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Characterization of Three Activity Domains from the Multifunctional RTX Toxin from <em> Vibrio cholerae

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The Gram-negative bacterium &lt;em&gt; Vibrio cholerae &lt;/em&gt; elicits disease through the export of enterotoxins. The &lt;em&gt; V. cholerae &lt;/em&gt; RTX toxin was identified due its ability to cause cell rounding. Characterization of the RTX toxin demonstrated that this cell rounding was due to the depolymerization of the actin cytoskeleton through the novel mechanism of covalent actin cross-linking. In this study, three functional domains were identified and characterized within the RTX toxin. Expression of a 47.8 kDa domain from the RTX toxin mediated the covalent cross-linking of actin. An in-frame deletion of this domain within the RTX toxin abrogated the cross-linking activity of the toxin but not cell rounding. Further investigation revealed that this second cell rounding activity was due to RTX inactivation of the Rho GTPases. This activity was mapped to a 548 amino acid region within RTX. Characterization of a third domain from RTX demonstrated that it was a novel cysteine protease domain, which is speculated to autoproces the toxin. Mutation of the catalytic cysteine on the &lt;em&gt; V. cholerae &lt;/em&gt; chromosome inhibited the actin cross-linking activity of the toxin suggesting that autoprocessing of the RTX toxin is important for activity. Analysis of this cysteine protease domain revealed that it is conserved in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from &lt;em&gt; V. vulnificus &lt;/em&gt;, &lt;em&gt; Yersinia sp. &lt;/em&gt;, &lt;em&gt; Photorhabdus sp. &lt;/em&gt;, and &lt;em&gt; Xenorhabdus sp. &lt;/em&gt;; and a filamentous/hemagglutinin-like protein FhaL from &lt;em&gt; Bordetella sp. &lt;/em&gt; which suggests that autoprocessing is a common mechanism utilized by these proteins. Altogether, the studies presented in this thesis have further characterized the mechanism of the &lt;em&gt; V. cholerae &lt;/em&gt; RTX toxin

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  • 05/09/2018
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