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

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Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Doctor of Philosophy / Department 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.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a Gram-negative coccobacillus of the Pasteurellaceae family. It is implicated in periodontitis, a common low-grade bacterial infection, but it can also cause non-oral infections. The main aim of this project was to identify and characterize in A. actinomycetemcomitans novel cell surface components bearing virulence potential that could contribute to systemic immunoinflammatory burden. We first established and evaluated a method for preparing homogeneous cell suspensions of autoaggregating clinical isolates of A. actinomycetemcomitans. The chosen method is based on a gradual dispersion of bacterial colonies into solution, which generated homogeneous suspensions without losing cell viability or fimbriation. When sera from two patients with A. actinomycetemcomitans-associated infections were used to probe A. actinomycetemcomitans outer membrane protein (OMP) preparations in western blot, strong reactions were found at 17 kDa. Interestingly, antiserum against CsgA, a major subunit of Eschirichia coli curli, also reacted with A. actinomycetemcomitans OMP preparations at 17 kDa size, that is the size of E. coli CsgA, suggesting antigenic crossreactivity. The 17 kDa A. actinomycetemcomitans OMP was subsequently identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by using immunoproteomics methods. Studies on the pal gene and its gene product showed that they were conserved among the clinical A. actinomycetemcomitans isolates representing all currently known serotypes. AaPAL expression was shown under different nutritional and atmospheric conditions that resembled those in periodontal pockets. PAL deficiency in turn led to pleiotropic effects on the phenotype of A. actinomycetemcomitans, such as cell elongation and decreased growth rate. To purify AaPAL we employed affinity chromatography using anti-AaPAL peptide antibodies. The extensive characterization of the purified AaPAL by SDS-PAGE gel staining and mass spectrometry demonstrated that the final purification product did not contain other bacterial proteins than AaPAL. The protein had not lost its antigenicity during purification, since it was recognized by sera from patients with A. actinomycetemcomitans-associated oral and nonoral infections. AaPAL also appeared to be a strongly immunoreactive antigen in patients with periodontitis whose serum IgG antibodies recognized in western blot a 17 kDa OMP in the parental strain but not in the pal-deficient mutant. In addition to its immunogenicity, AaPAL also induced proinflammatory cytokine and chemokine response from human whole blood as determined by a cytokine antibody array. A cell culture insert model was designed to study how bacterial components could be introduced to the host in infections. The experiments demonstrated that live bacteria released extracellularly free-soluble AaPAL, but also other components, via an unknown outer membrane vesicle-independent mechanism. The immunogenicity and proinflammatory potential of the previously uncharacterized outer membrane lipoprotein of A. actinomycetemcomitans, AaPAL, suggests that it contributes to the pathogenicity of this bacterium. That live A. actinomycetemcomitans cells released free-soluble cell components may represent a new pathogenic mechanism.

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.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "August 2001." Typescript. Vita. Includes bibliographical references (leaves 135-149). Also available on the Internet.

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.