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

Streptococcus pyogenes is a leading cause of infection-related mortality in humans globally. The characteristic cell wall-anchored group A carbohydrate (GAC) is expressed by all S. pyogenes strains and consists of a polyrhamnose backbone with alternating N-acetylglucosamine (GlcNAc) side chains, of which 25% are decorated with glycerol phosphate (GroP). The genes in the gacA-L cluster are critical for GAC biosynthesis, with gacH-L being responsible for the characteristic GlcNAc-GroP decoration, which confers the agglutination in rapid test diagnostic assays and contributes to S. pyogenes pathogenicity. Historical research papers described S. pyogenes 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 gacI, explaining the described loss of the GlcNAc side chain. Subsequently, we analysed the genetic variation of the 12 gacA-L genes in a collection of 2021 S. pyogenes genome sequences. Although all gac genes (gacA-L) displayed genetic variation, we only identified 26 isolates (1.3%) with a premature stop codon in one of the gac genes. Twelve out of 26 (46%) isolates contained a premature stop codon in gacH, 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 gacH variants in a S. pyogenes gacH-deficient strain. Cell wall analysis confirmed GacH loss of function for the studied gacH 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 gacA-L cluster in a global population of S. pyogenes strains and the functional consequences of rare inactivating mutations in gacH for host interaction.