MxiE-dependent Gene Regulation in Shigella flexneri

Shigella spp. are the causative agents of bacillary dysentery, in which the hallmark symptom is bloody diarrhea. According to a recent estimate, there are approximately 270 million and nearly 200,000 deaths per year due to Shigella infection and there is currently no effective vaccine available. S. flexneri is the most widespread and studied species. Necessary for its virulence, the large virulence plasmid of S. flexneri encodes a type 3 secretion system, virulence effector proteins, as well as virulence regulators. Expression of virulence genes follows a sophisticated regulatory cascade involving three transcriptional regulators: i) VirF, ii) VirB, and iii) MxiE. Prior to my thesis work, the factors involved in MxiE-dependent gene expression were still unclear. My thesis work has improved our understanding of MxiE- and H-NS-dependent gene regulation in S. flexneri (Chapter 2). We identified novel chromosomal MxiE-dependent genes (yccE and yfdF) that require an intact MxiE box sequence in their promoter regions for gene expression. In addition, based on the AT-rich nature of MxiE-dependent genes, I hypothesized that H-NS, a protein involved in nucleoid organization, is mediating their transcriptional repression. We used a dominant negative H-NS to show that representative MxiE-dependent virulence plasmid and chromosome genes are expressed when H-NS is sequestered. Additionally, we show that a known anti-silencer of H-NS, VirB, is not required for MxiE-dependent gene expression when MxiE and its co-activator, IpgC, are overexpressed. Finally, we demonstrate that MxiE is no longer necessary for gene expression when H-NS is sequestered. These data led us to propose a model of MxiE-mediated counter-silencing of H-NS, which is a previously unreported role for MxiE. In summary, my work has provided insights into previously unknown layers of MxiE-dependent gene regulation, which contributes to the overall understanding of S. flexneri virulence.