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

Streptococcus pyogenes (S. 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 gacI-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. We performed genomic analysis of a single viable historic parent/A-variant strain pair to reveal a premature inactivating stop codon in gacI, explaining the described loss of the GlcNAc side chain. Subsequently, we analyzed the genetic variation of the 12 gacA-L genes in a collection of 2,021 S. pyogenes genome sequences. Although all gac genes (gacA-L) displayed genetic variation, we only identified 25 isolates (1.2%) with a premature stop codon in one of the gac genes. Twelve out of 25 (48%) of these isolates contained a premature stop codon in gacH. 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 through the significant reduction of GroP. Complementary, we showed that strains expressing gacH loss-of-function variants were completely resistant to the human bactericidal enzyme group IIA-secreted phospholipase. Overall, our data provide a comprehensive overview of the genetic variation of the gacA-L gene cluster in a global population of S. pyogenes strains and the functional consequences of gacH variation for immune recognition and clearance.