Abstract
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 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. In the present study, we synthesized the fully modified Fap1 glycan in Escherichia coli by incorporating all six genes from the cluster. This study represents the first reconstitution of an exogenous stepwise O-glycosylation synthetic pathway in E. coli. In addition, 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. The Fap1 glycosylation system offers an excellent model to engineer glycans using different permutations of glycosyltransferases and to investigate biosynthetic pathways of SRRPs because SRRP genetic loci are highly conserved.
Highlights
Protein glycosylation is an important post-translational modification that mediates a variety of biological processes
Serine-rich repeat glycoproteins (SRRPs)3 are a family of glycosylated adhesins highly conserved in Gram-positive bacteria that mediate bacterial-host interactions [3]
GalT2 and Gly Catalyze the Sequential Glycosylation of Fap1 Following dGT1—To determine whether GalT2 and Gly can further modify Fap1, we engineered an in vivo glycosylation system in E. coli by co-expressing a recombinant Fap1 with a series of glycosyltransferases and examined the modification of rFap1 variants by monitoring their migration using SDS-PAGE analysis
Summary
Serine-rich repeat glycoproteins (SRRPs) are conserved in Gram-positive bacteria They are crucial for modulating biofilm formation and bacterial-host interactions. Serine-rich repeat glycoproteins (SRRPs) are a family of glycosylated adhesins highly conserved in Gram-positive bacteria that mediate bacterial-host interactions [3]. They have been implicated in the bacterial pathogenesis in a variety of infectious diseases such as pneumonia, infective endocarditis, meningitis, and oral infectious diseases [3]. Two New Glycosyltransferases Involved in Fap Glycosylation dGT1, which is referred as DUF1792 (domain of unknown function 1792), mediates the third step of Fap glycosylation by adding an additional Glc to Glc-GlcNAc modified Fap1 [15, 23]. Inactivation of each glycosyltransferase gene led to differentially impaired biofilms of S. parasanguinis, indicating the importance of the Fap glycosylation in the biofilm formation
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