Engineering and Dissecting the Glycosylation Pathway of a Streptococcal Serine-rich Repeat Adhesin

Abstract

Edited by Gerald HartSerine-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.

O-acetylation of the serine-rich repeat glycoprotein GspB is coordinated with accessory Sec transport

“…The GtfAB complex mediates the O-linked transfer of GlcNAc to the SRR regions of GspB [ 7 ] [ 8 ]. Two additional Gtfs (Nss and Gly) sequentially add glucose residues to this glycan moiety ( Fig 1B , S1 Fig and S2 Fig ) (consistent with glycosylation of the SRR adhesin Fap1 [ 14 ]). Based on these findings, we first examined which components of the glycan were affected by an Asp2 S362A mutation.…”

“…Other Gtfs sequentially further modify the SRR domains by adding other glycan moieties to the GlcNAc core. The number and type of these Gtfs varies considerably between species, as do the resulting extent of glycan modification [ 12 ] [ 13 ] [ 14 ].…”

Osteopontin adsorption to Gram-positive cells reduces adhesion forces and attachment to surfaces under flow

“…[17], the streptococcal antigen I/II [18], and serine-rich repeat protein (SRRP) families [19][20][21][22][23]. In SRRP adhesins, which have been identified in both streptococci and lactobacilli [23], glycoconjugates play an important role for adhesion [21,24]. Moreover, glycoconjugates are essential structural elements of lipoteichoic acids [25], which also mediate adhesion to saliva [26,27].…”

Biofilms and their properties

“…Bacterial glucosyltransferases producing extracellular polysaccharides, either soluble or insoluble, were associated with adhesion of S. mutans to tooth surfaces and with cariogenicity Glucosyltransferases have also been identified in other oral bacteria. Moreover, in addition to the production of extracellular polysaccharides, glucosyltransferases have been assumed to glycosylate proteins important for adhesion and biofilm formation .…”