secretion system. Nevertheless, many chromosomal genes whose products are directly involved in virulence have also been identified. Some of these critical virulence determinants are outer membrane proteins. Outer membrane proteins of Gram-negative bacteria often have important physiological roles; however, some have also been found to be important for pathogenesis. In this thesis, we investigated two Yersinia. pestis outer membrane proteins, Ail and OmpA, and their roles in virulence. We provide evidence that Y. pestis Ail is a highly expressed outer membrane protein that is absolutely essential for Y. pestis to resist the killing action of the complement system present in human blood and tissues, as well as the blood and tissues of other mammalian hosts. Furthermore, Ail was important for virulence in a Y. pestis-Canorhabditis elegans model of infection.The work in this thesis also provided the first evidence that another surface-exposed outer membrane protein, termed OmpA, is required for both Yersinia pseudotuberculosis and Y. pestis to survive and proliferate intracellularly in macrophages. Finally, we provide evidence that Y. pestis has a functional small RNA MicA that controls the expression of OmpA. This is the first demonstration of sRNA-mediated regulation of a Yersinia virulence factor. This work has paved the way for future studies on the role of outer membrane proteins in virulence, particularly the role of Ail and OmpA.
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Findlay, Heather E. "Expression and folding of recombinant chlamydial outer membrane proteins." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/24570.
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The Chlamydiaceae are obligate intracellular pathogens of eukaryotic cells. These few species are responsible for a wide variety of human and animal diseases, including trachoma, sexually transmitted genital infections, pneumonia and abortion. Female infertility secondary to chronic pelvic inflammation is a particularly important and relatively common complication of C. trachomatis infection. The cysteine-rich Major Outer Membrane Protein (MOMP) is the immunodominant chlamydial protein and a primary vaccine target. It is predicted to function as a general diffusion porin that may also, through intra- and/or intermolecular disulphide bond formation, play an important structural role in maintaining stability of the organism in the absence of a peptideoglycan layer. However, vaccine development and examining the specific biochemistry of MOMP in situ is problematic due to difficulties involved in culturing the organisms and the presence of other chlamydial membrane proteins. A dual approach was taken to develop a recombinant system to express MOMP in E. coli. Full length MOMP expressed with the E. coli ompT signal sequence was shown to be successfully transported to the outer membrane of the cell. A proportion of the protein solubilised from the membrane eluted as trimers during size exclusion chromatography, and formed porin-like channels in planar lipid bilayers. Mature MOMP lacking a signal sequence accumulated in inclusion bodies when expressed in E. coli. These were denatured and refolded in vitro to produce higher order complexes of MOMP composed of SDS resistant trimers. Cysteine residues were found to play a critical role in the stabilisation of both secondary and tertiary structures. The ability to express properly folded recombinant MOMPs, and other chlamydial outer membrane proteins, will allow more detailed analysis of the structural and functional roles of MOMPS, and will contribute to the drive for vaccine development.
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Lou, Hubing. "Structural and functional studies of bacterial outer membrane proteins." Thesis, University of St Andrews, 2010. http://hdl.handle.net/10023/995.
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This thesis studies two particular bacterial outer membrane proteins called OmpC and Wzi, focusing on their expression, purification, crystallization and X-ray structure determination. A series of four naturally occurring OmpC mutants were isolated from a single patient with an E. coli infection of liver cysts. The isolated E. coli strains progressively exhibited increasing breadth of antibiotic resistance in which OmpC was predicted to take a partial role. We carried out an assay in which a strain of E. coli lacking OmpC was used to express the first (antibiotic sensitive) and the last (antibiotic resistant) of the clinical OmpC mutants and drug permeation assessed. Single channel conductance measurements were carried out and the X-ray structures for all the isolates were determined. Protein stability was assessed. With these data we propose that changes in the transverse electric field, not the pore size, underlie the clinically observed resistance to the antibiotics. This is the first demonstration of this strategy for antibiotic resistance. Wzi is a novel outer membrane protein involved in the biosynthesis and translocation mechanism of the K30 antigen from E. coli. The mechanism is a complicated process that requires several proteins including outer and inner membrane proteins. The protein Wzi was expressed, purified and crystallized. Initial crystals were tested and diffracted to 15Å. After optimization, a crystal diffracting to 2.4Å has been obtained.
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Ilkavets, Iryna. "Membrane proteins in the outer mebrane of plastids and mitochondria." Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-50439.
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Donald, James William. "The assembly of outer membrane proteins in gram-negative bacteria." Thesis, University of East Anglia, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492961.
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Visudtiphole, Virak. "Folding and topological studies of Escherichia coli outer membrane proteins." Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420000.
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Ye, Cui. "STABILITY STUDIES OF MEMBRANE PROTEINS." UKnowledge, 2014. http://uknowledge.uky.edu/chemistry_etds/33.
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The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. Gram-negative bacteria such as Escherichia coli are intrinsically more resistant to antimicrobials. There are very few drugs either on the market or in the pharmaceutical pipeline targeting Gram-negative pathogens. Two mechanisms, the protection of the outer membrane and the active efflux by the multidrug transporters, play important roles in conferring multidrug resistance to Gram-negative bacteria. My work focuses on two main directions, each aligning with one of the known multidrug resistance mechanisms.
The first direction of my research is in the area of the biogenesis of the bacterial outer membrane. The outer membrane serves as a permeability barrier in Gram-negative bacteria. Antibiotics cross the membrane barrier mainly via diffusion into the lipid bilayer or channels formed by outer membrane proteins. Therefore, bacterial drug resistance is closely correlated with the integrity of the outer membrane, which depends on the correct folding of the outer membrane proteins. The folding of the outer membrane proteins has been studied extensively in dilute buffer solution. However, the cell periplasm, where the folding actually occurs, is a crowded environment. In Chapter 2, effects of the macromolecular crowding on the folding mechanisms of two bacterial outer membrane proteins (OmpA and OmpT) were examined. Our results suggested that the periplasmic domain of OmpA improved the efficiency of the OmpA maturation under the crowding condition, while refolding of OmpT was barely affected by the crowding.
The second direction of my research focuses on the major multidrug efflux transporter in Gram-negative bacteria, AcrB. AcrB is an obligate trimer, which exists and functions exclusively in a trimeric state. In Chapter 3, the unfolding of the AcrB trimer was investigated. Our results revealed that sodium dodecyl sulfate induced unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild type AcrB. In Chapter 4, the correlation between the AcrB trimer stability and the transporter activity was studied. A non-linear correlation was observed, in which the threshold trimer stability was required to maintain the efflux activity. Finally, in Chapter 5, the stability of another inner membrane protein, AqpZ, was studied. AqpZ was remarkably stable. Several molecular engineering approaches were tested to improve the thermal stability of the protein.
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Sharma, Meenakshi [Verfasser]. "Facilitating folding of outer membrane proteins, roles of the periplasmic chaperone Skp and the outer membrane lipoprotein BamD / Meenakshi Sharma." Kassel : Universitätsbibliothek Kassel, 2014. http://d-nb.info/1060470454/34.
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Nilsson, IngMarie. "Conformational properties of transmembrane polypeptide segments in the ER membrane /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3613-7/.
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Setchfield, Kerry Jane. "Identification of Neisseria lactamica outer membrane proteins protective against meningococcal disease." Thesis, Open University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.395260.
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Blackburn, Paul Edward. "Characterisation of the virulence-related, outer-membrane proteins of B. pertussis." Thesis, Connect to e-thesis, 2000. http://theses.gla.ac.uk/1095/.
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Thesis (Ph.D.) -- University of Glasgow, 2000.Thesis submitted to the Institute of Biomedical and Life Sciences, University of Glasgow, in fulfilment of the degree of Doctor of Philosophy. Includes bibliographical references (p. 187-215). Print version also available. Mode of access : World Wide Web. System requirements : Adobe Acrobat reader required to view PDF document.
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Hamburger, Jonathan. "Isolation of antibodies to outer membrane proteins from nontypable Haemophilus influenza." Thesis, Boston University, 1988. https://hdl.handle.net/2144/37159.
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Thesis (M.A.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Outer membrane proteins from unencapsulated(nontypable) Haemophilus influenza were prepared by incubation with EDTA and shearing followed by molecular sieve chromatography in a lipooligosaccharide (LOS) disaggregating deoxycholate buffer. Outer membranes proteins collected were determined to be free of LOS by polyacrylamide gel electrophoresis and then covalently attached to cyanogen bromide activated Sepharose. Antibodies to outer membrane proteins were then isolated by affinity chromatography and characterized by enzyme-linked immunosorbant assay. These antibodies will prove useful for passive immunization experiments to establish the role of outer membrane proteins in host protection from infection with nontypable Haemophilus influenza.
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See, Sarah Bihui. "Outer membrane protein immunity to Pasteurella pneumotropica and the interaction of allergy." University of Western Australia. School of Paediatrics and Child Health, 2010. http://theses.library.uwa.edu.au/adt-WU2010.0103.
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[Truncated abstract] Infectious and allergic diseases of the respiratory tract are major contributors to global mortality, morbidity and economic burden. Bacterial infections such as pneumonia and otitis media are important diseases, especially in children, while allergic diseases such as asthma and allergic rhinitis afflict up to 30% of the world's population. A confounding aspect of respiratory disease is the evidence of a complex relationship between respiratory allergy and respiratory infection, with infection suggested to both promote and prevent the pathogenesis of allergic disease. Additionally, allergy is a risk factor for bacterial infection such as otitis media, pneumonia and sinusitis, while respiratory infection can exacerbate allergic symptoms. Given the burden of bacterial respiratory disease and respiratory allergy, the development of preventative treatments for these diseases is needed and will benefit from clearer knowledge of the underlying immune mechanisms. This thesis aimed to to extend current knowledge by using Pasteurella pneumotropica, a similar bacteria to the human pathogen nontypeable Haemophilus influenzae (NTHi), to study respiratory infection and protective anti-outer membrane protein (OMP) immunity as well as the interaction of respiratory infection and allergic inflammation. Homologues of the important NTHi vaccine candidates P4, P6, P26 and D15 were found to be encoded by P. pneumotropica and a high level of amino acid sequence identity was noted between the different P. pneumotropica strains, as well as between other Pasteurellaceae members. ... In contrast, anti-P6his serum antibodies transferred to naïve mice did not confer protection. These results suggested that T-cellmediated mechanisms were involved in P6his-mediated protection, and showed that the P. pneumotropcia model was useful for elucidating protective mechansims. The interaction of P. pneumotropica infection and papain-induced allergy was studied to investigate immune mechanisms underlying respiratory infection and allergy. Mice with ongoing allergic inflammation were intranasally challenged with bacteria and exhibited reduced pulmonary bacterial numbers, prolonged eosinophilia in the lungs and the induction of Th2 cytokines in the BALF, compared to nonallergic, infected mice. This suggested a protective role for allergic inflammation in this model. The effect of papaininduced inflammation on mice colonised by P. pneumotropica was also examined and allergic inflammation appeared to worsen infection in colonised mice. This suggested that allergic inflammation may also have a role in promoting infection in this model. In conclusion, this thesis explored mechanisms involved in vaccine-mediated immunity and the interaction of respiratory infection and allergy using a P. pneumotropica infection in its natural host. It was shown that intranasally administered recombinant P6 and P4 protected mice from lung infection, which justifies the inclusion of these OMPs as NTHi vaccine candidates. Additionally, it was demonstrated that the interaction of allergy and respiratory infection modulated immune responses. Overall, these results emphasize that a clearer understanding of the complex mechanisms underlying these interactions is required, and may be aided by the development of suitable animal models.
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Vitali, Daniela Giulia [Verfasser], and Doron [Akademischer Betreuer] Rapaport. "Sorting and membrane integration of mitochondrial outer membrane proteins / Daniela Giulia Vitali ; Betreuer: Doron Rapaport." Tübingen : Universitätsbibliothek Tübingen, 2018. http://d-nb.info/1199115800/34.
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Sawyer, Janet Gail. "Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas Aeruginosa." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26531.
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Purified macrophage cationic proteins were used in functional assays to determine their interactions with the outer membrane and lipopolysaccharide of Pseudomonas aeruginosa. A fluorescent derivative of polymyxin B (dansyl-polymyxin) was found to bind to saturation to purified lipopolysaocharide, with similar affinity for the aminoglycoside supersensitive strain H215 and wild type strain H103 lipopolysaocharide. MCP-1 could displace more dansyl-polymyxin bound to the lipopolysaocharide of both strains, and bound with greater affinity than MCP-2. When whole cells were used, MCPs also displaced bound dansyl-polymyxin. Effects on the outer membrane of whole cells were examined by determining the initial rate of uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine. Uptake was enhanced in the presence of MCPs, indicating permeabilization of the outer membrane. MCP-1 caused maximal uptake of the probe at 40 µg/ml, MCP-2 at 70 µg/ml, and crude extract at only 20 µg/ml. Uptake of the probe was found to be enhanced at add pH, with maximal uptake occurring with only 7.5 µg/ml MCP-1 at pH 6.5. The data suggested that MCPs act to permeabilize the outer membranes of P. aeruginosa in a manner analagous to that defined for other polycationic agents.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Oliveira-Fry, Anna Maria, and s9911120@student rmit edu au. "Identification of Legionella outer membrane proteins for the development of a biosensor." RMIT University. Applied Sciences, 2007. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080513.140744.
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Legionella spp. can cause a life threatening form of pneumonia, which is observed world-wide. Outbreaks of the disease are, unfortunately, not a rare event, despite the introduction of government regulations which enforce the mandatory testing of cooling towers to ensure that they contain levels of the organism which are regarded as being within safe limits. Therefore, cooling towers should be monitored for Legionella spp. by using a biosensor. These could potentially save the community from a great deal of morbidity and mortality due to legionellosis. This study identified and investigated novel outer membrane proteins in L. pneumophila, and analysed their potential for use in a Legionella biosensor. A combination of bioinformatics and laboratory investigations was used to identify the Omp87, an outer membrane protein of L. pneumophila which had not been previously described in this organism. Sequence analysis of the protein showed that it shares similarity with various other members of the Omp85 protein family, including the D15 antigen of Haemophilus influenzae and the Oma87 of Pseudomonas aeruginosa. The omp87 gene of L. pneumophila was amplified and cloned, and was found to encode a protein of 786 amino acids, with a molecular weight of 87 kDa. Distribution studies revealed that the gene is present in most, but not all species and serogroups of Legionella. To investigate the function of the Omp87 protein in L. pneumophila, the omp87 gene was insertionally inactivated with the use of a kanamycin resistance gene. Amplicons of this disrupted gene were then introduced into L. pneumophila, and a double-cross over event occurred, integrating the inactivated gene into the genome of the organism. This resulted in non-viable cells, indicating that the gene is essential in L. pneumophila. The expression vector pRSETA was used to express the Omp87 protein in E. coli, and four truncates of varying sizes were designed, through the use of different PCR primers. Two of the protein truncates were then expressed and purified by gravity flow chromatography using columns packed with Ni-NTA sepharose resin. Following analysis of the proteins by SDS-PAGE and Western blotting, polyclonal antibodies were raised against the truncates. Distribution studies were then performed using the antiserum with different strains and species of Legionella. This study demonstrated that most serogroups of L. pneumophila, and most other Legionella species reacted with the polyclonal anti-Omp87 L. pneumophila antisera. Cross-reactivity was also observed with most other Legionella related organisms tested. The results presented in this thesis demonstrated that the Omp87 protein or the omp87 gene can be used to construct a biosensor. In addition other novel outer membrane proteins were identified which could also serve as potential targets for a biosensor. These biosensors will be able to identify Legionella spp. in water reservoirs and in clinical samples and hopefully reduce the number of infections and deaths caused by this organism.
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Morgan, Cecilia A. "Treponema pallidum repeat protein K and heterologous protection against syphilis /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/9300.
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Kowal, Maria Theresa. "Characterisation of proteins secreted in the outer membrane vesicles of Bacteroides fragilis." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/23471.
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Bacteroides fragilis is an important, anaerobic commensal of the human gastro-intestinal tract. As a Gram-negative bacterium, B. fragilis produces a large number of outer membrane vesicles (OMV), spherical globules consisting of outer membrane and periplasmic material, which have a range of potential functions and which are known to be able to deliver their cargo to host dendritic cells (DCs). One of the proteins believed to be packaged into the OMV of B. fragilis is BfUbb (encoded by the ubb gene) which shares 63% homology with human ubiquitin. Ubiquitin is a small, common, eukaryotic protein modifier, which is conjugated to target proteins via a series of activating, conjugating and ligating enzymes, and which has known roles in a wide range of eukaryotic cell processes. Due to key differences between the two proteins, BfUbb has the potential to act as a suicide substrate mimic of ubiquitin. BfUbb was therefore assayed for its ability to interact with ubiquitin E2 conjugating enzymes of the ubiquitylation cascade in vitro, and was found to covalently bind the majority of available enzymes in a DTT-sensitive manner. BfUbb showed a preference for three specific E2 enzymes, all of which are involved in the degradation of mitotic check point proteins, suggesting a role for BfUbb in the inhibition of cell cycle progression and, consequently, tumorigenesis. No binding partners of BfUbb were identified outside of the ubiquitylation cascade, however BfUbb was found to form spontaneous multimers in vitro, the biological function of which is unknown. This study also describes the construction of two sets of plasmids. The first set will allow the expression of untagged and fluorescently tagged forms of BfUbb for purification and use in biochemical assays. The second set will allow the expression of his-tagged and fluorescently tagged forms of BfUbb in mammalian cells, so that the effects of BfUbb on the host epithelial cells may be studied. The proteome of the OMV of B. fragilis was solved using LTQ-Orbitrap mass spectrometry. The identified proteins indicated several putative roles for B. fragilis OMV, including nutrient acquisition and protease inhibition. The suitability of techniques used during the isolation and proteomic analysis of OMV in different studies is discussed. BfUbb-carrying B. fragilis OMV were able to inhibit growth of Salmonella enterica Typhimurium, thus indicating a role for BfUbb in the inhibition of competing, pathogenic bacteria in the gastro-intestinal tract. The conclusions of this study are that the putative roles of both BfUbb and the OMV of B. fragilis may promote both survival of the bacterium and the gastro-intestinal health of the host.
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Ahanotu, Ejemihu Ndu. "Immune Response of the Rat to Outer Membrane Proteins of Legionella pneumophila." Thesis, North Texas State University, 1985. https://digital.library.unt.edu/ark:/67531/metadc935780/.
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Outer membrane proteins (OMPs) were recovered from eleven strains (eight serogroups) of Legionella pneumophila by sequential treatment with Tris buffer (pH 8), citrate buffer(pH 2.75) and Tris buffer (pH 8). Transmission electron microscopy revealed clearly the separation of the outer membrane from the bacteria. The development of delayed hypersensitivity was also noted by measuring the area of arythema and induration produced by intradermal injections of the MPSs from Chicago 8 strain. The adjuvants enhanced greatly both active and cell-meditated immunity (CMI). Transient lymphocytopenia with a slight rise in neutrophils was noted in each of the immunized groups. Intraperitoneal challenge, seven days after the OMP booster, of one LD (1.5 x10^6) of legionellae resulted in lymphocytopenia with elevated neutrophils. All immunized rats survived the challenge, although those in the saline-OMP group were clearly the sickest. Post-challenge, legionella antibody titers rose greatly and CMI was heightened. Passive immunization (homologous and heterologous) was found to protect the rats from a challenge of on LD. Actively-immunized rats retained their immunity for at least six months as determined by their resistance to a second challenge.
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Kaye, Elena Cortizas. "The Function of Outer Membrane Protein A (OmpA) in Yersinia pestis." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_theses/58.
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The outer membrane protein OmpA is one of the major outer membrane proteins in many species of bacteria, including the Yersiniae. Our goal was to explore the role of OmpA in Y. pestis. This encompasses the ability of Yersinia to infect and survive within macrophages, as well as to resist antimicrobial compounds. Our laboratory found that a delta ompA mutant is impaired in a macrophage-associated infectivity assay. We also found that OmpA might play a role in the ability of the bacteria to resist antimicrobial peptides, specifically polymyxin B. Aditionally, we assessed the differences in OmpA of Y. pestis and E. coli, and determined that the characteristics we have observed in Y. pestis are unique compared to what has previously been described in E. coli. Our results indicate that Y. pestis OmpA might act through known pathways of antimicrobial resistance such as the PhoPQ two-component regulatory system, although further experiments are needed to determine the precise mechanism of function OmpA. Overall, our project characterizes the different functions of OmpA in Y. pestis, both as a key player in intracellular survival and as a necessary component in conferring resistance to antimicrobial peptides.
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Markovski, Monica. "Bacterial Cell Wall Synthases Require Outer Membrane Lipoprotein Cofactors." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10146.
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To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton. The PG synthases that build this structure are called penicillin-binding proteins (PBPs). Since they are the targets of penicillin and related antibiotics, the structures and in vitro biochemical functions of the PBPs have been extensively studied. However, the in vivo functions of the PBPs and the factors they work with to build the PG meshwork remain poorly understood. PBPs work in the context of multicomponent complexes organized by cytoskeletal elements. A major outstanding question has been whether or not these complexes contain factors required for PBP function. I addressed this using Escherichia coli as a model system by taking advantage of the synthetic lethal phenotype resulting from simultaneous inactivation of the major PG synthases: PBP1a and PBP1b. Using a screen for mutants synthetically lethal with the inactivation of PBP1b, I identified LpoA as a factor required for PBP1a function. A colleague in the lab performed the analogous screen for mutants synthetically lethal with the inactivation of PBP1a and identified LpoB as a factor required for PBP1b function. We showed that the Lpo factors are outer membrane lipoproteins that form specific trans-envelope complexes with their cognate PBPs in the inner membrane and that LpoB can stimulate the activity of PBP1b in vitro. Our results reveal unexpected complexity in the control of PBP activity and indicate that they likely receive regulatory input from the outer membrane in addition to cytoskeletal elements in the cytoplasm. To investigate the role of LpoB in morphogenesis further, I took a genetic approach that has identified PBP1b* variants capable of functioning in vivo in the absence of LpoB. Preliminary characterization of these variants indicates that LpoB has cellular functions in addition to PBP1b activation and that LpoB may be important for coordinating the two different catalytic activities of PBP1b. Future study of these mutants is likely to uncover important insights into PBP function and their control by the Lpo factors. These insights may open new avenues for the development of novel therapeutics that target the PBPs.
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Yunkun, Wu. "X-ray crystal structures of yeast heat shock proteins and mitochondrial outer membrane translocon member Tom70p." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2009r/wu.pdf.
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Huff, Jason. "Functional determinants of the porin MspA and its role in permeabilizing mycobacterial outer membranes." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2010. https://www.mhsl.uab.edu/dt/2010p/huff.pdf.
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Webb, Dianne, and n/a. "Nontypeable Haemophilus influenzae outer membrane protein analysis, isolation, characterisation and vaccine potential." University of Canberra. Applied Science, 1998. http://erl.canberra.edu.au./public/adt-AUC20061110.105953.
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Heterogeneity in immunodominant outer membrane proteins has been
proposed as a significant factor in the failure of an NTHi infection to induce
immune protection against subsequent infections. This study has examined
the vaccine potential of three outer membrane proteins in an attempt to
identify conserved regions that could be targeted by an immune response
after vaccination. The three proteins investigated were: TbpB, P5 and P48
(HI0164). The optimal route of immunisation in clearing a bolus inoculum
of NTHi to the lung in the rat has been shown to be a combination of gut
sensitisation with a respiratory boost and this regime was used in the present
study.
A panel of NTHi isolates was assessed to determine the frequency with
which strains were able to bind transferrin and thus be targeted by a TbpBspecific
immune response. A high proportion of strains was able to bind
transferrin with similar frequencies in isolates associated with infection and
those from normal throat swabs. A protocol was developed to purify
nonlipidated recombinant TbpB from NTHi using a glutathione-Stransferase
(GST)-rTbpB fusion protein and Glutathione-Sepharose affinity
chromatography. Mucosal-directed immunisation with rTbpB significantly
enhanced clearance of an NTHi challenge to the lung, however, whilst
rTbpB-specific antibodies were cross-reactive on Western immunoblots, the
cross-reactivity was variable in both transferrin binding inhibition assays and
bactericidal activity. This suggested that the rTbpB-specific humoral response
would be variable in the recognition of heterologous NTHi isolates.
The secondary structure of P5 has been controversial with several reports
suggesting that P5 was a fimbrin protein composed of coiled coils. In this
present study the interstrain variation in P5 amongst isolates from diverse
anatomical sites, as well as computer prediction methods and
spectrophotometric analysis, generated a model of P5 based on the
homologous E. coli protein, OmpA. This model suggested a B-barrel
conformation with no evidence of coiled coils. Synthetic peptides
corresponding to conserved regions of P5 that were thought to be surface
exposed, as well as a region (H3) with some homology to a protective epitope
in the P. aeruginosa protein, OprF, were then combined with a
"promiscuous" T cell epitope from the measles virus F protein (MVF) and
used for immunisation studies. Whilst variable protection was seen with the
peptides, the MVF/H3 peptide was the most efficacious of the antigens
assessed in this study in enhancing clearance of NTHi. This occurred in the
absence of detectable peptide- or PS-specific antibody leading to the
suggestion that cell mediated responses may have played an important role
in enhancing clearance in this model. The highly conserved nature of the
region in P5 represented by the H3 peptide suggests that further study should
be focused on this peptide as a potential NTHi vaccine candidate.
The last antigen, P48, is homologous to a A. pleuropneumoniae antigen,
AopA, which has been proposed to have potential as a vaccine component
against pleuropneumonia in pigs. Sequence analysis of the gene encoding
P48 from several isolates showed that this protein was well conserved.
Recombinant P48 was purified from a GST-rP48 fusion protein and used for
immunisation, which also conferred significant protection. However,
immunisation with rP48 was not as efficacious as immunisation with the
MVF/H3 peptide. Whilst immunisation with rP48 induced high antibody
titres, no bactericidal activity could be detected indicating that bactericidal
antibody had not contributed to the observed clearance. In addition, the rP48-
specific serum IgG was predominantly of the IgG2a isotype suggesting that
Thl cell mediated responses had been induced by immunisation with rP48.
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Dyer, Adam. "Identification and structural characterisation of novel outer membrane proteins in the genus Borrelia." Thesis, University of Huddersfield, 2013. http://eprints.hud.ac.uk/id/eprint/19265/.
Full textAbstract:
Although considered Gram-negative the outer membrane (OM) of Borrelia is unique, consisting of a variety of glycolipids, a significant number of surface exposed lipoproteins and a smaller number of integral membrane-spanning β-barrels. Many of these proteins are known to act as adhesins and invasins binding to regulators of the host immune response and extracellular matrix proteins. Many Gram-negative bacteria have the evolutionary conserved OmpA-like transmembrane domain consisting of an eight-stranded membrane spanning β- barrel. The prototypical example is E. coli OmpA; a multifunctional protein involved in a wide variety of physiological and pathological functions. To date the OmpA-like transmembrane domain has not been identified in the Spirochaete phylum. An approach based on hidden Markov models and topology and fold prediction identified a group of homologous genes in the genus Borrelia (BAPKO_0026, BAPKO_0422, BAPKO_0423 and BAPKO_0571), which are predicted to form an eight stranded OM-spanning β-barrel structure with similar structure to E. coli OmpA/W. One of these orthologues in B. afzelii (BAPKO_0422) has been expressed, purified and characterised in vitro; circular dichroism studies show a large percentage of β-strand. A low resolution molecular envelope generated from small-angle X-ray scattering data is consistent with an eight-stranded β-barrel.
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Naylor, Kathryn Lucy. "The role of outer membrane proteins of porphyromonas gingivalis in host-pathogen interactions." Thesis, University of Sheffield, 2016. http://etheses.whiterose.ac.uk/15759/.
Full textAbstract:
The keystone periodontal pathogen, P. gingivalis, is a Gram-negative oral anaerobe that is strongly implicated as the prime etiological agent in the initiation and progression of periodontal disease. P. gingivalis contains a plethora of virulence factors, including fimbriae, proteases, lipopolysaccharides and outer membrane proteins that contribute to the pathogenesis of the disease. The bacterium also displays the ability to interact with host cells through the adherence and invasion both in vitro and in vivo. In this thesis new light has been shed on the role of the heterotrimeric outer membrane protein A (OmpA). Through the creation of ompA mutants and recombinant complementation plasmids, alongside the use of standard antibiotic protection assays in an oral epithelial cell line, this research has demonstrated the importance of OmpA, specifically the OmpA2 subunit, in the invasion of the host and in the ability of the bacteria to form biofilms. Structural analysis of the protein identified extracellular loops, which when synthetic versions were applied to host cells, demonstrated successful interruption of wild-type P. gingivalis adherence and invasion of the host, indicating a direct interaction of OmpA2 with oral epithelial cells. In particular, this research demonstrates that OmpA2-loop 4 plays an important role in the interaction with the host through significantly increased binding to host cells when applied to fluorescent latex beads. This work also characterised the putative periplasmic chaperone protein OmpH, identifying potential proteins that act as clients to this chaperone, including OmpA. Through the creation of an ompH1H2 knock out mutant, enzymatic analysis of various virulence factors was assessed, demonstrating a loss of gingipain activity, whilst also identifying a loss of membrane stability through the observation of an increase in outer membrane vesicle formation. Evidence is also presented for the role of OmpH protein in chaperoning OmpA across the periplasm to the outer membrane and this work has opened up the potential for novel work identifying other clients of this chaperone system. Overall, the work in this thesis has demonstrated that the OmpA protein, specifically OmpA2, is directly involved in the interaction with host oral epithelial cells and in biofilm formation, specifically demonstrating the direct role of the extracellular surface loops in this interaction. This work also hypothesises a role for the OmpH protein in chaperoning outer membrane proteins, with the identification of potential clients, such as OmpA and the gingipains. Finally, this work has developed an improved mutagenesis technique for the creation of P. gingivalis mutants based on a modified natural competence method.
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Fischer, Steven Harold. "Interactions of Neisseria gonorrhoeae with human neutrophils: Gonococcal outer membrane protein II modulates neutrophil responses." Diss., The University of Arizona, 1988. http://hdl.handle.net/10150/184364.
Full textAbstract:
The disease gonorrhea has plagued mankind at least as long as written records have been kept (Black and Sparling, 1985). N. gonorrhoeae is still an important cause of suffering, infertility, and occasional mortality despite the fact that treatment with antibiotics is relatively easy and highly effective, even with the recent increase in penicillin-resistant isolates (Jephcott, 1986). The continued existence of this public health problem is partly the result of a reservoir of asymptomatic carriers within the community who normally don't seek treatment and continue their usual sexual practices (Handsfield, 1983; Kavli et al., 1984). Asymptomatic carriers do not have the purulent discharge characteristic of gonococcal urethritis and cervicitis in which the neutrophil is such a prominent element. Since IgM is present in only trace amounts on genital mucosa (Schumacher, 1973), and this is the "naturally occurring" antibody against gonococci (Rich and Kasper, 1982); it is not unreasonable to assume that non-opsonic chemotaxis and non-opsonic phagocytosis by PMN may play important roles in initiating the inflammatory response and symptomatology seen with gonorrhea. Further, non-opsonic phagocytic killing may be important in eventually clearing gonococcal infection since the role of specific humoral immunity is limited by the ability of gonococcus to constantly vary its antigenic facade (Zak et al., 1984). I have found that three different gonococcal strains express certain outer membrane proteins of the protein II (P.II) family which stimulate neutrophil phagocytic killing and oxidative metabolism in a highly efficient, dose-dependent manner. Other P.IIs expressed by two of the strains are non-stimulatory. Since all P.IIs have very similar physicochemical properties, these results suggest that a specific receptor-ligand interaction occurs between the gonococcal P.II and some element of the neutrophil plasma membrane. The presence or absence of pili on the gonococcal surface has no apparent effect on the ability of certain P.IIs to stimulate neutrophils. Changes in gonococcal outer membrane protein I and lipopolysaccharide, which are thought to confer serum resistance, also have no apparent effect on P.II stimulation of human PMN. Therefore, gonococcal outer membrane P.II may be an important mediator in the inflammatory response to gonococcal infection. Once gonococci are phagocytized by human PMN killing occurs rapidly and there is no evidence of significant intracellular survival. Non-oxidative killing by human chronic granulomatous disease neutrophils is as effective as the killing seen with normal PMN. Extracellular killing of gonococci does not occur to any appreciable extent.
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Huang, Haibin. "Analysis of lipoproteins, outer membrane proteins, and genetic diversities of Ehrlichia and Anaplasma species." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1155154668.
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Elliott, Roslyn Marie Ann. "Studies involving calcium binding proteins associated with the outer acrosomal membrane of ram spermatozoa." Thesis, Royal Veterinary College (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322378.
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Kumar, Amit. "Outer membrane proteins of Fusobacterium necrophorum and their role in adhesion to bovine cells." Diss., Kansas State University, 2011. http://hdl.handle.net/2097/16695.
Full textAbstract:
Doctor of PhilosophyDepartment of Diagnostic Medicine/Pathobiology
Sanjeev K. Narayanan
Fusobacterium necrophorum is a Gram-negative, anaerobic, and rod-shaped to pleomorphic bacterium. It is frequently associated with necrotic infections of animals and humans. It is a major bovine pathogen and causes hepatic abscesses, foot rot, and necrotic laryngitis (calf-diphtheria). Liver abscesses in feedlot cattle and foot rot in beef and dairy cattle are of significant economic importance to the cattle industry. Fusobacterium necrophorum is classified into two subspecies, subsp. necrophorum and subsp. funduliforme. The subsp. necrophorum is more virulent and isolated more frequently from bovine hepatic abscesses than subsp. funduliforme. Outer membrane proteins (OMPs) of Gram-negative bacteria play an important role in their adhesion to host eukaryotic cells and hence, help in the establishment of infection and disease. Our objectives were to characterize OMPs of the two subspecies of F. necrophorum and assess their role in adhesion to bovine cells. Electrophoretic separation of extracted OMPs of subsp. necrophorum showed a total of 19 bands. Four bands of 38, 40, 60 and 74 kDa were more prominent than others. The OMPs of subsp. funduliforme showed a total of 20 proteins bands, of which, five were prominent (37.5, 58, 70, 140 and 150 kDa). The 40 kDa band was prominent in subsp. necrophorum while 37.5 kDa band was prominent in subsp. funduliforme. The human strains of F. necrophorum subsp. funduliforme had more heterogeneous banding patterns than the bovine strains of subsp. funduliforme. The role of OMPs in adhesion was studied using bovine endothelial cell line (EJG cells). A significant decrease in the attachment of subsp. necrophorum and subsp. funduliforme to bovine endothelial cell line (EJG cells) was observed when the cell line was preincubated with the OMPs of each subspecies. Treatment of the bacterial cells with trypsin also decreased their binding. In addition, when each subspecies was incubated with the polyclonal antibody produced against their OMPs before adding them to endothelial cells, there was a significant reduction in the bacterial attachment and the inhibition was subspecies specific. A 40 kDa OMP of subsp. necrophorum was identified that binds to the bovine endothelial cells with high affinity. The protein when preincubated with the endothelial cells, lead to a significant decrease in the bacterial binding to the endothelial cells. The N-terminal sequencing of the protein indicated similarity to FomA, an outer membrane protein of Fusobacterium nucleatum, an oral pathogen of humans. In summary, OMPs of F. necrophorum subsp. necrophorum and subsp. funduliforme differ from each other and they play a significant role in binding to bovine endothelial cells. We identified a 40 kDa OMP in subsp. necrophorum that binds to the bovine endothelial cells with high affinity and have a potential role as adhesin.
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Al-Akash, Ahmed M. "Increased expression of ompA, ompX, dedA, and gutS genes in Enterobacter sp. YSU in the presence of selenite." Youngstown State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1607517925584702.
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Vasfi, Marandi Mehdi. "Identification and characterization of outer membrane proteins of Pasteurella multocida involved in serotyping and pathogenesis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq26750.pdf.
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Wan, Yao. "From Powerhouse to Processing Plant: Conserved Roles of Mitochondrial Outer Membrane Proteins in tRNA Splicing." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1531410494675571.
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Leader, Brandon Troy. "Immune responses during experimental Treponema pallidum infection /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/9277.
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Furrer, Jason L. "Enterobactin export in escherichia coli via P43 (ents) and associated components." Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/4392.
Full textAbstract:
Thesis (Ph. D.)--University of Missouri-Columbia, 2006."December 2006" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.
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Borrow, R. "The role of outer membrane proteins and lipooligosaccharide in the immunogenicity and pathogenicity of Neisseria meningitidis." Thesis, Manchester Metropolitan University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386713.
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Leary, Dagmar Hajkova. "CIRCADIAN PROTEOME CHANGES IN PHOTORECEPTOR OUTER SEGMENTS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1264276011.
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