Genetic insight into zinc and antimicrobial toxicity uncovers a glycerol phosphate modification on streptococcal rhamnose polysaccharides

Phosphate-containing polyanionic glycopolymers, such as wall teichoic acid (WTA), play important roles in many fundamental aspects of bacterial physiology and infection biology. Interestingly, the cell walls of many streptococcal species express glycopolymers that are considered to be WTA homologues but surprisingly lack the characteristic and biologically-relevant anionic charge. The human pathogen Group A Streptococcus (GAS; Streptococcus pyogenes) expresses the species-defining Group A Carbohydrate (GAC), which consists of a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side-chains that are important for pathogenesis. We identified gacH, a gene of unknown function located in the GAC biosynthesis cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA secreted phospholipase A2 (hGIIA). To understand the underlying mechanism of these phenotypes, we expressed and crystallized the extracellular domain of GacH and demonstrate that it represents a new family of glycerol phosphate (GroP) transferases that is evolutionarily distinct from lipoteichoic acid synthase. Importantly, we demonstrate that GAC and also the homologous Serotype C Carbohydrate (SCC) from the dental pathogen Streptococcus mutans contain previously unidentified GroP modifications conferred by gacH and sccH products, respectively. NMR analysis of purified GAC revealed that approximately 30% of the GlcNAc side-chains of GAC are modified by GroP at the C6 hydroxyl group. In conclusion, our findings pinpoint a previously unrecognized structural modification of streptococcal cell wall polysaccharides that has important biological consequences, conferring negative charge to the bacterial cell wall. Furthermore, considering that GAC and homologous structures in other streptococcal pathogens are highly regarded targets for vaccine development, our discovery has implications for vaccine design.