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1
Barlow, Ann Katherine. "Neisseria meningitidis : the class 1 outer membrane protein." Thesis, University of Southampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280415.
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McBride, Heidi May. "Protein import into and across the mitochondrial outer membrane." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40395.
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Protein import into the mitochondria is a result of a series of sequential binding interactions between a mitochondrial targeting signal and the translocation machinery of both mitochondrial membranes. The targeting signals contained within protein of the outer membrane are distinct from those which target proteins to other subcompartments. The transmembrane domain of the yeast outer membrane receptor protein yTom70 is capable of both targeting and inserting the protein into the outer membrane. The efficiency of this process is increased by the addition of a positively-charged region preceding the transmembrane region. These two structural domains co-operate to form a signal-anchor sequence selective for the outer membrane, since this is the first membrane encountered by the targeting signal.Consistent with this model, the signal-anchor sequence of the outer membrane protein yTom70 is also capable of importing into the inner membrane of mitochondria when the outer membrane is selectively removed. Import into the inner membrane requires the presence of an electrochemical potential across the lipid bilayer. Import proceeds in the absence of $ Delta Psi$ only when constructs are used which lack the positively-charged amino terminal region of the signal-anchor sequence. These results suggest that the positively-charged presequence leads the transmembrane domain into the import machinery and that $ Delta Psi$ is required to clear this region in order that the distal transmembrane region can enter the translocation pathway.
The charged N-terminal 10 residues of yTom70 are incapable of directing import into intact mammalian mitochondria, however, are able to efficiently direct import into the matrix of yeast mitochondria or mammalian mitoplasts. This potentially cryptic signal is excluded from intact mammalian mitochondria due to the presence of the receptor protein Tom20, since replacement of yeast Tom20 with mammalian Tom20 confers the mammalian phenotype onto yeast. These results suggest that receptor proteins may also have the ability to screen potentially cryptic signals from distal components of the outer and inner membrane translocation machinery.
3
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.
4
Juodeikis, Rokas. "Engineering membranes in Escherichia coli : the magnetosome, LemA protein family and outer membrane vesicles." Thesis, University of Kent, 2016. https://kar.kent.ac.uk/61062/.
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Magnetosomes are membranous organelles found in magnetotactic bacteria (MTB). The organelle consist of ferromagnetic crystals housed within a lipid bilayer chained together by an actin-like filament and allows MTB to orient within magnetic fields. The genetic information required to produce these organelles has been linked to four different operons, encoding for 30 genes. These membranous organelles and the magnetic minerals housed within have various biotechnological applications, therefore enhanced recombinant production of such structures in a model organism holds significant potential. The research described in this thesis is focuses on the production of recombinant magnetosomes in the model organism Escherichia coli. Cloning the genes involved in the generation of the organelle individually or in various combinations resulted in the construction of over 100 different plasmids, compatible with the model organism. SDS-PAGE and electron microscopy analysis was used to characterise E. coli cells harbouring these constructs. The observation of electron dense particles, arranged in a chain structure, show that magnetosome generation in the model organism is possible, but is highly dependent on the growth conditions used. The need for specific growth conditions is later backed up by the analysis of the maturation of the cytochrome c proteins involved in magnetosome biomineralisation, which can only be correctly processed under certain conditions. Individual production of two different magnetosome proteins, MamQ or MamY, allowed the generation of various membranous structures in E. coli observed in 48.9% and 56.2% of the whole population of cells respectively. Combinations of these with MamI, MamL or MamB in a variety of combinations led to a variation in the phenotype observed. Bioinformatics analysis of MamQ led to the discovery of a novel membrane restructuring protein family, the LemA protein family, present in a broad range of bacteria. Four different LemA proteins from Bacillus megaterium, Clostridium kluyveri, Brucella melitensis or Pseudomonas aeruginosa were then produced in E. coli and the analysis of the resulting strains revealed the presence of novel intracellular membranous structures which vary in size, form and localisation. Furthermore, when attempts were made to target these proteins for the modification of the outer membrane, a mechanism for increased outer membrane vesicle generation was serendipitously discovered and different effects of these proteins were once again observed. Together, the results described shows good evidence for recombinant magnetosome production in E. coli and opens a new avenue of membrane engineering in this commonly used organism. Such membranous structures have various biotechnological applications, such as enhanced metabolic engineering potential or specialised lipid vesicle production.
5
Menon, Sailesh. "Characterization of a Fusobacterium necrophorum subspecies necrophorum outer membrane protein." Kansas State University, 2014. http://hdl.handle.net/2097/18128.
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Master of ScienceDepartment of Biomedical Sciences
Sanjeev K. Narayanan
Fusobacterium necrophorum is an anaerobic Gram-negative non spore forming rod shaped bacteria that is a normal inhabitant of the alimentary tract of humans and animals. Two subspecies of F. necrophorum have been recognized- subspecies necrophorum and subspecies funduliforme. Subspecies necrophorum is an opportunistic pathogen in animals causing diseases such as bovine hepatic abscesses and sheep foot rot while as subspecies funduliforme is linked with human oral and hepatic infections such as sore throats, Lemierre’s syndrome and hepatic abscesses. The pathogenic mechanisms of F. necrophorum are complex and are not well understood or defined. Several virulence factors such as leukotoxin, haemolysin, haemagglutinin and adhesin have been described. One of the most important factors in F. necrophorum bacterial pathogenesis is the adhesion of the bacteria to the host cell. The adhesion of the bacteria to the host cell helps it colonize the host tissue and this is followed by intracellular multiplication with dissemination to other tissues, which could ultimately lead to septicemia and death. Bacteria use adhesins which are proteins found in the outer membrane which help them bind with host receptors and this helps with the adhesion of the bacteria to the host cell. Not much is known about F. necrophorum adhesins. Here, we describe and characterize a novel adhesin.
6
Shand, Geoffrey H. "Antibiotic resistance and outer membrane protein antigens of Pseudomonas aeruginosa." Thesis, Aston University, 1985. http://publications.aston.ac.uk/12475/.
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7
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.
8
Ferris, Shirley. "Antibody responses to the major outer membrane protein of Chlamydia trachomatis." Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295880.
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Schiffrin, Robert. "Roles of periplasmic chaperones and BamA in outer membrane protein folding." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15952/.
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A defining feature of living things is that they have an inside and an outside, and in order for all living cells to survive, whether they are part of a blue whale or a unicellular microscopic organism, they must have mechanisms to mediate exchange with their environment. Food and energy enters the cell, and material must also leave, such as the waste products of metabolism, or virulence factors from pathogenic organisms. Lipid membranes define these boundaries, but it is membrane proteins that mediate the exchange. Although lipid bilayers can self-assemble in vitro, the assembly of complex biological membranes containing proteins requires energy and careful coordination. The work presented in this thesis examines the biogenesis pathway of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. OMPs are synthesised on cytosolic ribosomes, translocated across the inner membrane, then chaperoned across the periplasm, before folding and insertion into the OM. While OMPs can fold spontaneously into lipid membranes, this process is too slow to be biologically relevant, so a dedicated folding catalyst, the β-barrel assembly machinery (BAM) complex, is required at the OM. Recent genetic, structural and biochemical investigations have increased our understanding of OMP assembly, but key questions remain, including: How do periplasmic chaperones bind and release OMP substrates? What are the roles and interactions of BAM subunits? What is the molecular mechanism by which BAM folds and inserts OMPs? Here, an assay was developed to monitor OMP folding kinetics in vitro using intrinsic fluorescence in low concentrations of urea (0.24 M). This allowed comparison of the real-time folding behaviour of different OMPs under the same conditions for the first time (Chapter 3). The assay was then successfully extended to include OMP assembly factors, including the periplasmic chaperones Skp and SurA, and BamA, the principal component of the BAM complex, to obtain the following key results: Investigations into the interactions between Skp and OMPs of varying size (tOmpA, PagP, OmpT, OmpF and tBamA) revealed that greater Skp:OMP ratios are required to prevent the folding of 16-stranded OMPs compared with smaller 8-stranded OMPs. Supported by ion mobility spectrometry-mass spectrometry (IMS-MS) data, computer modelling and molecular dynamics simulations, the results imply a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp to protect a single substrate from aggregation (Chapter 4). Addition of further folding factors to the assay demonstrated that the model OMP tOmpA can be released and folded from its complex with Skp by BamA, possibly recapitulating an in vivo assembly pathway. BamA consists of a β-barrel membrane-embedded domain and soluble periplasmic domains, and while the release activity was shown to located in the membrane domain, the activity was greatest when full-length BamA was present. By contrast, SurA was not able to release tOmpA from Skp under the conditions employed, arguing against a sequential chaperone model (Chapter 5). Next, kinetic studies were used to investigate the mechanism of OMP folding catalysis by the BAM complex. The effect of hydrophobic mismatch between the BamA β-barrel and the membrane was examined by monitoring the folding of tOmpA into liposomes containing lipids of different chain lengths in the presence or absence of BamA. The results showed that BamA has a greater catalytic effect in lipids with longer chain lengths, with the largest rate enhancement achieved in bilayers with a hydrophobic thickness close to that of the OM. The results establish the importance of hydrophobic mismatch in the mechanism by which OMPs are folded in vivo, which may be influenced by local thinning of the membrane and increases in lipid disorder in the vicinity of the BAM complex (Chapter 5). Finally, based on the results obtained in this project, and consideration of the currently available literature, a new 'barrel elongation' model is proposed for the mechanism of OMP assembly by the BAM complex (Chapter 6). The OMP assembly pathway is an attractive target for novel antibacterials given that it is surface located, highly conserved, and essential in clinically important pathogens. Understanding the molecular mechanisms of OMP biogenesis factors will facilitate the development of drugs targeting this pathway. The work described in this thesis provides new insights into the mechanisms of OMP assembly, using a wide range of biochemical and biophysical techniques, thereby contributing to the development of this fast-moving and fascinating field.
10
Huysmans, Gerard Herman Marleen. "On the folding mechanism of the bacterial outer membrane protein PagP." Thesis, University of Leeds, 2008. http://etheses.whiterose.ac.uk/6752/.
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Membrane proteins represent an important class of macromolecules that play critical roles in many biological processes. The energetic principles underlying their stability, however, are not well understood. To address this deficiency, the kinetics and thermodynamics of the folding of the Escherichia coli outer membrane protein PagP were investigated by exploiting the ability of this polypeptide to refold into detergent micelles and into artificial lipid membranes. Investigations using the latter enabled the contributions to the folding process of both the protein sequence and of the bilayer lipid oomposition to be discerned.
11
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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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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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.
14
Arbing, Mark A. "Electrophysiological properties of porin, the major outer membrane protein of Haemophilus influenzae." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38146.
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Haemophilus influenzae (Hi) is a Gram-negative bacteria that is the causative agent of bacterial meningitis. The outer membrane (OM) of Gram-negative bacteria functions as a selectively permeable barrier. The exchange of small hydrophilic solutes between the external environment and the periplasm is mediated by large water-filled channels, porins. Charged residues of the pore determine the functional properties of the protein, which include: ion conductance, ionic selectivity, and voltage gating.To study the properties of the porin (341 amino acids; Mr 37,782) of Haemophilus influenzae type b (Hib), purified porin was subjected to chemical modification. The covalent modification of lysine residues with succinic anhydride (SA; Mr 100.08) results in charge reversal. The addition of up to 12 succinate groups per porin molecule was identified using electrospray ionization mass spectrometry (MS). Tryptic digestion of the modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight MS identified the sites of succinylation. The majority of modified lysines were positioned in surface-located loops, numbers 1 and 4 to 7. When the electrophysiological properties of SA-modified porin were analyzed in planar lipid bilayers (PLBs) and compared to Hib porin it was found that the single channel conductance was increased, while the threshold for voltage gating was decreased. The addition of extra negative charges increase the single channel conductance of Hib porin and function as voltage sensors.
Selected lysine residues that were found to be modified with SA were substituted with glutamic acid using site-directed mutagenesis. Single point mutations were made in a residue assigned to the barrel lumen and to three residues in each of loops 4 and 6. The mutant Hib porins had increased single channel conductances relative to wild-type Hib porin. Voltage gating of mutant Hib porins was altered by the introduction of negative charges into loops 4 and 6 and in the barrel lumen. Previous experiments had implicated surface-exposed loop 4 in voltage gating. This study ascribes a role for residues in loop 6 and a residue within the barrel lumen in the changes that accompany pore closure.
Hi strains causing infection in cystic fibrosis patients are capable of persistent infection despite prolonged antibiotic treatment with beta-lactam antibiotics. During the course of infection porin properties may be altered due to the changes in porin sequences that are attributed to antigenic drift. The electrophysiological properties of four porins from CF patient-derived Hi strains were characterized to examine changes in porin properties arising from persistent infection of the CF lung. The clinical Hi porins displayed altered channel properties that included increased voltage sensitivity and single channel conductances that were either greater or smaller than that of Hib porin. The decreased single channel conductance of one of the porins was associated with an increase in the minimal inhibitory concentration of the antibiotics novobiocin and streptomycin. These results demonstrate a porin-mediated decrease in OM permeability as an antibiotic resistance mechanism for Hi.
15
Carter, Michael William. "The major outer membrane protein gene of Chlamydia as a phylogenetic chronometer." Thesis, University of Southampton, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336071.
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Eneroth, Elina, and Astrid Karlsson. "A role of Aggregatibacter actinomycetemcomitans Outer Membrane Protein 100 in Serum Resistance?" Thesis, Umeå universitet, Institutionen för odontologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-128759.
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The disease periodontitis is an inflammatory response to the oral bacterial microflora. The Gram-negative bacterium Aggregatibacter actinomycetemcomitans has been specifically associated with the aggressive type of periodontitis. This species has a number of virulence factors that can contribute to host cell death, tissue inflammation, bone resorption, and colonization advantage relative to other microbes. A. actinomycetemcomitans has defense mechanisms against killing by the complement system (a part of our immune system). In the alternative pathway of complement activation, a protein called Factor H is the main regulator by having the ability to inhibit the complement reactions in three separate ways. Binding of Factor H to the bacterial surface was earlier shown to promote survival in human serum of an A. actinomycetemcomitans serotype d strain. This binding was caused by the outer membrane protein Omp100. The aim of this study was to establish whether Omp100 has a similar role in other serotypes of A. actinomycetemcomitans. For this we examined the serotype a strains D7S and D7SS, and their omp100 knockout mutants, which were generated in this laboratory. Western immunoblotting was used to compare a possible amount difference of the protein in each serotype. By incubating the bacterial strains in human serum, our results showed that lack of Omp100 did not have an obvious negative effect on the serum survival rate in strain D7S nor D7SS. We therefore concluded that omp100 was not required for serum resistance in these serotype a strains, and suggest that there might be another protein or factor in this serotype that is more important for serum resistance.
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Rios, Rosa Elvira. "Characterisation of the physiological, chemical and pathogenic changes arising from the adaptation of Campylobacter jejuni to aerotolerant growth." Thesis, Manchester Metropolitan University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243715.
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Dahan, David. "Biophysics of porin, the major outer membrane protein of Haemophilus influenzae type b." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0025/NQ29916.pdf.
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Srikumar, Ramakrishnan. "Topology of porin, the major outer membrane protein of Haemophilus influenzae type b." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40261.
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Haemophilus influenzae type b (Hib) is a Gram-negative bacterium that causes meningitis in infants. A protein called porin of 341 amino acids, M$ rm sb{r}$ 37,782 daltons, is located in the outer membrane of Hib and allows for the diffusion into the periplasmic space of small solutes up to a molecular mass of 1400 daltons.Based on parameters of hydrophilicity and amphiphilicity a model for Hib porin was generated. The model proposed an organization of sixteen anti-parallel $ beta$-strands that traverse the outer membrane, eight long loops that connect the $ beta$-strands on one side and short turns on the other side.
By flow cytometry, six out of a panel of nine monoclonal antibodies against Hib porin recognized amino acid sequences at the cell surface. Hib porin was purified and subjected to chemical and enzymatic digestions. The fragments were immunoblotted; N-terminal sequencing identified boundaries of fragments. C-terminal deletions of Hib porin generated in the baculovirus expression system identified C-terminal boundaries of monoclonal antibody reactivities.
To map precisely the primary sequences to which these monoclonal antibodies bound, overlapping hexapeptides for the entire sequence of Hib porin were synthesized. These studies identified two surface-exposed regions in the mature sequence of Hib porin, amino acid residues 162-172 and 318-325. In the Hib porin model, these regions correspond to loops 4 and 8, respectively. Two regions between residues 112-126 (loop 3) and residues 148-153 were buried or inaccessible at the surface of the outer membrane.
Recombinant Hib porin was expressed in Bacillus subtilis. The biophysical and immunological properties of this lipooligosaccharide-free recombinant Hib porin were compared with those of native Hib porin.
In order to examine the role of loop 3, site-directed mutagenesis of the cloned Hib porin gene was undertaken. Six or twelve amino acid deletions in loop 3, expressed in a porin deletion strain, showed significant increase in sensitivities to several anti-microbial agents as compared to wild-type Hib porin. Deletion of twelve amino acids showed more pronounced phenotypes than deletion of six amino acids. Such mutagenesis experiments provided support to the notion that loop 3 in Hib porin folds back into the pore and produces a constriction of the channel.
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Dahan, David. "Biophysics of porin : the major outer membrane protein of Haemophilus influenzae type b." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=42009.
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Haemophilus influenzae type b (Hib) is a Gram-negative bacterium that causes meningitis in infants. Located in the outer membrane, Hib porin (342 amino acids, M$ rm sb{r}$ 37,782 daltons) forms channels that allow for the diffusion into the periplasm of small solutes up to a molecular mass of 1400 daltons.Based on predictions of porin secondary structure, a model of Hib porin was generated. The model proposed sixteen membrane spanning $ beta$-strands connected by eight long loops on one side and eight short $ beta$-turns on the other side. To refine further this model, the epitopes of nine monoclonal antibodies specific for Hib porin were mapped precisely. Surface-exposure of these epitopes was determined by flow cytometry of intact Hib cells. Our studies identified two surface-exposed regions of Hib porin: amino acids 162 to 172, and 318 to 325. These two regions correspond to loops 4 and 8 in the Hib porin model.
Three naturally occurring Hib porin variants were purified, reconstituted into planar bilayers, and analyzed for their voltage dependency. Substitutions at Arg166 residue, which is localized to loop 4, were associated with a lowered threshold potential for the voltage gating of Hib porin. This surface-exposed loop was therefore implicated in the conformational changes that are postulated to occur during voltage gating.
Lipooligosaccharide (LOS) binds tightly to Hib porin. To generate large amounts of Hib porin devoid of LOS, the ompP2 gene was cloned into an expression vector of Bacillus subtilis. Recombinant Hib porin was produced in large quantities and it aggregated into inclusion bodies. Under denaturing conditions, recombinant porin was purified to homogeneity and subsequently refolded. To assess the fidelity of refolding of recombinant porin, four criteria were used: spectroscopic properties, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by monoclonal antibody Hb-2. We conclude that in the absence of LOS, recombinant porin was refolded into a functional form, its structure closely resembling the native state.
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McCallan, Lyanne Mary. "Differentiation of Campylobacter jejuni on the basis of outer membrane protein (OMP) patterns." Thesis, University of Ulster, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422191.
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Ward, Stephen John. "Characterisation and immunogenicity of recombinant class 1 outer membrane protein of Neisseria meningitidis." Thesis, University of Southampton, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296140.
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Huang, Shouxiong. "Genetic and Antigenic Characterization of the Major Outer Membrane Protein of Campylobacter Jejuni." The Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1046991783.
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Wyllie, Susan. "Structural and functional characterisation of the major outer membrane protein of Chlamydia psittaci." Thesis, University of Edinburgh, 1999. http://hdl.handle.net/1842/22762.
Full textAbstract:
The major outer membrane protein (MOMP) of Chlamydia shares several biochemical properties with classical porin proteins. To directly test the "porin channel" hypothesis at the molecular level, MOMP was reconstituted into planar lipid bilayers where it gave rise to "triple-barrelled" channels which were modified by an anti-MOMP neutralising monoclonal antibody. These observations are consistent with the well characterised homo-oligomeric nature of MOMP previously revealed by biochemical analysis, and the "triple-barrelled" behaviour of other porins. MOMP channels were weakly anion selective (PCl/PK ~ 2) and permeable to ATP. They may therefore be a route by which Chlamydia can take advantage of host nucleoside triphosphates, and explain why some anti-MOMP antibodies neutralise infection. In order to undertake more detailed studies of the MOMP structure/function relationship, recombinant MOMP from both C. psittaci and C. pneumoniae have been cloned and expressed. The recombinant proteins were functionally reconstituted in planar lipid and analysed at the single channel level. Both form porin-like ion channels that are functionally similar to the native protein. The C. psittaci recombinant porin was modified by the same anti-MOMP neutralising monoclonal antibody that effected the native protein. In contrast to the native protein, both recombinant C. psittaci (PCl/K ~ 0.38) and C. pneumonia (PCl/PK ~ 0.49) proteins were marginally cation selective. This is the first time native function has been demonstrated for recombinant chlamydial MOMP and will have an important impact on the future development of subunit vaccines.
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Ye, Cui. "STABILITY STUDIES OF MEMBRANE PROTEINS." UKnowledge, 2014. http://uknowledge.uky.edu/chemistry_etds/33.
Full textAbstract:
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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Muhammad, Noor [Verfasser]. "Insertion of Cecropin A and reconstitution of bacterial outer membrane protein FhuA variants in polymeric membranes / Noor Muhammad." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2011. http://d-nb.info/1018204458/34.
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au, T. La@murdoch edu, and Tom La. "Characterisation, Recombinant Expression and Immunogenicity of BHLP29.7, An Outer Membrane Lipoprotein of Brachyspira Hyodysenteriae." Murdoch University, 2006. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20070830.141953.
Full textAbstract:
Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study, the gene encoding a 29.7 kDa outer membrane lipoprotein of the causative intestinal spirochaete Brachyspira hyodysenteriae, was identified and sequenced. An 816 bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site, and putative 10 and 35 promoter regions upstream from the start of the ORF. The 29.7 kDa outer membrane lipoprotein was designated Bhlp29.7 and the encoding gene named bhlp29.7.
The amino acid sequence of Bhlp29.7 included a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. In silico analysis of this protein together with lipidation studies further supported its probable outer membrane localisation. Comparison of the Bhlp29.7 sequence with public sequence databases showed that it had up to 40% similarity with the D-methionine substrate-binding outer membrane lipoprotein (MetQ) of a number of bacterial pathogens. The Bhlp29.7 gene was detected in all 48 strains of B. hyodysenteriae examined, and in Brachyspira innocens strain B256T, but not in 10 other strains of B. innocens or in 42 strains of other Brachyspira spp. The gene was sequenced from B. innocens strain B256T and from 11 strains of B. hyodysenteriae. The B. hyodysenteriae genes shared 97.9-100% nucleotide sequence identity and had 97.5-99.5% identity with the gene of B. innocens strain B256T. The Bhlp29.7 gene was subsequently cloned and expressed as a histidine fusion protein in an Escherichia coli expression system.
An ELISA test using recombinant his-tagged Bhlp29.7 (His6-Bhlp29.7) as the detecting antigen was developed and evaluated. The threshold value of the test was chosen to provide a highly stringent assessment of the disease status of a herd. The sensitivity and specificity of the test was 100%. When the test was applied to sera from eight herds with suspected SD, four gave ELISA values indicating that the herds were diseased. The remaining four herds gave ELISA values below the threshold value. These results indicated that the Bhlp29.7-ELISA was useful as an indirect test for exposure of a herd to B. hyodysenteriae and may be a helpful complement to current methods of SD diagnosis.
Recombinant His6-Bhlp29.7 was evaluated as a vaccine subunit for prevention of SD. The His6-Bhlp29.7 was shown to be immunogenic in mice following two intramuscular injections. Vaccination of mice with His6-Bhlp29.7 provided full protection after oral challenge with B. hyodysenteriae. In two experiments, intramuscular and oral vaccination of pigs with the His6-Bhlp29.7 resulted in a 50% reduction in incidence of SD compared to unvaccinated control pigs (P=0.047). This is the first subunit vaccine shown to provide pigs with protection from SD. Further work is needed to optimise delivery routes and adjuvants for commercial development of the vaccine.
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McGuinness, Brian Timothy. "Immunochemistry and structural variation of the class 1 outer membrane protein of Neisseria meningitidis." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386663.
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Humes, Julia Rose. "The mechanism of the ATP-independent periplasmic chaperone SurA in outer membrane protein biogenesis." Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/22526/.
Full textAbstract:
Outer membrane proteins (OMPs) of Gram-negative bacteria travel from their site of synthesis in the cytoplasm, across the inner membrane and through the periplasm to the outer membrane (OM) prior to their folding to a functional form. To protect OMPs from misfolding or aggregation while traversing the periplasm a network of chaperones are employed. The OMPs must then reach the essential β-barrel assembly machinery (BAM) complex, which is involved in inserting OMPs into the OM. This process occurs in the absence of chemical energy as the periplasm is devoid of ATP and in a highly dynamic environment as the 'leaky' OM allows small molecules (< 600 Da) to enter from the extracellular milieu. The major periplasmic chaperone for OMPs, SurA, is known to interact with a number of substrates and has been crosslinked to the BAM complex in vivo. SurA is composed of four domains, an N-terminal domain, two peptidyl-prolyl isomerase (PPIase) domains and a short C-terminal helical domain. In this work wild-type E. coli SurA and SurA truncation variants lacking one (P2) or both (N-Ct) of the PPIase domains have been studied. Using microscale thermophoresis, light scattering, native mass spectrometry and other biophysical techniques how each domain is involved in OMP binding, chaperoning and delivery to BAM is investigated. The results demonstrate that SurA binds unfolded OMPs, tOmpA and OmpT with μM affinity, agreeing with previous findings. The core domain (SurA N-Ct) is sufficient for this interaction, but the addition of the PPIase domains leads to a tighter binding. Light scattering experiments shows that SurA WT can prevent aggregation of the two model OMPs, but the removal of the PPIase domains reduces the chaperoning ability for the larger, more aggregation-prone OMP, OmpT. These observations demonstrate that the acquisition of the PPIase domains is advantageous for both OMP binding and chaperoning. An interaction between SurA and the BAM complex is also observed for the first time in vitro. Overall, the results reveal new insights into how SurA binds and chaperones OMPs before delivering them to the BAM complex for folding in the OM.
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McMorran, Lindsay Madeline. "Mechanisms of outer membrane protein folding : effects of the lipid environment and periplasmic chaperones." Thesis, University of Leeds, 2013. http://etheses.whiterose.ac.uk/5906/.
Full textAbstract:
In contrast with the wealth of information on the folding of soluble, cytosolic proteins, little is known about the folding of integral membrane proteins. The outer membrane proteins (OMPs) of Gram-negative bacteria have a β-barrel structure and are essential for cell survival. The mechanisms of OMP transport across the periplasm and how these proteins subsequently fold and insert into the outer membrane remain to be elucidated. The work presented herein examines the folding and membrane insertion of four different OMP constructs. Two homologous bacterial OMPs, OmpT and OmpP, were cloned, over-expressed and purified before biochemical and biophysical methods were employed to examine their folding properties. This work demonstrates that small differences in primary sequence can have large effects on folding efficiency and stability. In spite of both OmpT and OmpP being able to fold under a variety of conditions, it was not possible to establish conditions under which folding was completely reversible. Examination of the origins of irreversible OMP folding was carried out using OmpT, as well as both hexa-histidine tagged and untagged constructs of the outer membrane acyltransferase enzyme, PagP. This study revealed evidence that lipid adhesion of the protein in the unfolded state may be important in preventing aggregation and promoting reversibility. Finally, conditions were established to promote the folding of untagged PagP in low urea concentrations to allow the study of OMPs in the presence of the periplasmic chaperones, SurA and Skp. SurA was shown to have little effect on the folding of PagP into liposomes with zwitterionic or negatively charged membrane surfaces, while Skp was shown to exhibit holdase activity and to modulate PagP folding rate, dependent on the lipid composition. The results present the first detailed insights into the mechanism by which Skp and SurA act to facilitate PagP folding in vitro.
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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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Easton, Donna Meredith, and n/a. "Functional and Antigenic Characterisation of the Moraxella catarrhalis protein M35." University of Canberra. n/a, 2008. http://erl.canberra.edu.au./public/adt-AUC20081217.083105.
Full textAbstract:
This thesis reports the characterisation of a novel outer membrane protein (OMP)
from M. catarrhalis, designated M35, with a molecular mass of 36.1 kDa. This protein is
structurally homologous to classic Gram-negative porins, such as OMP C from E. coli and
OMP K36 from K. pneumoniae, with a predicted structure of 8 surface loops connecting 16
antiparallel -sheets. Comparison of the DNA sequences of the M35 genes from 18 diverse
clinical isolates showed that the gene was highly conserved (99.6-100 % of nucleotides)
with only one isolate (ID78LN266) having base variations that resulted in amino acid
substitutions.
A single amino acid mutation in the 3rd external loop of M35 in isolate ID78LN266
significantly affected antibody recognition, indicating that loop 3 contains an immunodominant
B-cell epitope. The reduction in antibody-binding to M35 from ID78LN266
was similar to that caused by complete removal of loop 3. Since loop 3 folds into the porin
channel in the classic structure, the antibody specificity to loop 3 was hypothesised to be
a potential mechanism for evasion of host immune responses targeted to M35, potentially
explaining the high degree of conservation across isolates.
A series of recombinant proteins were constructed to analyse the binding to M35 of
antibodies specificity for loop 3 or the remainder of the protein. It was found that loop 3-
specific antibodies were not able to bind to M35 on the surface of M. catarrhalis and
that this corresponds both with a lack of ability to enhance opsonophagocytosis in vitro
and bacterial clearance in vivo. Additionally, antibodies raised against a version of M35
lacking loop 3 and M35 from the variant isolate ID78LN266 were both no less effective
than the full consensus M35 by both these measures. It therefore appears that while the
majority of antibodies raised against M35 are specific for loop 3 these antibodies do not
mediate anti-M. catarrhalis actions.
Two deletion mutant strains of M. catarrhalis that do not contain the outer membrane
protein M35 were created by insertional inactivation of the M35 gene. Growth comparisons
between these mutant strains and their wildtype parent strains initially led to the
hypothesis that M35 is necessary for efficient glutamic acid uptake by M. catarrhalis,
however this hypothesis was later shown to be incorrect. Efficient uptake of glutamic acid
seemed to be mediated by a novel 40 kDa protein that was up-regulated in the deletion
mutant strains, presumably to compensate for the lack of M35. M35 was also found to
be essential for in vivo survival of M. catarrhalis in the nasal cavities of mice, indicating
that it is an essential functional protein for colonisation of the mucosal surface.
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Sukumaran, Suja. "Spectroscopic investigation of stability, unfolding and refolding of outer membrane protein porin from Paracoccus denitrificans." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=974953849.
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Bell, Angus. "Structure, function, and role in antibiotic resistance of outer membrane protein H1 in Pseudomonas aeruginosa." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/29008.
Full textAbstract:
A divalent cation-regulated outer membrane protein of Pseudomonas aeruginosa, HI, was purified by selective solubilizations of outer membranes in detergent and ethylenediaminetraacetate (EDTA) followed by either two cycles of anion-exchange chromatography or sodium dodecylsulphate-polyacrylamide gel electrophoresis. Protein purified by the former method was contaminated with an equi-molar or higher concentration of lipopolysaccharide , (LPS) that was enriched in 0 side chain-containing molecules, suggesting an association between protein Hi and smooth LPS. Electrophoresis gave higher yields of purified protein Hi, and this product was used for N-terminal amino acid sequencing, amino acid analysis, and polyclonal antiserum production.
Oligodeoxyribonucleotides complementary to the upstream end of the structural gene for protein Hi, oprH, were designed using the N-terminal sequence of the protein. Probing of Southern blots of chromosomal DNA digests with the oligonucleotides revealed that oprH was probably a single-copy gene, and allowed it to be cloned in Escherichia coli. Nucleotide sequence analysis confirmed the cloning of the correct gene, and the derived amino acid sequence indicated a slightly basic protein (in agreement with its proposed function of interacting with anionic sites on LPS) of 178 residues, with two hydrophobic segments.
Protein H1 was produced, proteolytically processed and probably exported to the outer membrane in E. coli cells carrying the complete oprH gene on plasmids. The oprH gene could be expressed weakly from a promoter on the cloned DNA provided that a particular downstream sequence was not deleted. This suggested that the downstream region was involved in regulation of expression of the cloned oprH gene. Protein H1 was produced at levels much higher than background when extra copies of the oprH gene were present in an expression vector in P. aeruginosa. This overproduction of protein H1 caused decreased susceptibility of cells to killing by EDTA, but not by polymyxin B or gentamicin. This partly confirmed the hypothesis (T.I. Nicas and R.E.W. Hancock, J. Bacteriol. 143; 872-878, 1980) that protein HI causes resistance to these agents by inhibiting their self-promoted uptake across the outer membrane. However, an
additional alteration (possibly an increase in cationic
substituents on LPS) was apparently required for the
fully resistant phenotype. This idea was supported by
the observed suppression by LPS mutations of the
polymyxin B resistance of protein H1-overproducing cells,
and by the properties of a P. aeruginosa strain
apparently lacking protein H1.
Several species of bacteria related to P. aeruginosa
produced envelope proteins that were inducible by growth
in Mg²⁺-deficient medium, and one (a protein of apparent
molecular weight 20,000 from P. chloraphis) cross-reacted
immunologically with protein Hi. P. chloraphis, like P.
aeruginosa, was polymyxin B resistant when it was grown
in Mg²⁺-deficient medium.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Berry, Jody Douglas. "Antibody gene markers and their relationship to chlamydial major outer membrane protein (MOMP) immune response." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0006/NQ41602.pdf.
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Butt, Neil James. "The complete nucleotide sequence and immunochemistry of the major outer membrane protein of Neisseria gonorrhoeae." Thesis, University of Southampton, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316059.
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Bulieris, Paula Vasilichia. "Kinetic Studies on the Folding and Insertion of Outer Membrane Protein A from Escherichia Coli." [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-48784.
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Ujwal, Rachma. "Structural and fuctional characterization of the outer mitochondrial membrane protein voltage-dependent anion channel 1/." Diss., Restricted to subscribing institutions, 2009. http://proquest.umi.com/pqdweb?did=1930281371&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Königer, Verena [Verfasser], and Rainer [Akademischer Betreuer] Haas. "CEACAMs as novel receptors for Helicobacter pylori outer membrane protein HopQ / Verena Königer ; Betreuer: Rainer Haas." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1119073731/34.
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Khursigara, Cezar M. "Interactions between the energy-transducing protein TonB and the outer membrane receptor FhuA from Escherichia coli." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85563.
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Iron is an essential element required by most organisms. To acquire iron, Gram-negative bacteria utilize siderophores; compounds that bind iron and form soluble complexes. Escherichia coli imports siderophores via specialized transport systems. A model siderophore receptor system requires the integral outer membrane (OM) receptor FhuA, which facilitates transport of hydroxamate siderophores ferricrocin (Fc) and ferrichrome. Transport of siderophores by FhuA to the periplasm and into the cell requires energy provided by the proton motive force and delivered by the cytoplasmic membrane protein complex TonB-ExbB-ExbD. Energy is transduced from this complex by protein-protein interactions between TonB and FhuA. Although this system has been extensively studied, much remains unclear about interactions between TonB and FhuA.Sedimentation velocity analytical ultracentrifugation (AUC) experiments involving mixtures of recombinant TonB and FhuA were conducted to determine the oligomeric state of TonB-FhuA complexes. These experiments demonstrated that wild-type TonB-FhuA proteins form a 2:1 (TonB:FhuA) complex, and that this complex is more abundant when FhuA is pre-incubated with the siderophore Fc. Deletion of the N-terminal region of TonB also demonstrated a 2:1 complex stoichiometry with FhuA, but in much lower abundance. Deletion of a highly conserved proline-rich region within TonB resulted in the formation of a 1:1 complex, suggesting a novel role for this domain in the formation of a functional 2:1 complex.
Affinities of interaction between wild-type and mutant TonB and FhuA proteins were measured by surface plasmon resonance (SPR). SPR data revealed that wild-type TonB-FhuA interactions are comprised of multiple interaction sites, and undergo an intermediate rearrangement event prior to the formation of the 2:1 complex. Deletions of TonB domains do not disrupt this rearrangement, but do decrease affinity. FhuA cork domain deletions also identified novel sites important for stable interactions.
Information of TonB-FhuA interactions derived by AUC and SPR described herein has contributed greatly to the purification and crystallization of TonB-FhuA co-complexes. Overall, these complementary biophysical techniques have provided important mechanistic details into the protein-mediated transport of iron into Gram-negative bacteria, and will provide a conceptual framework of TonB-FhuA that will help in elucidating aspects of the high-resolution structure of the complex.
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Malia, Thomas J. 1977. "NMR structural and functional studies of the mithochondrial outer membrane protein VDAC by Thomas J. Malia." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/37889.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006."September 2006." Vita.
Includes bibliographical references.
Apoptosis is a mechanism of programmed cell death required by multicellular organisms during development and for tissue maintenance. Bcl-2 family proteins are central regulators of apoptosis and many of their primary roles are carried out in the outer mitochondrial membrane. Anti-apoptotic Bcl-xL has been found previously to interact with VDAC, an outer mitochondrial membrane protein responsible for metabolite trafficking. Here, the NMR-based investigation of VDAC and its interaction with Bcl-xL in detergent micelles is described. As an integral membrane protein VDAC presented a challenge for producing a folded form amenable to solution NMR studies. Methods developed for expression and purification of human VDAC for structural studies by NMR spectroscopy were developed. Optimal sample conditions were explored in a screen of various detergents and buffers. Suitable NMR sample conditions for VDAC were identified and allowed further characterization of VDAC and its interactions by NMR and other methods. Obtained through various means, evidence of a concentration dependent self-association of VDAC is also presented. Chemical cross-linking, analytical size exclusion chromatography, and NMR spectroscopic studies strongly support an equilibrium model for LDAO-purified VDAC between monomer and trimer.
(cont.) Methods that were necessary to carry out NMR structural studies of VDAC and results from those studies are described. In addition to various methods previously developed for solution NMR spectroscopy of very large proteins, extensive labeling approaches and unconventional experimental NMR methods were necessary to obtain a high level of chemical shift assignment for micelle-bound VDAC. Chemical shift assignment has allowed the preliminary characterization of ATP and Bcl-xL binding to VDAC. Binding of Bcl-xL, a central regulator of apoptosis, to VDAC is demonstrated here. Using NMR spectroscopy, the VDAC-binding region of Bcl-xL has been mapped to a putative helical hairpin motif, which also mediates insertion into membranes. The stoichiometry of the VDAC/Bcl-xL complex is shown to be a heterotrimer of two VDAC monomers and one Bcl-xL. The demonstration of VDAC/Bcl-xL complex formation supports the concept that components of apoptosis and metabolism are integrally connected, and that interplay between the two processes is required for regulation of cell survival and cell death.
Ph.D.
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Dai, Xian-zhu. "Studies on the folding of an outer membrane protein from a psychrotrophic bacterium, Shewanella livingstonensis Ac10." Kyoto University, 2012. http://hdl.handle.net/2433/157718.
Full textAbstract:
Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第16927号
農博第1943号
新制||農||1001(附属図書館)
学位論文||H24||N4688(農学部図書室)
29602
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能
学位規則第4条第1項該当
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Peddireddi, Lalitha. "Transcriptional analysis and promoter characterization of two differentially expressed outer membrane protein genes of Ehrlichia chaffeensis." Diss., Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/1499.
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White, Paul. "Bacterial protein import mediated by an iron transporter." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:20298bb4-0998-4dad-9dfa-dd9e52854dec.
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Multidrug resistant bacteria (MDR) have the potential to push back society to the pre-antibiotic era. Although discovered before penicillin, the inexorable rise in antibiotic resistance has revitalised interest in bacteriocins as treatments for bacterial infections. Bacteriocins are protein antibiotics principal to competition amongst pathogens and commensals, but the mechanisms by which they translocate across the Gram-negative cell envelope are poorly understood. The work presented in this thesis demonstrates how the endonuclease bacteriocin pyocin S2 (pyoS2) exploits the iron transporter FpvAI to translocate across the outer membrane (OM) of Pseudomonas aeruginosa. FpvAI is a 22-strand β-barrel and virulence factor in P. aeruginosa that transports iron into the cell in the form of a small siderophore, ferripyoverdine (Fe-Pvd). Uptake of Fe-Pvd requires the proton motive force (PMF), which is transduced to the ligand-bound receptor by TonB1 and its partner proteins ExbB-ExbD in the inner membrane (IM). The crystal structure of the high affinity complex (Kd = 240 pM) formed between the N-terminal domain of pyoS2 (pyoS2NTD) and FpvAI is presented, which shows pyoS2NTD mimics Fe-Pvd, and induces the same conformational changes in the receptor. Fluorescently-labelled pyoS2NTD was actively imported into P. aeruginosa PAO1 cells and this import was dependent on the PMF, TonB1 and a TonB1-box motif at the N-terminus of pyoS2NTD. Finally, photo-activated crosslinking of stalled translocation intermediates demonstrated pyoS2NTD translocates through the FpvAI β-barrel lumen by a process analogous to that of Fe-Pvd. Following binding to FpvAI, translocation begins by the unfolding of a force-labile portion of the plug domain, opening a narrow channel through FpvAI. This enables pyoS2 to deliver its own TonB1-box to the periplasm where contact with TonB1 activates its import through the same channel, most likely as an unfolded polypeptide. Hence, this study demonstrates that bacteria possess a rudimentary protein import system that exploits energised nutrient transporters in the OM.
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Romero-Ruiz, Mercedes [Verfasser]. "Interactions of Polypeptides with the Protein Translocation Channel of the Outer Membrane of Mitochondria / Mercedes Romero-Ruiz." München : Verlag Dr. Hut, 2011. http://d-nb.info/1012431703/34.
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Taiarol, Natasha Sabrina Tamara. "The effect of environmental conditions on the antigenicity of the outer membrane 35K protein present in Salmonella." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0021/MQ53121.pdf.
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Lai, Tzung-Huei. "Two-Component Regulatory Systems of Anaplasma phagocytophilum and Outer Membrane Protein P44 Expression Locus of Anaplasma platys." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1276709646.
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Mohan, Kumar Dipu. "Insights into the Host Cell Entry of Ehrlichia chaffeensis: Roles of the Bacterial Outer Membrane Protein EtpE." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397229647.
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Saul-McBeth, Jessica. "Characterization of SipA, A Protein Important for Stress Responses in Vibrio cholerae." University of Toledo Health Science Campus / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=mco1544540466901883.
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Gaddy, Jennifer Angeline. "Acinetobacter baumannii Virulence Attributes: The Roles of Outer Membrane Protein A, Acinetobactin-mediated Iron Acquisition Functions, and Blue Light Sensing Protein A." Miami University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=miami1289178632.
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