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In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.

ABSTRACT: Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria-host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach that enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared undecaprenyl-diphospho-N-acetyl-D-galactosamine, the E. coli O86 oligosaccharide repeating unit was assembled by means of sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy using a chaperone coexpression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of more substrates revealed a defined mode of recognition for Wzy toward the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well defined system for further underpinning molecular details of this biosynthetic pathway.

SUBMITTER: Woodward R

PROVIDER: S-EPMC2921718 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.

Woodward Robert R Yi Wen W Li Lei L Zhao Guohui G Eguchi Hironobu H Sridhar Perali Ramu PR Guo Hongjie H Song Jing Katherine JK Motari Edwin E Cai Li L Kelleher Patrick P Liu Xianwei X Han Weiqing W Zhang Wenpeng W Ding Yan Y Li Mei M Wang Peng George PG

Nature chemical biology 20100425 6

Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria-host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach that enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared undecaprenyl-diphospho-N-acetyl-D-ga ...[more]

PMID: 20418877

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Project description:Shigella flexneri implements the Wzy-dependent pathway to biosynthesize the O antigen (Oag) component of its surface lipopolysaccharide. The inner membrane polymerase WzySF catalyzes the repeat addition of undecaprenol-diphosphate-linked Oag (Und-PP-RUs) to produce a polysaccharide, the length of which is tightly regulated by two competing copolymerase proteins, WzzSF (short-type Oag; 10 to 17 RUs) and WzzpHS-2 (very-long-type Oag; >90 RUs). The nature of the interaction between WzySF and WzzSF/WzzpHS-2 in Oag polymerization remains poorly characterized, with the majority of the literature characterizing the individual protein constituents of the Wzy-dependent pathway. Here, we report instead a major investigation into the specific binding interactions of WzySF with its copolymerase counterparts. For the first time, a region of WzySF that forms a unique binding site for WzzpHS-2 has been identified. Specifically, this work has elucidated key WzySF moieties at the N- and C-terminal domains (NTD and CTD) that form an intramolecular pocket modulating the WzzpHS-2 interaction. Novel copurification data highlight that disruption of residues within this NTD-CTD pocket impairs the interaction with WzzpHS-2 without affecting WzzSF binding, thereby specifically disrupting polymerization of longer polysaccharide chains. This study provides a novel understanding of the molecular interaction of WzySF with WzzSF/WzzpHS-2 in the Wzy-dependent pathway and, furthermore, detects the Wzy/Wzz/Und-PP-Oag complex for the first time. Beyond S. flexneri, this work may be extended to provide insight into the interactions between protein homologues expressed by related species, especially members of Enterobacteriaceae, that produce dual Oag chain length determinants. IMPORTANCE Shigella flexneri is a pathogen causing significant morbidity and mortality, predominantly devastating the pediatric age group in developing countries. A major virulence factor contributing to S. flexneri pathogenesis is its surface lipopolysaccharide, which is comprised of three domains: lipid A, core oligosaccharide, and O antigen (Oag). The Wzy-dependent pathway is the most common biosynthetic mechanism implemented for Oag biosynthesis by Gram-negative bacteria, including S. flexneri. The nature of the interaction between the polymerase, WzySF, and the polysaccharide copolymerases, WzzSF and WzzpHS-2, in Oag polymerization is poorly characterized. This study investigates the molecular interplay between WzySF and its copolymerases, deciphering key interactions in the Wzy-dependent pathway that may be extended beyond S. flexneri, providing insight into Oag biosynthesis in Gram-negative bacteria.

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In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.

Publications

In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.

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