Shigella flexneri is a Gram negative, intracellular pathogen responsible for bacillary dysentery in humans. To achieve infection of the human colonic epithelium, S. flexneri must adapt to varying environmental conditions, including fluctuations in pH, osmolarity, and nutrient and oxygen availabilities. The plaque assay is commonly used to measure the ability of S. flexneri to invade epithelial cells, grow intracellularly, and spread intercellularly to adjacent cells. However, as traditionally performed, this assay is of limited use in testing the virulence of Shigella in response to many of the conditions encountered in the host. I have modified the plaque assay to identify factors contributing to the virulence of S. flexneri under the anaerobic conditions present in the colon. This assay demonstrated that the Feo transport system that acquires anaerobically abundant ferrous iron, as well as the transcription factors ArcA, Fnr, and Fur, impact Shigella plaque formation in anoxic environments. Transcriptional analyses indicated that anaerobic conditions activated expression of feoABC in S. flexneri. Anaerobiosis also repressed genes encoding two other iron transport systems that allow plaque formation by S. flexneri in aerobic environments, the ABC transporter Sit and the Iuc/Iut aerobactin siderophore synthesis and acquisition system that binds ferric iron, the dominant form of iron under aerobic conditions. The anaerobic regulators ArcA and Fnr induced expression of feoABC. Additionally, ArcA represses transcription of iucABCDiutA and fur. fur encodes a transcriptional regulator that is activated in the presence of iron and is responsible for repression of genes encoding iron acquisition systems and the sRNA RyhB that affects synthesis of iron-storage proteins including many TCA cycle enzymes. ArcA is a redox regulator known for redirecting metabolism upon oxygen depletion by down-regulating TCA cycle and aerobic respiratory enzymes and inducing fermentation and anaerobic respiratory complexes. However, ArcA regulation of fur and its downstream targets offers S. flexneri a means to coordinate energy and carbon metabolism with that of iron availability in response to environmental redox conditions. / text

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 1997. / Includes bibliographical references (leaf 129-150).

Thesis (Ph.D.)--University of Hong Kong, 1997. / Includes bibliographical references (leaf 129-150) Also available in print.

Thesis (M.A.)--Central Connecticut State University, 2002. / Thesis advisor: Michael A. Davis. " ... in partial fulfillment of the requirements for the degree of Master of Arts in Biology." Includes bibliographical references. Also available via the World Wide Web.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Vita. Includes bibliographical references.

Zugl.: Berlin, Humboldt-Univ., Diss., 2009

Shigella flexneri is a Gram negative facultative anaerobe that infects millions world-wide each year. The route for infection of a host is through the intestinal and rectal epithelium layers, but it also can survive in the environment. Different genes have been found to be up regulated depending upon its presence in the intracellular or extracellular environment, as shown in previous work in the lab. This thesis seeks to examine the role these upregulated genes, bioA and lysA, play in the intracellular activity of S. flexneri. Knock-out mutations in the bioA and lysA genes were created using P1 transduction. To test the effects of these mutations on S. flexneri, plaque, invasion, and attachment assays were performed. It was found that the bioA mutation resulted in fewer plaques being formed, while the lysA mutation resulted in slower forming and incompletely lysed plaques being formed. / text

The gram negative bacterium Shigella flexneri is an etiological agent of bacillary dysentery, and causes destruction of the human intestinal epithelium. S. flexneri is primarily transmitted via the fecal-oral route to its primary infective site in the colon. The bacterium invades and replicates within colonic epithelial cells, ultimately ulcerating the mucosal epithelium. To successfully establish infection, S. flexneri must quickly adapt to different environments in the host, including adjusting metabolism in response to changes in available carbon sources. In this study, the importance of the glycolytic and gluconeogenic pathways in S. flexneri pathogenesis was examined. The metabolic regulators CsrA and Cra reciprocally regulate the glycolytic and gluconeogenic pathways. The post-transcriptional regulator Cra activates expression of genes involved in gluconeogenesis and represses glycolysis. Conversely, CsrA activates glycolysis and represses gluconeogenesis. The absence of Cra increased S. flexneri attachment and invasion of cultured epithelial cells. In contrast, the csrA mutant was significantly impaired in both adherence and invasion. Both the csrA and cra mutants formed small, turbid plaques, suggesting that both regulators are required for plaque formation. The opposing phenotypes of the csrA and cra mutants suggested a correlation between invasion and glycolysis. The role of glycolysis in S. flexneri pathogenesis was confirmed by directly examining the first committed step in the pathway. The glycolytic enzyme phosphofructokinase I (PfkI, encoded by pfkA) is repressed by Cra and activated by CsrA. Glycolysis was critical for S. flexneri pathogenesis, as a mutation in pfkA rendered the bacterium noninvasive. The invasion defect of the csrA and pfkA mutants was due to reduced expression and secretion of the Shigella invasion plasmid antigen (Ipa) effectors. Expression of the master virulence regulators virF and virB was significantly reduced in the pfkA mutant, and is the principle reason for decreased invasion. The data presented show that glycolysis is required for invasion, but that plaque formation requires both glycolysis and gluconeogenesis. Because expression of the master virulence regulators is repressed in the pfkA mutant, S. flexneri may use carbon as an environmental regulator of virulence gene expression. / text