Investigation of core machinery for biosynthesis of Vi antigen capsular polysaccharides in Gram-negative bacteria

Salmonella enterica serovar Typhi causes typhoid fever. It possesses a Vi antigen capsular polysaccharide coat that is important for virulence and is the basis of a current glyco-conjugate vaccine. Vi antigen is also produced by environmental Bordetella isolates, while mammal-adapted Bordetella species (such as Bordetella bronchiseptica) produce a capsule of undetermined structure that cross-reacts with antibodies recognizing Vi antigen. The Vi antigen backbone is composed of poly-alpha-(1 -> 4)-linked N-acetylgalactosaminuronic acid, modified with O-acetyl residues that are necessary for vaccine efficacy. Despite its biological and biotechnological importance, some central aspects of Vi antigen production are poorly understood. Here we demonstrate that TviE and TviD, two proteins encoded in the viaB (Vi antigen production) locus, interact and are the Vi antigen polymerase and O-acetyltransferase, respectively. Structural modeling and site-directed mutagenesis reveal that TviE is a GT4-family glycosyl-transferase. While TviD has no identifiable homologs beyond Vi antigen systems in other bacteria, structural modeling suggests that it belongs to the large SGNH hydrolase family, which contains other O-acetyltransferases. Although TviD possesses an atypical catalytic triad, its O-acetyltransferase function was verified by antibody reactivity and C-13 NMR data for tviD-mutant polysaccharide. The B. bronchiseptica genetic locus predicts a mode of synthesis distinct from classical S. enterica Vi antigen production, but which still involves TviD and TviE homologs that are both active in a reconstituted S. Typhi system. These findings provide new insight into Vi antigen production and foundational information for the glycoengineering of Vi antigen production in heterologous bacteria.

Automated summary

This study reveals the production mechanism of Vi antigen. Two proteins, TviE and TviD, encoded in the viAB locus, are identified as Vi antigen polymerase and O-acetyltransferase, respectively. Their functions and mechanisms are determined through structural modeling and mutagenesis experiments. These findings contribute to understanding the production process of Vi antigen and provide foundational information for glycoengineering of Vi antigen production in heterologous bacteria.