A conserved domain of previously unknown function in Gap1 mediates protein–protein interaction and is required for biogenesis of a serine‐rich streptococcal adhesin

Abstract

SummaryFap1-like serine-rich proteins are a new family of bacterial adhesins found in a variety of streptococci and staphylococci that have been implicated in bacterial pathogenesis. A gene cluster encoding glycosyltransferases and accessory Sec components is required for Fap1 glycosylation and biogenesis in Streptococcus parasanguinis. Here we report that the glycosylation-associated protein, Gap1, contributes to glycosylation and biogenesis of Fap1 by interacting with another glycosylation-associated protein, Gap3. Gap1 shares structural homology with glycosyltransferases. The gap1 mutant, like the gap3 mutant, produced an aberrantly glycosylated Fap1 precursor and failed to produce mature Fap1, suggesting that Gap1 and Gap3 might function in concert in the Fap1 glycosylation and biogenesis. Indeed, Gap1 interacted with Gap3 in vitro and in vivo. A Gap1 N-terminal motif, within a highly conserved domain of unknown function (DUF1975) identified in many bacterial glycosyltransferases, was required for the Gap1-Gap3 interaction. Deletion of one, four and nine amino acids within the conserved motif gradually inhibited the Gap1-Gap3 interaction and diminished production of mature Fap1 and concurrently increased production of the Fap1 precursor. Consequently, bacterial adhesion to an in vitro tooth model was also reduced. These data demonstrate that the Gap1-Gap3 interaction is required for Fap1 biogenesis and Fap1-dependent bacterial adhesion.

Canonical SecA Associates with an Accessory Secretory Protein Complex Involved in Biogenesis of a Streptococcal Serine-Rich Repeat Glycoprotein

“…mAbD10 is a glycan-specific antibody, and mAbF51 reacts with mature Fap1. Previous studies had revealed that the gap1 and gap3 mutants exhibited similar phenotypes in Fap1 biogenesis; both produce a 470-kDa high-molecular-weight (HMW) Fap1 precursor (18). A similar HMW Fap1 protein was also detected in the secA2 mutant by Western blot analyses using mAbE42 (Fig.…”

“…A number of accessory Sec components have been identified and implicated in secretion of SRRPs (1,7,18,19,22,24,26,30,35,36); however, the precise roles of each component have yet to be defined.…”

“…All strains were grown to exponential growth phase (A 470 Ϸ 0.6). Cell lysates were prepared as described previously (18). Prepared cell lysates (400 g) were incubated with anti-GFP monoclonal antibody or anti-SecA2 polyclonal antibody for 4 h at 4°C on a rotator prior to incubation with 50 l of a 50% slurry of immobilized protein A/G agarose (Invitrogen) overnight at 4°C.…”

“…Mature Fap1 is not detected in the membrane and cytoplasm fractions of a secA2 mutant (7). Gap1 and Gap3 interact with each other, and mutagenesis of either one blocks Fap1 maturation (18,20). Study of Gap1 and Gap3 homologues in Streptococcus gordonii has also indicated that Asp2 and Asp3 interact with SecA2 to modulate export of GspB (22).…”

Structure of a Novel O-Linked N-Acetyl-d-glucosamine (O-GlcNAc) Transferase, GtfA, Reveals Insights into the Glycosylation of Pneumococcal Serine-rich Repeat Adhesins

“…Previous studies have proven that TPR, a scaffold for the recognition of partner proteins or acceptor peptides, is crucial for the activity of OGTs (9,60). Similar to TPR, the DUF1975 domain of GtfA has also been implicated in mediating protein-protein interactions and is essential for the glycosylation of SRRPs (25,61). In our docking model, we found that the two most N-terminal residues of SRR1, Asn-1Ј and Ser-2Ј, form sidechain hydrogen bonds with residues Asn-98, Arg-103, and Tyr-116 of DUF1975 (Fig.…”