Browsing by Author "Stephen J. Libby, Committee Chair"

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    Evolutionary and Functional Analysis of SlyA in Salmonella typhimurium
    (2002-08-27) Boutt, Elizabeth Ann; Stephen J. Libby, Committee Chair
    Salmonella species cause a variety of infections in humans and domestic animals, ranging from mild food poisoning-gastroenteritis Salmonella typhimurium to severe systemic disease Salmonella typhi. Salmonella is used as a paradigm to understand how intracellular pathogens withstand the onslaught of the host innate immune system, namely the professional macrophage. SlyA is a global transcriptional regulator that is necessary for the virulence of Salmonella typhimurium. In a mouse model system, a slyA mutant is profoundly attenuated for virulence and is unable to survive in macrophages. To understand the evolutionary history of SlyA in Eubacteria, a phylogenetic analysis was performed. Several alignments of amino acid sequences were constructed with MarR and also with known SlyA homologues. Searches for other SlyA homologues were undertaken using databases of unfinished and finished microbial genomes and more than 50 putative homologues were found. SlyA has been classified as a MarR-like transcriptional regulator by homology. This classification may not be appropriate given the differences in function. Therefore it is suggested that a new class of SlyA-like regulators be formed incorporating all of the homologues found in this study. In order to determine the extent of the SlyA regulon, studies were conducted to analyze the DNA-binding properties of SlyA. The target promoters from this study included genes that were shown to be either activated or repressed by SlyA by utilizing a Salmonella genomic DNA microarray. SlyA was shown to bind specifically to the promoters of clyA, pagC, pagK and mig-14. The approximate Kd values appear to be similar between each of the promoters indicating a similar propensity for SlyA to bind.
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    The Role of SlyA-dependent Genes in Salmonella Pathogenesis
    (2003-09-12) Halsey, Thomas Allen; Stephen J. Libby, Committee Chair
    The purpose of this research has been to examine the contribution of the transcriptional regulator, SlyA, to the expression of virulence genes in Salmonella enterica serovar Typhimurium. SlyA is a small molecular weight transcriptional regulatory protein that is required for oxidative stress resistance, intramacrophage survival, and for virulence in the murine model of salmonellosis. This work demonstrates that SlyA is required for survival in aerobic environments. A mutation in slyA causes profound loss of viability during prolonged stationary phase and slyA mutant Salmonella grow more slowly as compared to wild type under aerobic conditions. Conversely, under anaerobic conditions, a slyA mutant shows a similar phenotype to that of wild type suggesting that a mutation in slyA renders it more susceptible to oxidative damage. The transcription of slyA was also found to not be significantly affected by other known oxidative stress loci. A transposon insertion into STM2359, however, completely abolished slyA expression and this mutant demonstrated many of the phenotypic characteristics of a slyA mutant. This work also demonstrates that slyA expression can be induced under conditions of low pH and low magnesium ion concentration. The contribution of the PhoP/Q two-component regulatory system was also examined. As a result of these studies, the pagC locus, which was previously thought to be PhoP-dependent, was found to be directly activated by SlyA. Electrophoretic mobility shift analysis and DNase I protection assays showed that SlyA physically interacts with the pagC promoter. Microarray analysis of a slyA mutant showed reduced pagC expression as compared to wild type. Further analysis by quantitative real time PCR also demonstrated that pagC expression is profoundly reduced in a slyA mutant Salmonella. The greatest reduction in pagC expression, however, was illustrated by a mutation in both slyA and phoP. A pagC::lacZ promoter fusion combined with a mutation in slyA, phoP, or both also confirmed these observations. These studies also demonstrated that PhoP contributes to pagC expression indirectly by possibly influencing the specificity of SlyA. The work presented here also illustrated a role of SlyA in the activation of Salmonella pathogenicity island 2 gene expression. These studies demonstrated that SlyA binds to the ssrB promoter by electrophoretic mobility shift analysis and DNase I protection assays. Furthermore, these studies showed that SlyA directly induces ssrB expression as determined by microarray analysis, quantitative real time PCR, and promoter fusions. The competitive infection data also suggests that SPI2 gene expression and SlyA are part of the same pathway. Collectively, these data show a direct connection between SPI2 and SlyA.