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bioRxiv · 10.1101/2024.11.04.621835

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

Tamminga, S. M.·Schipper, K.·Murner, N.·Davies, M. A.·Berkhout, P.·Bessen, D. E.·Korotkova, N.·Pannekoek, Y.·van Sorge, N. M.
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Abstract

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 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 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 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.

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Tamminga, S. M., Schipper, K., Murner, N., Davies, M. A., Berkhout, P., Bessen, D. E., Korotkova, N., Pannekoek, Y., van Sorge, N. M.. 2024-11-05. Natural variation of the streptococcal Group A carbohydrate biosynthesis genes impacts host-pathogen interaction. https://doi.org/10.1101/2024.11.04.621835

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