Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 77-93).

Background: Escherichia coli (E.coli) O86a:K61 belongs to the Enteropathogenic E. coli (EPEC) group of pathogens. Acute gastroenteritis affects 2-4 billion people annually and EPEC is associated with 10-40% of hospitalized diarrhea cases globally. Coagulation Factor (F) V circulates as an inactive procofactor (Mr 330kDa) which upon thrombin activation to the active cofactor, FVa, functions in prothombinase to accelerate prothrombin to thrombin conversion by 300,000-fold. The ability of E.coli O86a:K61 to cause intestinal hemorrhage is of interest because previous research demonstrated that during E.coli O86a:K61 sepsis in baboons, a dose-dependent inactivation of FV was observed as the bacterial dose increased. These results suggested a secreted E.coli protease may have mediated this effect on FV. This research has focused on the purification, identification, and characterization of a secreted E. coli O86a:K61 protease that inactivates FV. The final partially-purified protease inactivated FV to a 250kDa product by immunoblotting, and possessed a 900-fold increase in specific activity versus FV in human plasma compared to the culture supernatant. At least 3 proteins were observed upon SDS-PAGE. Proteolytic inactivation of FV was activated by up to 500-fold with β-mercaptoethanol and 2-fold with 1M urea. The protease was heat stable retaining all of its activity versus FV after 1h at 70°C or 80°C, and partial activity (50%) at 95°C. Proteolysis of FV was blocked by 90% with alpha-1-protease inhibitor; however, the protease was resistant to 1.5 mM PMSF, and unaffected by E64, or iodoacetamide. FV is a major regulator of the coagulation process and its inactivation by the secreted E.coli protease would be expected to result in a net bleeding tendency which may contribute to the mucosal hemorrhage observed in humans with associated hemorrhagic colitis. Proteolytic inactivation of FV is predicted to result in decreased bacterial containment by host fibrin thereby increasing pathogen survival and growth. FV inactivation by the secreted E.coli protease may be part of a novel pathogenic virulence mechanism that deregulates the blood coagulation process to enhance bacterial infectivity and transmission. / UOIT

The hallmark of Gram-negative bacteria is the presence of an outer membrane (OM) surrounding the cytoplasmic membrane (here called the inner membrane [IM]) and the cell wall. The OM is a unique asymmetric bilayer with an inner leaflet consisting of phospholipid and an outer leaflet consisting of lipopolysaccharide (LPS). LPS is a large anionic molecule that typically contains six fatty acyl chains and up to several hundred sugar residues. This chemical structure explains why the OM is relatively impermeable to large hydrophobic molecules, such as detergents, bile salts, and high molecular weight antibiotics, which readily cross a normal phospholipid bilayer. LPS and the OM are essential to the viability of most Gram-negative organisms, including major human pathogens. LPS molecules are biosynthesized at the IM and subsequently exported out of the IM, across the intermembrane space (the periplasm) and through the OM to their final position at the cell surface. In Escherichia coli, the essential LPS transport proteins, LptA-G, are required for this process. This Lpt pathway includes an IM adenosine triphosphate binding cassette (ABC) transporter, LptBFG, which is associated with an additional IM protein, LptC; a periplasmic protein, LptA; and an OM complex consisting of the lipoprotein LptE and the transmembrane \(\beta\)-barrel protein LptD. All seven Lpt proteins associate as a single complex that spans the cell envelope. However, little is known about how these proteins work together to transport LPS. Here, we use in vivo and in vitro biochemical studies to probe the organization, function, and assembly of the Lpt machine. In Chapter 2, we show that LptE forms a plug within the LptD \(\beta\)-barrel and present a model for how this unusual structure can move LPS from the periplasm directly into the outer leaflet of the OM. In Chapter 3, we demonstrate that the Lpt transenvelope bridge consists of a series of structurally homologous domains – LptC, LptA, and the N-terminal domain of LptD – stacked in a head-to-tail orientation, providing a route for LPS from the IM to the OM. Finally, in Chapter 4, we connect these two sets of results by showing how the assembly of the Lpt transenvelope bridge is regulated by that of the LptD/E complex in the OM. Together, these findings explain how the functions of the Lpt proteins are coordinated to ensure delivery of LPS to the correct cellular compartment. A fundamental understanding of LPS biogenesis will contribute to the development of new therapies against Gram-negative infections.

<p>Outer membrane vesicles are an important constitutive product of all Gram-negative bacteria. Bacteria have evolved many responses to alleviate all different types of stress. The primary objective of this dissertation is to investigate the role of outer membrane vesicles (OMVs) as a method by which Gram-negative bacteria can quickly act to protect themselves against particular threats. Generally, we find that stressors whose primary effect is on the outer membrane can be protected against by OMVs. Throughout this study, a variety of different microbiological and biochemical methods are used to answer key questions in the innate ability of OMVs to protect against particular antimicrobials. Using Escherichia coli as well as Pseudomonas aeruginosa as model organisms we tested the ability of purified vesicles from each species to protect themselves and other hosts. Using bacteriophage T4, we investigated the ability of OMVs purified from E. coli to adsorb phage as well as how this interaction affected the efficiency of infection. We found that OMVs are protective against antimicrobial peptides, as well as bacteriophage. In the course of understanding this protection we also observed and characterized the cross species effects of both OMV protection as well as phage infection. Where typically a phage infects a specific species, we found that T4 associated OMVs treating a non-native host P. aeruginosa resulted in the production of a novel prophage. Upon further examination, we determined that this induction was occurring via a novel pathway that we attempted to further characterize by performing a genetic screen to identify genes important to this induction. The work within this dissertation fully supports the hypothesis of a regulated response to outer membrane acting stimuli, resulting in the induction of vesiculation and the adsorption of stressor in the extra-cellular milieu. This model of protection agrees with the idea of a bacterial innate defense system, which acts in the short term before the adaptive response can fully occur, resulting in a bridge between the untreated to the treated and resistant culture.</p> / Dissertation

Despite being amongst the most common zoonotic diseases in the world, brucellosis is a neglected disease for which an approved vaccine for human use does not exist. Thus far, the traditional approaches to Brucella antigen selection for subunit vaccine development have yielded unacceptable results. In this work, we evaluated the predictive ability of a multistep Brucella antigen selection process with in vitro immunological and invasion assays and in vivo protection experiments. Initial in silico screening for antigens was performed via genomic sequence analysis where 27 Brucella melitensis open reading frames (ORF) coding for outer membrane proteins bearing MHC epitopes, adhesin and conserved properties were identified. Evidence for a role in any aspect of Brucella virulence (i.e., invasion, co-regulation/expression with known Brucella virulence factors, intracellular adaptation) was then used to narrow the list of candidate antigens. To further increase confidence in the candidate ORF putative role in Brucella pathogenesis, differential expression of candidate ORF was evaluated using previously generated global transcriptomics data in in vitro HeLa and in vivo bovine models of acute Brucella infection. Protein expression in the E. coli heterologous system resulted in the successful expression of OmpW, BtuB, Omp22, Hia, and FlgK. With regards to virulence, the two proteins with the highest predicted adhesin scores conferred an invasive phenotype to the non-invasive BL-21 E. coli strain in alveolar epithelial cells. From an immunogenicity standpoint, all proteins elicited IgG production in Brucella-exposed goats, mouse and humans. Antigen-specific recall responses in splenocytes from C57BL/6 mice immunized with a cocktail of the three proteins with highest MHC scores revealed a mixed Th1/Th2 response with a comparatively greater Th1 response. In protection studies, subcutaneous (SQ) immunization with BtuB, Hia and FlgK, individually, promoted bacterial clearance following a robust intraperitoneal challenge dose of Brucella melitensis 16M. In addition, single SQ inoculation of FlgK enhanced protective efficacy of the vaccine strain B. abortus S19. In contrast, immunization of mice with the three protective antigens in a cocktail formulation elicited immune responses but no protection against intraperitoneal challenge with Brucella melitensis 16M in the spleen and liver. In conclusion, our results indicate that our combinatorial in silico, in vitro and in vivo antigen selection and identification modeling approach provides strong evidence for prediction of Brucella protective antigens, and represent a novel strategy with broad application to other major pathogens.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 134-165.

The Salmonella enterica subsp. enterica serovar Typhimurium PhoPQ two-component system is activated within the intracellular phagosome environment, where it promotes remodeling of the outer membrane and resistance to innate immune antimicrobial peptides. Maintenance of the PhoPQ-regulated outer membrane barrier requires PbgA, an inner membrane protein with a transmembrane domain essential for growth, and a periplasmic domain required for PhoPQ-activated increases in outer membrane cardiolipin. Here, we report the crystal structure of cardiolipin-bound PbgA, adopting a novel transmembrane fold that features a cardiolipin binding site in close proximity to a long and deep cleft spanning the lipid bilayer. The end of the cleft extends into the periplasmic domain of the protein, which is structurally coupled to the transmembrane domain via a functionally critical C-terminal helix. In conjunction with a conserved putative catalytic dyad situated at the middle of the cleft, our structural and mutational analyses suggest that PbgA is a multifunction membrane protein that mediates cardiolipin transport, a function essential for growth, and perhaps catalysis of an unknown enzymatic reaction. IMPORTANCE Gram-negative bacteria cause many types of infections and have become increasingly resistant to available antibiotic drugs. The outer membrane serves as an important barrier that protects bacteria against antibiotics and other toxic compounds. This outer membrane barrier function is regulated when bacteria are in host environments, and the protein PbgA contributes significantly to this increased barrier function by transporting cardiolipin to the outer membrane. We determined the crystal structure of PbgA in complex with cardiolipin and propose a model for its function. Knowledge of the mechanisms of outer membrane assembly and integrity can greatly contribute to the development of new and effective antibiotics, and this structural information may be useful in this regard.

No description available.

Neste trabalho foi avaliado o potencial antigênico das vesículas de membrana externa, OMV, de N. meningitidis, das OMV e componetes protéicos selecionados de N. lactamica. Para tal foram realizados cultivos de ambas as espécies em biorreatores, ensaios de imunização em camundongos, análises de espectrometria de massas, e análises de tamanho das partículas, polidispersabilidade e potencial zeta. As análises da cinética de cultivos levaram a dados inéditos possibilitando uma nova discussão sobre o metabolismo e sobre a produtividade de OMV destas bactérias. N. lactamica obteve valores 5 vezes maiores de concentração máxima de OMV de 152 mg/L e 2,5 vezes maiores de produtividade de OMV de 0,32 g/L.h comparado aos obtidos para N. meningitidis nas mesmas condições de cultivo. OMV obtidas nos cultivos de ambas e componentes proteicos de N. lactamica foram utilizadas para imunizar camundongos com 3 doses subcutâneas. Ensaios de imunoblote e ELISA demonstraram que soros gerados contra as proteínas isoladas de N. lactamica foram reativos com proteínas de N. meningitidis, assim como o soro anti-OMV de N. lactamica que reagiu com 6 proteínas de N. meningitidis, as proteínas de membrana App, Omp85, PilQ, PorA, PorB, e ComL ou Opa/Opc. Análises de espectrometria de massas identificaram 229 proteínas na OMV de N. lactamica, sendo 77 proteínas de membrana e 243 proteínas de N. meningitidis, sendo 54 proteínas de membrana. Os resultados obtidos neste trabalho sugerem a possibilidade do uso das OMV de Neisseria lactamica como abordagem alternativa para o desenvolvimento de vacinas contra a doença meningocócica. / In these studies we evaluated the antigenic potential of outer membrane vesicles (OMV) from N. meningitidis, OMV from N. lactamica and proteic components from N. lactamica. Outer membrane vesicles were obtained from cultures of both species in bioreactors. Immunization tests were conducted in mice. Western-blotting and ELISA techniques were used to evaluate the cross-reactivity of murine sera against outer-membrane proteins from Neisseria meningitidis and Neisseria lactamica. Analysis of mass spectrometry and determination of particle size, polydispersity and zeta potential were also performed. Analysis of bacterial growth kinetics led to new data enabling a discussion about metabolism and OMV productivity. When both species were cultured in the same medium OMV concentration of N. lactamica (152 mg/L) is 5 times higher than that in N. meningitidis and OMV productivity of N. lactamica (0.32 g/L.h) is 2.5 times higher. Mice were vaccinated subcutaneously with 3 doses of OMV from both species and proteins from N. lactamica. These vaccines induced antibodies against N. meningitidis proteins. By mass spectrometry it was possible to identify these membrane proteins as App, Omp85, PilQ, PorA, PorB, and ComL or Opa/Opc. Mass spectrometry analyses also identified 229 proteins in N. lactamica OMV, with 77 predicted as membrane proteins and 243 in N. meningitidis OMV with 54 predicted as membrane proteins. Ours results suggested that OMV from Neisseria lactamica provides protection against N. meningitidis and could be used as an alternative approach for the development of a vaccine against meningococcal disease.