Group A, B, C, and G Streptococcus Lancefield antigen biosynthesis is initiated by a conserved α-d-GlcNAc-β-1,4-l-rhamnosyltransferase

Abstract

Group A carbohydrate (GAC) is a bacterial peptidoglycan-anchored surface rhamnose polysaccharide (RhaPS) that is essential for growth of Streptococcus pyogenes and contributes to its ability to infect the human host. In this study, using molecular and synthetic biology approaches, biochemistry, radiolabeling techniques, and NMR and MS analyses, we examined the role of GacB, encoded in the S. pyogenes GAC gene cluster, in the GAC biosynthesis pathway. We demonstrate that GacB is the first characterized α-d-GlcNAc-β-1,4-l-rhamnosyltransferase that synthesizes the committed step in the biosynthesis of the GAC virulence determinant. Importantly, the substitution of S. pyogenes gacB with the homologous gene from Streptococcus agalactiae (Group B Streptococcus), Streptococcus equi subsp. zooepidemicus (Group C Streptococcus), Streptococcus dysgalactiae subsp. equisimilis (Group G Streptococcus), or Streptococcus mutans complemented the GAC biosynthesis pathway. These results, combined with those from extensive in silico studies, reveal a common phylogenetic origin of the genes required for this priming step in >40 pathogenic species of the Streptococcus genus, including members from the Lancefield Groups B, C, D, E, G, and H. Importantly, this priming step appears to be unique to streptococcal ABC transporter–dependent RhaPS biosynthesis, whereas the Wzx/Wzy-dependent streptococcal capsular polysaccharide pathways instead require an α-d-Glc-β-1,4-l-rhamnosyltransferase. The insights into the RhaPS priming step obtained here open the door to targeting the early steps of the group carbohydrate biosynthesis pathways in species of the Streptococcus genus of high clinical and veterinary importance.

N-glycan chitobiose core biosynthesis by Agl24 strengthens the hypothesis of an archaeal origin of the eukaryal N-glycosylation

“…The C4 signal of the -GlcNAc residue is shifted to 79.6 ppm, strongly suggesting that the terminal β-GlcNAc was linked at this position. This was further supported by a relative increase in shift of the H4 proton of the -GlcNAc residue compared to the un-modified acceptor (Zorzoli et al, 2019). Relative shifts of other protons reported for the acceptor aligned with our experimental data.…”

Disruption of l -Rhamnose Biosynthesis Results in Severe Growth Defects in Streptococcus mutans

“…The rmlD gene resides in an interesting location in the S. mutans genome as the first gene in the 10-gene rgp operon (27). The location of rmlD is conserved in other streptococcal species as well, including Streptococcus pyogenes and Streptococcus agalactiae (43,44). As the first gene in an operon that is cotranscribed (28), regulation of the rmlD promoter can have significant effects on the expression of downstream genes in the operon.…”

Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin

“…To test this hypothesis, we recombinantly produced the pRha backbone in E. coli cells. We and others have previously reported that the S. mutans sccABCDEFG gene cluster, when transformed into E. coli cells, functionally produces the pRha backbone attached to the lipid A [23, 28]. Additionally, to understand if PlyCB recognizes a specific pRha backbone, we engineered E. coli cells expressing the GAC gene cluster gacABCDEFG required for the GAC pRha production.…”

“…Bacterial strains E. coli CS2775 were transformed with pRGP1 plasmid [28], gacABCDEFG [23] to produce pRha or empty plasmid control (pHD0131). The bacterial cells were grown overnight in LB containing erythromycin (150 µg/ml) at 37°C and used next day for whole cell Western blots and FACS and microscopy analysis.…”