Human Group IIA-secreted phospholipase A2 (sPLA2-IIA), an enzyme present in plasma and tissue fluids, exerts potent bactericidal effects by hydrolyzing membrane phospholipids. The importance of sPLA2-IIA in host defense against Gram-positive pathogens, including Group A Streptococcus (GAS), has been demonstrated in animal models. Compared to other Gram-positive bacteria, GAS is remarkably resistant to sPLA2-IIA activity. We set out to uncover mechanisms conferring GAS resistance towards sPLA2-IIA. By screening a random GAS mutant library, we identified 17 mutants with altered sPLA2-IIA susceptibility. Interestingly, three of these genes (gacH, I, and J) clustered within the Group A Carbohydrate (GAC) biosynthesis locus. The GAC is a glycopolymer of the GAS cell wall, composed of a polyrhamnose backbone with alternating N-acetylglucosamine (GlcNAc) side chains. We have previously shown that the GlcNAc side chain contributes to GAS virulence in animal models.
We validated the contribution of gacH and gacI to sPLA2-IIA resistance by generating specific deletion mutants and complemented strains in different GAS backgrounds. In contrast to gacI, mutation of gacH does not affect GAC GlcNAc expression and likely confers resistance through a different mechanism. We present evidence that GacH is responsible for incorporation of glycerolphosphate in the GAC, a yet unrecognized structural modification of the GAC. Our findings justify a thorough redetermination of Lancefield antigen structures in species expressing a GacH homologue. Furthermore, the presence of glycerolphosphate likely has important biological consequences since it provides a negative charge to the bacterial cell wall. Finally, our structural findings also have consequences for vaccine design.