Biosynthesis of the Pseudomonas aeruginosa Common Polysaccharide Antigen by D-Rhamnosyltransferases WbpX and WbpY
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Authors
Melamed, Jacob
Date
Type
thesis
Language
eng
Keyword
Pseudomonas aeruginosa , Lipopolysaccharide , O antigen , Glycosyltransferase , D-Rhamnose , D-Rhamnosyltransferase , Common Polysaccharide Antigen , Polymerization , Glycobiology
Alternative Title
Abstract
The Gram-negative bacterium Pseudomonas aeruginosa is responsible for chronic and antibiotic-resistant infection in cystic fibrosis patients and immunocompromised individuals and is one of the leading organisms associated with nosocomial infection. Unlike other species, P. aeruginosa simultaneously expresses two glycoforms of lipopolysaccharide (LPS), differing in their O antigens, the most distal outer membrane polysaccharides. While the O-specific antigen (OSA) is variable in composition, the common polysaccharide antigen (CPA) is highly conserved and is composed of a homopolymer of D-rhamnose (D-Rha) in trisaccharide repeating units [D-Rhaα1-2-D-Rhaα1-3- D-Rhaα1-3]n. It has previously been established that D-Rha-transferase WbpZ adds the first D-Rha residue to the natural acceptor analogue GlcNAcα-PO3-PO3-(CH2)11-O-phenyl. Genes encoding two more D-Rha- transferases are found in the CPA biosynthesis gene cluster (wbpX and wbpY). This research seeks to biochemically characterize these two glycosyltransferases and understand their functions in the assembly of CPA. These studies show that WbpX and WbpY differ in their donor and acceptor specificities and have properties of GT-B folded enzymes of the GT4 glycosyltransferase family. Using mass spectrometry, we showed that recombinant His6-tagged WbpX and WbpY individually transferred a single D-Rha residue to the WbpZ product D-Rhaα1-3GlcNAcα-PO3-PO3-(CH2)11-O-phenyl. However, the mixture of WbpX and WbpY was able to catalyze the synthesis of longer D-Rha polymers. Furthermore, WbpX was found to possess polymerase activity in isolation under certain conditions. Nuclear magnetic resonance (NMR) analysis of the WbpX and WbpY D-Rha products revealed that WbpY is an α1-3-D-Rha-transferase, adding a single D-Rha residue to the WbpZ product. In contrast, WbpX adds several D-Rha residues to synthetic acceptor D-Rhaα-benzyl, forming both α1-2 and α1-3 linkages. Since CPA, and O antigens in general, are important virulence factors in bacterial infection, these findings open the door to advancing technology for antibacterial drug discovery and vaccine development against P. aeruginosa and other D-Rha-harbouring pathogenic bacteria.
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Attribution 3.0 United States
ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Attribution 3.0 United States