Natural variation of the streptococcal Group A carbohydrate biosynthesis genes impacts host-pathogen interaction.

() is a leading cause of infection-related mortality in humans globally. The characteristic cell wall-anchored Group A Carbohydrate (GAC) is expressed by all strains and consists of a polyrhamnose backbone with alternating -acetylglucosamine (GlcNAc) side chains, of which 25% are decorated with glycerol phosphate (GroP). The genes in the cluster are critical for GAC biosynthesis with being responsible for the characteristic GlcNAc-GroP decoration, which confers the agglutination in rapid test diagnostic assays and contributes to pathogenicity. Historical research papers described isolates, so-called A-variant strains, that lost the characteristic GlcNAc side chain following serial animal passage. Genomic analysis of a single viable historic parent/A-variant strain pair revealed a premature inactivating stop codon in , explaining the described loss of the GlcNAc side chain. Subsequently, we analyzed the genetic variation of the 12 genes in a collection of 2,021 genome sequences. Although all genes () displayed genetic variation, we only identified 26 isolates (1.3%) with a premature stop codon in one of the genes. Twelve out of 26 (46%) isolates contained a premature stop codon in , which encodes the enzyme responsible for the GroP modification. To study the functional consequences of the different premature stop codons for GacH function, we plasmid-expressed three variants in a -deficient strain. Cell wall analysis confirmed GacH loss-of-function for the studied variants through the significant reduction of GAC GroP, complete resistance to killing by the human bactericidal enzyme group IIA-secreted phospholipase, and susceptibility to zinc toxicity. Overall, our data provide a comprehensive overview of the genetic variation of the cluster in a global population of strains and the functional consequences of rare inactivating mutations in for host interaction.