Pathogenesis of enterotoxigenic Escherichia coli (ETEC) and Shigella flexneri relies predominantly on members of the AraC/XylS family of transcriptional regulators, Rns (or its homolog, CfaD) and MxiE, respectively. Rns/CfaD regulate the expression of pili, which allow the bacteria to attach to the intestinal epithelium. Better understanding of the role Rns plays in virulence was attained by expanding our knowledge of the Rns regulon, revealing that it functions as an activator of cexE, a previously uncharacterized gene. By in vitro DNase I footprinting two Rns-binding sites were identified upstream of cexEp, both of which are required for full activation of cexE. The amino terminus of CexE also contains a secretory signal peptide that is removed during translocation to the periplasm. Though the function of CexE remains unknown, these studies suggest that CexE is a novel ETEC virulence factor since it is regulated by Rns/CfaD. In Shigella flexneri, the expression of a subset of virulence genes (including, ipaH9.8 and ospE2) is dependent upon the activator MxiE and a cytoplasmic chaperone IpgC. To define the molecular mechanism of transcriptional activation by this chaperone-activator pair, an in vitro pull down assay was performed revealing that MxiE specifically interacts with IpgC in a complex. Additionally, IpgC recognizes three polypeptide regions in MxiE: within MxiE(1-46), MxiE(46-110) and MxiE(196-216). Furthermore, it seems that MxiE and IpgC regulate transcription of ipaH9.8 and ospE2 promoters differently. In the bacterium, the formation of the MxiE-IpgC complex is initially prevented because IpgC is sequestered in individual complexes with effector proteins, IpaB and IpaC. Upon contact with an eukaryotic host cell the effector proteins are secreted, thereby freeing IpgC to form a complex with MxiE and activate the expression of virulence genes. This new characterization of the role of Rns and MxiE in virulence gene regulation in ETEC and S. flexneri, respectively will give new insights into the pathogenesis of the regulators.

Shigella flexneri attacks cells of the intestinal tract, causing over 1 million deaths annually from bacterial dysentery. A type III secretion system (T3SS) initiates the host-pathogen interaction and transports virulence factors directly into host cells via a needle complex (NC) comprising an extracellular needle and membrane-spanning basal body. Rings formed by the single-pass membrane proteins MxiG and MxiJ are arranged concentrically within the inner membrane ring (IMR) of the NC. The Neterminal domain of MxiG (MxiG-N) is the predominant IMR cytoplasmic structure, however it was structurally and functionally uncharacterised. Determination of the solution structure of MxiG-N in this study revealed it to be a forkhead associated (FHA) domain, although subsequent analyses of conserved residues suggested it does not have the canonical role in cell-signalling via phospho-threonine recognition. Subsequent positioning of the structure in the electron microscopy (EM) density for the S. flexneri NC supported models with 24-fold symmetry in the IMR. Both MxiG and MxiJ also have significant periplasmic domains, which were purified to homogeneity in this study, facilitating preliminary characterisation of their structures and intermolecular interactions. In addition, the entire IMR within the context of intact basal bodies was isolated and visualised in vitro by EM. The essential function of MxiG-N could be to localise the putative cytoplasmic ring (Cering) at the base of the T3SS. Although absolutely required for secretion, the Csring component, Spa33, was structurally uncharacterised. The crystal structure of the Cvterminal domain of Spa33 (Spa33-C) was determined in this study, showing an intertwined dimer that aligned with homologous structures and exhibited a novel interaction with the N-terminus of the ATPase regulator, MxiN. Subsequently, Spa33-C was identified as an altemative translation product of spa33 that formed a 2: 1 complex with Spa33 in vitro. This complex oligomerised further, demonstrating for the first time that Spa33 has the propensity to form the ordered, high molecular weight assemblies that would be required for C-ring formation in S. flexneri.

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MSc (Microbiology) / Department of Microbiology / See the attached abstract below