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Characterization of the Biosynthesis of Klebsiella pneumoniae O-antigen Polysaccharides and the Origin of Ribofuranose in Bacterial Polysaccharides

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Title: Characterization of the Biosynthesis of Klebsiella pneumoniae O-antigen Polysaccharides and the Origin of Ribofuranose in Bacterial Polysaccharides
Author: Kelly, Steven
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Whitfield, Chris
Abstract: Klebsiella pneumoniae is a Gram-negative opportunistic bacteria pathogen of global concern due to extensive and growing antibiotic resistance. Cell surface polysaccharides can be used to produce glycoconjugate-based immunogens for vaccines or production of prophylactic antibodies to combat resistant infections. Lipopolysaccharide O-antigen polysaccharides (O-PSs) are candidates but the use of O-PS in glycoconjugate vaccine production requires a detailed understanding of the structures of O-PSs. This research aims to fill some important gaps in current understanding. The K. pneumoniae O2a O-PS is prevalent in clinical isolates but is diversified by various structural modifications to generate different antigenic forms. In one example, the O2c antigen is a structurally and antigenically distinct polysaccharide added to the non-reducing terminus of O2a chains. A three gene locus (wbmVWX) is implicated in the biosynthesis of the O2c antigen, but the precise biochemical activities encoded by these genes are unknown. Here I show that WbmV and WbmW are glycosyltransferase enzymes sufficient to produce the O2c disaccharide repeat-unit structure. Together, WbmV and WbmW interact to form the active, membrane associated O2c biosynthesis complex. This work provides the first comprehensive biochemical insight into an unusual form of polysaccharide modification. The O-PSs of K. pneumoniae O4 and O7 serotypes share ribofuranose in their repeat units, but the identity of the activated donor for ribofuranose residues in any bacterial polysaccharide is unknown, as are the corresponding ribofuranoslytransferase enzymes. Here I unequivocally show the activated donor is 5'-phospho-ᴅ-ribosyl-alpha-1-diphosphate (PRPP) and characterize the first known ribosyltransferase, a bifunctional protein containing a glycan phosphoribosyltransferase (gPRT) and a phosphoribose phosphatase (PRP). The crystal structure of a thermophilic ortholog of these enzymes revealed a novel glycosyltransferase fold shared by a well distributed collection of ribofuranoslytransferases. Using the PRP domain as a probe for bioinformatics, I identified four groups of ribofuranosyltransferase enzymes differing in the sequence of the gPRT components and enzyme modularity. While 3 groups transfer ribofuranose to cytoplasmic O-PS biosynthetic intermediates, one system represented a periplasmic based modification system for the addition of α-linked ribofuranose side chains. This has established several new and novel prototype systems for further investigation of the principles of glycosylation machinery.
URI: https://hdl.handle.net/10214/27266
Date: 2022-09
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Related Publications: Kelly, S.D., and Clarke, B.R., Ovchinnikova, O.G., Sweeney, R.P., Williamson, M.L., Lowary, T.L., Whitfield, C. Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification. Journal of Biological Chemistry 294(28) 10863- 76 (2019). 10.1074/jbc.RA119.008969.Kelly, S.D., Williams, D.M., Nothof, J.T., Kim, T., Lowary, T.L., Kimber, M.S., Whitfield, C. The biosynthetic origin of ribofuranose in bacterial polysaccharides. Nature Chemical Biology 18 530-537 (2022). 10.1038/s41589-022-01006-6.


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