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 analysed the genetic variation of the 12 genes in a collection of 2021 . 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.