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Molecular and Biochemical Investigation of Mechanisms that propel O-antigen Diversity in Pseudomonas aeruginosa

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Title: Molecular and Biochemical Investigation of Mechanisms that propel O-antigen Diversity in Pseudomonas aeruginosa
Author: Taylor, Véronique Louise
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Lam, Joseph S.
Abstract: Lipopolysaccharide of Pseudomonas aeruginosa is capped by highly diverse O-specific antigens (OSA). Differences in the OSA are the basis for classifying P. aeruginosa into 20 distinct serotypes. OSA is synthesized by the Wzx/Wzy-dependent pathway wherein O-units are polymerized by Wzy with either α- or β- intramolecular linkages to chain-lengths regulated by Wzz1 or Wzz2 whereas the polymer is ligated by WaaL a lipid-anchor forming mature LPS. Infection of P. aeruginosa by the D3 bacteriophage results in serotype conversion by altering the intramolecular linkage from α- to β- due to the expression of an inhibitor of α-polymerase (Iap), and a β-polymerase (Wzyβ). The inner membrane topologies of WaaL and Wzyβ were determined experimentally using a dual-reporter capable of PhoA-LacZ activity to screen the subcellular localization of random and site-targeted 3’ truncations. Twelve transmembrane segments (TMS) and a large periplasmic loop were identified for WaaL. Wzyβ contained 10 TMS and two large periplasmic loops (PL3 and PL4). Both WaaL and Wzyβ possessed residues essential for an inverting glycosyltransferase reaction. The topology of Wzyβ supports the proposed “catch-and-release” mechanism of Wzy proteins. Wzyβ is therefore resistant to Iap inhibition due to using a different reaction mechanism.By titrating Iap expression it was determined that Iap inhibition occurs after Wzyα was inserted into the inner membrane. Specificity of the Iap to the O2 serogroup is supported by sequence similarity between Iap and the N’ terminal TMS of only cognate Wzz proteins. Iap appears to function by disrupting the proposed Wzz/Wzy interaction. In a separate project, we developed an algorithm for in silico serotyping of P. aeruginosa based on unique sequences in the OSA clusters. This method was used to type >80 P. aeruginosa strains with published genome sequences. We discovered a unique genomic island among O12 strains with a multidrug-resistance phenotype, and collected evidence of concomitant transfer of the O12 OSA cluster and antibiotic resistance genes from an ancestral clone, PA7. The presence of this island accounts for the geographic dissemination of the O12 serotype in hospital settings. In summary, this research furthered the understanding of mechanisms which drive O-antigen diversity in P. aeruginosa.
Date: 2016-02

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