Investigation of Two Polysaccharide ‘Polymerases’ Involved in the Synthesis of Bacterial Cell-Surface Glycans
Cell-surface polysaccharides serve as essential virulence determinants for many bacteria. These molecules are structurally diverse, as are the activities of the glycosyltransferase (GT) enzymes involved in their synthesis. Most GTs catalyze a single transfer/linkage, whereas those which transfer multiple sugars are referred to as processive GTs or polymerases and are the focus of the research herein. Some polymerases, often referred to as synthases, have the additional capacity to both polymerize and export glycans across the membrane. One example is the O:54 antigen synthase, WbbF, from Salmonella enterica serovar Borreze. WbbF differs from prototypical synthases in the requirement for a lipid carrier. I examined the membrane topology of WbbF and used site-directed mutagenesis to confirm that residues important for catalysis and processivity in known synthases are conserved in WbbF. These findings indicate that the GT mechanism of WbbF and classic synthases are likely conserved, despite the use of a lipid acceptor. Regardless of the biological and biotechnological importance of the virulence capsular polysaccharide (Vi antigen) of S. enterica serovar Typhi, some central aspects of its production are poorly understood. The production of Vi antigen was hypothesized to employ a single polymerase. Here I demonstrate that TviE and TviD are the Vi antigen polymerase and O-acetyltransferase, respectively. Structural modeling and properties of site-directed mutants reveal TviE to be a GT4 enzyme. TviD contains an N-terminal O-acetyltransferase, verified by the structure of the product made by a tviD-mutant polysaccharide. Site-directed mutagenesis determined that TviD possesses an atypical catalytic triad compared to members of the SGNH hydrolase family. Tetratricopeptide repeats at the C-terminus of TviD mediate interactions between TviD and TviE. Vi antigen is also produced by environmental Bordetella isolates, while mammal-adapted Bordetella species (like B. bronchiseptica) produce Vi antigen-related capsule of undetermined structure that cross-reacts with antibodies recognizing Vi antigen. The B. bronchiseptica genetic locus predicts a different mode of synthesis from classical Vi antigen but still involves TviD and TviE homologs that are both active in a reconstituted S. Typhi system. This research provides foundational information for biosynthesis of important classes of polysaccharides and the structures and activities of polymerases with different properties.
Whitfield, C., Wear, S.S., and Sande, C. (2020). Assembly of bacterial capsular polysaccharides and exopolysaccharides. Annu. Rev. Microbiol. 74, 521-543. DOI 10.1146/annurev-micro-011420-075607.
Wear, S.S., Hunt, B.A., Clarke, B.R., and Whitfield, C. (2020). Analysis of the topology and active-site residues of WbbF, a putative O-polysaccharide synthase from Salmonella enterica serovar Borreze. J. Bacteriol. 202, e00625-19. DOI 10.1128/JB.00625-19.
Sande, C., Wear, S., and Whitfield, C. (2019). Capsules and secreted extracellular polysaccharides. In Encyclopedia of Microbiology, T.M. Schmidt, ed. (Cambridge, MA: Academic Press), pp. 604-612. DOI 10.1016/b978-0-12-801238-3.99215-8.