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Biosynthesis and Assembly of the Vi Antigen Capsule Produced by Salmonella enterica serovar Typhi

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Title: Biosynthesis and Assembly of the Vi Antigen Capsule Produced by Salmonella enterica serovar Typhi
Author: Liston, Sean
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Whitfield, Chris
Abstract: Capsules are bacterial cell-surface structures composed of hydrated capsular polysaccharides (CPS). The human pathogen Salmonella enterica serovar Typhi produces the ‘Vi antigen’ CPS, which contributes to virulence. This CPS is a component of some current vaccines. Vi antigen is delivered to the cell surface by an ATP-binding cassette (ABC) transporter. CPS ABC transporters participate in heteroligomeric protein complexes, that are proposed to form enclosed translocation conduits to the cell surface. This Thesis describes the identification of Vi antigen biosynthesis genetic loci in the Burkholderiales that are paradoxically distinguished from the S. Typhi by encoding a predicted pectate lyase I named VexL. Biochemical analyses of a representative VexL from Achromobacter denitrificans demonstrated that it is a Vi antigen-specific endo-lyase enzyme. A 1.2-Å crystal structure of the VexL-Vi antigen complex revealed determinants of specificity and features which distinguish common secreted catabolic pectate lyases from VexL, which participates in cell-surface assembly. When introduced into S. Typhi, VexL localized to the periplasm and degraded nascent Vi antigen. In contrast, a cytosolic derivative had no effect unless Vi antigen export was disrupted. This provides the first evidence that CPS assembled using ABC transporters are periplasm-exposed during translocation. Vi antigen-producers lack enzymes to build a terminal glycolipid residue that is conserved in all other ABC transporter-dependent CPS assembly systems. VexL was used to depolymerize Vi antigen to facilitate isolation of intact Vi antigen chain termini. Mass spectrometry revealed Vi antigen oligosaccharides with a reducing terminal N-acetylhexosamine residue decorated with two β-hydroxyacyl chains. The VexE protein, which is uniquely encoded by Vi antigen biosynthesis loci, shares similarity with acyltransferases from lipid A biosynthesis. A ΔvexE mutant of S. Typhi produced Vi antigen with altered physical properties; its export was impaired, the glycan was not attached at the cell surface, and the glycolipid was not identified. Biochemical assays demonstrate that VexE is a UDP-N-acetylglucosamine C6 β-hydroxyacyltransferase that prefers 14-carbon acyl chains. The structure of the terminal glycolipid dictates a unique assembly mechanism and has potential implications in pathogenesis, immunology, and vaccine production. This thesis provides novel insight into mechanisms for CPS production conserved in diverse and clinically-relevant bacteria.
URI: http://hdl.handle.net/10214/12955
Date: 2018-04
Rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
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Attribution-NonCommercial-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 2.5 Canada