Engineering and Dissecting the Glycosylation Pathway of a Streptococcal Adhesin

Objectives: Serine-rich repeat glycoproteins (SRRPs) are conserved in Gram-positive bacteria. They are crucial for modulating biofilm formation and bacterial-host interactions. Glycosylation of SRRPs plays a pivotal role in the process, thus understanding the glycosyltransferases involved is key to identifying new therapeutic drug targets. The glycosylation of Fap1, an SRRP of oral commensal Streptococcus parasanguinis, is mediated by a gene cluster consisting of six genes, gtf1, gtf2, gly, gtf3, dGT1 and galT2. Mature Fap1 glycan possesses the sequence of Rha1-3Glc1-(Glc1-3GlcNAc1-) 2, 6 Glc1-6GlcNAc. Gtf12, Gtf3 and dGT1 are responsible for the first four steps of the Fap1 glycosylation, catalyzing the transfer of GlcNAc, Glc, Glc and GlcNAc residues to the protein backbone sequentially. The role of GalT2 and Gly in the Fap1 glycosylation is unknown.

Methods: We synthesized the fully modified Fap1 glycan in E. coli by incorporating all six genes from the cluster. To determine how GalT2 and Gly contribute to the Fap1 glycosylation, differentially modified rFap1 were purified and subjected to glycan profiling by MALDI-TOF respectively. In vitro glycosyltransferase assays were also utilized to demonstrate the function of Gly. We also examined the effect of either dGT1 or gly mutation on the biofilm formation using microtiter plate assay and fluorescence microscopy.

Results: We have determined that GalT2 mediates the fifth step of the Fap1 glycosylation by adding a rhamnose residue, and Gly mediates the final glycosylation step by transferring glucosyl residues. Furthermore, inactivation of each glycosyltransferase gene resulted in differentially impaired biofilms of S. parasanguinis, demonstrating the importance of Fap1 glycosylation in the biofilm formation.

Conclusions: This study represents the first reconstitution of an exogenous stepwise O-glycosylation synthetic pathway in E. coli. The Fap1 glycosylation system offers an excellent model to engineer glycans using different permutations of glycosyltransferases in addition to investigating biosynthetic pathways of SRRPs as SRRP genetic loci are highly conserved.