Francisella tularensis, a Gram-negative coccobacillus, is an extremely virulent intracellular pathogen. Infection of humans with this pathogen results in tularemia, a life-threatening disease. An approximately 35 kb region found in the F. tularensis genome exhibits many features of a pathogenicity island. This region has a lower G+C content than the average G+C content of the F. tularensis genome, and is surrounded by transposable elements. Results from both a previous study and our present study demonstrated that at least four genes located on the Francisella pathogenicity island (FPI) are required for virulence. This represents the first description of a pathogenicity island in F. tularensis. The FPI-encoded proteins, however, have no significant similarities to any known bacterial proteins. Therefore, we believe that the FPI genes may encode a cluster of novel virulence factors, although the mechanism and their characteristics remain to be determined.

Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated which are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for the intracellular growth defect, colony morphology on LB (X-p) media, and virulence in mice. The locus consists of an apparent operon of two genes, designated mglAB, for macrophage growth locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 on LB (X-p) media. Sequencing of mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. Preliminary studies have also identified a convergently transcribed gene, tentatively designated mglC, immediately downstream of mglB. mglC null mutants are defective for intracellular growth and show the same phenotype on LB (X-p) agar as GB2. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Western blot analysis revealed the absence of MglA in an mglB null mutant, indicating MglB may influence MglA levels. Analysis of the regulation of mglA expression during growth in broth culture shows a decrease in expression upon entering late log-early stationary phase. mglA is also expressed during culture in macrophages. Cell fractionation studies revealed several differences in the protein profiles of mgl mutants compared with wild-type F. novicida, most notably the absence of a 70 kDa secreted protein. A candidate clone for the gene encoding this 70 kDa protein has been isolated. The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli, SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may control the expression of genes whose products contribute to survival and growth within macrophages. Roles for the putative MglC and possibly the 70 kDa secreted protein in this activity are also indicated. Acid phosphatases capable of inhibiting the respiratory burst of neutrophils have been identified in certain intracellular pathogens. The gene encoding AcpA, a respiratory burst-inhibiting acid phosphatase of Francisella , was cloned and sequenced. The deduced amino acid sequence of AcpA showed limited similarity to phospholipase C proteins present in Pseudomonas aeruginosa and Mycobacterium tuberculosis. An F. novicida acpA null mutant was found to exhibit wild-type growth kinetics in both cell-line and inflammatory mouse macrophages as well as remaining virulent for mice. These data suggest that AcpA is not essential for the intracellular growth or virulence of F. novicida, and that any role it may play in virulence is subtle. / Graduate

No description available.

Graduate

A survey was conducted, from 2001 to 2004, of viruses spreading within certified grapevine material in South Africa. As far as possible, viruses were identified and detection methods established. However, unknown spherical virus-like particles were observed in samples that also contained Grapevine Leafroll Associated Virus-Type 3. The unknown spherical particles were thought to most likely be Grapevine Fleck Virus, which was previously found in South Africa. A PCR method to be used locally for the routine detection of Grapevine Fleck Virus was established and first used to determine whether any of the greenhouse and field samples with the unknown spherical viruses were infected with Grapevine Fleck Virus. During the 2001 to 2004 survey, plants with leafroll and reddening symptoms unlike classical grapevine leafroll disease were also observed. No grapevine leafroll-associated viruses could be detected in these, but the symptoms observed resembled symptoms induced by phytoplasmas in Europe. A PCR method for the routine universal detection of phytoplasmas was established and this method was used to determine if phytoplasmas were associated with the symptomatic plants found. Sequence information from PCR amplicons suggest the presence of Candidatus phytoplasma solani, found for the first time in South Africa. This important finding however requires conformation by a second laboratory. / Dissertation (MSc (Microbiology))--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1). As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge. Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge. Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant. / Ph. D.

<p>The lungs and upper airways are mucosal surfaces that are common site for infection with an enormous variety of inhaled pathogens. Therefore, induction of immune responses in the respiratory tract is crucial for protection against respiratory diseases.</p><p>One of the pathogens infecting the host via the respiratory tract is <i>Mycobacterium Tuberculosis</i>. The reported efficacy of the currently used Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis is highly variable, ranging from 50% against pulmonary tuberculosis to 80% against disseminated tuberculosis. Recently, the current route of vaccination (intradermal) has been considered as a possible factor influencing the protective capacity of the BCG vaccine. In this regard, intradermal route most likely induces protective systemic responses while it fails to induce optimal responses in the lungs. Therefore, our working hypothesis is that vaccination should be directed towards the respiratory mucosal immunity in order to improve the degree of host protection in the lungs.</p><p>In this thesis we studied the effect of the route of immunization as well as of different mucosal adjuvants on the induction of mucosal immune responses against the mycobacterial surface antigen PstS-1. We found that, the intranasal (i.n.) route of immunization was a more favorable route inducing strong local immune responses, than intraperitoneal (i.p.) route. Indeed, i.n. route immunization, unlike the i.p. route, elicited strong IgA responses in the lungs accompanied by a major influx of CD4⁺ T cells and a significant local production of IFN-gamma.</p><p>IgA, being the predominant Ig isotype at mucosal tissues, is considered a major effector molecule involved in defense mechanisms against viral and bacterial pathogens at these sites. Therefore, we investigated the possible role of IgA in the protection of the respiratory mucosa against mycobacterial infections, using mice deficient in IgA and in the polymeric Ig receptor. We show that, deficient mice are more susceptible to mycobacterial infections than wild type mice, thereby demonstrating a role for IgA in protection against mycobacteria. Importantly, our studies revealed a reduced production of protective factors, such as INF-gamma and TNF-alpha in the lungs of deficient mice that was associated with the higher susceptibility seen in these mice compared to wild-type mice. We also conducted challenge experiments against another respiratory pathogen, <i>Chlamydia pneumoniae</i>, using IgA deficient mice. Likewise to mycobacteria, our data support a role for IgA in the protection of the respiratory tract against <i>C. pneumoniae</i> infection.</p><p>Finally, we investigated the possible mechanisms explaining the reduced pro-inflammatory responses in IgA deficient mice. Our data indicated that IgA deficient mice present a defective response to stimulation with LPS or 19kDa which appears to be both, essentially due to suboptimal stimulation of macrophages and restricted to the lungs.</p>

The lungs and upper airways are mucosal surfaces that are common site for infection with an enormous variety of inhaled pathogens. Therefore, induction of immune responses in the respiratory tract is crucial for protection against respiratory diseases. One of the pathogens infecting the host via the respiratory tract is Mycobacterium Tuberculosis. The reported efficacy of the currently used Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis is highly variable, ranging from 50% against pulmonary tuberculosis to 80% against disseminated tuberculosis. Recently, the current route of vaccination (intradermal) has been considered as a possible factor influencing the protective capacity of the BCG vaccine. In this regard, intradermal route most likely induces protective systemic responses while it fails to induce optimal responses in the lungs. Therefore, our working hypothesis is that vaccination should be directed towards the respiratory mucosal immunity in order to improve the degree of host protection in the lungs. In this thesis we studied the effect of the route of immunization as well as of different mucosal adjuvants on the induction of mucosal immune responses against the mycobacterial surface antigen PstS-1. We found that, the intranasal (i.n.) route of immunization was a more favorable route inducing strong local immune responses, than intraperitoneal (i.p.) route. Indeed, i.n. route immunization, unlike the i.p. route, elicited strong IgA responses in the lungs accompanied by a major influx of CD4+ T cells and a significant local production of IFN-gamma. IgA, being the predominant Ig isotype at mucosal tissues, is considered a major effector molecule involved in defense mechanisms against viral and bacterial pathogens at these sites. Therefore, we investigated the possible role of IgA in the protection of the respiratory mucosa against mycobacterial infections, using mice deficient in IgA and in the polymeric Ig receptor. We show that, deficient mice are more susceptible to mycobacterial infections than wild type mice, thereby demonstrating a role for IgA in protection against mycobacteria. Importantly, our studies revealed a reduced production of protective factors, such as INF-gamma and TNF-alpha in the lungs of deficient mice that was associated with the higher susceptibility seen in these mice compared to wild-type mice. We also conducted challenge experiments against another respiratory pathogen, Chlamydia pneumoniae, using IgA deficient mice. Likewise to mycobacteria, our data support a role for IgA in the protection of the respiratory tract against C. pneumoniae infection. Finally, we investigated the possible mechanisms explaining the reduced pro-inflammatory responses in IgA deficient mice. Our data indicated that IgA deficient mice present a defective response to stimulation with LPS or 19kDa which appears to be both, essentially due to suboptimal stimulation of macrophages and restricted to the lungs.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Metabolic networks are naturally represented as directed hypergraphs in such a way that metabolites are nodes and enzyme-catalyzed reactions form (hyper)edges. The familiar operations from set algebra (union, intersection, and difference) form a natural basis for both the pairwise comparison of networks and identification of distinct metabolic features of a set of algorithms. We report here on an implementation of this approach and its application to the procaryotes. We demonstrate that metabolic networks contain valuable phylogenetic information by comparing phylogenies obtained from network comparisons with 16S RNA phylogenies. We then used the same software to study metabolic innovations in two sets of organisms, free living microbes and Pyrococci, as well as obligate intracellular pathogens.