Interaction between two putative glycosyltransferases is required for glycosylation of a serine-rich streptococcal adhesin

Abstract

Fap1, a serine-rich glycoprotein, is essential for fimbrial biogenesis and biofilm formation of Streptococcus parasanguinis (formerly S. parasanguis). Fap1-like proteins are conserved in many streptococci and staphylococci and have been implicated in bacterial virulence. Fap1 contains two serine-rich repeat regions that are modified by O-linked glycosylation. A seven-gene cluster has been identified, and this cluster is implicated in Fap1 biogenesis. In this study, we investigated the initial step of Fap1 glycosylation by using a recombinant Fap1 as a model. This recombinant molecule has the same monosaccharide composition profile as the native Fap1 protein. Glycosyl linkage analyses indicated that N-acetylglucosamine (GlcNAc) is among the first group of sugar residues transferred to the Fap1 peptide. Two putative glycosyltransferases, Gtf1 and Gtf2, were essential for the glycosylation of Fap1 with GlcNAc-containing oligosaccharide(s) in both S. parasanguinis as well as in the Fap1 glycosylation system in Escherichia coli. Yeast two-hybrid analysis as well as in vitro and in vivo glutathione S-transferase pull-down assays demonstrated the two putative glycosyltransferases interacted with each other. The interaction domain was mapped to an N-terminal region of Gtf1 that was required for the Fap1 glycosylation. The data in this study suggested that the formation of the Gtf1 and Gtf2 complex was required for the initiation of the Fap1 glycosylation and that the N-terminal region of Gtf1 was necessary.

FIG. 1.

Western blot analyses of Fap1 expression by gtf1, gtf2, and gtf1 gtf2 mutants and their derivatives. Culture supernatants were prepared from wild-type FW213 (lane 1), VT1393 (fap1 mutant; lane 2), VT1583 (gtf1 mutant; lane 3), AL51 (gtf1 mutant, pVPT; lane 4), AL52 (gtf1 mutant, pVPT-gtf2; lane 5), AL50 (gtf1 mutant, pVPT-gtf1; lane 6), AL63 (gtf2 mutant; lane 7), AL64 (gtf2 mutant, pVPT; lane 8), AL65 (gtf2 mutant, pVPT-gtf1; lane 9), AL66 (gtf2 mutant, pVPT-gtf2; lane 10), AL74 (gtf1 gtf2 mutant; lane 11), AL75 (gtf1 gtf2 mutant, pVPT-gtf1; lane 12), AL76 (gtf1 gtf2 mutant, pVPT-gtf2; lane 13), and AL77 (gtf1 gtf2 mutant, pVPT-gtf1 and -2; lane 14); separated by electrophoresis through 4 to 12% polyacrylamide gradient gels; and subjected to Western blot analyses with peptide-specific MAb E42 (A) and glycan-specific MAb F51 (B). The culture supernatants prepared from VT1393 (fap1 mutant; lane 1), wild-type FW213 (lane 2), VT1583 (gtf1 mutant; lane 3), and AL63 (gtf2 mutant; lane 4) were probed with lectin sWGA (C), carbohydrate staining (D), and MAb E42 (D), respectively. MW, molecular mass.

FIG. 2.

Characterization of the recombinant Fap1 (Fap1ΔRII) that contains only repeat region I. (A) Diagram of Fap1 and Fap1ΔRII constructs. The fap1ΔRII construct was generated by deletion of the entire RII region from the intact fap1. (B) Fap1ΔRII is glycosylated by GlcNAc-containing sugar residues. The fap1ΔRII plasmid pAL81 was transformed into the fap1-null mutant VT1393 to generate a recombinant strain, AL81. AL81 (lane 1) and VT1393 (lane 2) were analyzed by Western blotting with MAb E42 and lectin sWGA (B). (C) Purification of Fap1ΔRII. Fap1ΔRII was purified by MAb E42 affinity chromatography from culture supernatants of AL81 and analyzed by Coomassie blue staining. (D) Glycosyl linkage analysis of Fap1ΔRII. The purified Fap1ΔRII protein was subjected to β-elimination, and the released oligosaccharides were analyzed by MALDI-TOF MS.

FIG. 3.

Analyses of Fap1ΔRII expression in an E. coli glycosylation system. E. coli strains carrying pAL81 were cotransformed with plasmids pGEX6p1 (lane 1), pAL201 (lane 2), pAL202 (lane 3), and pAL200 (lane 4) and subjected to immunoblot analysis with MAb E42 (A) and GlcNAc-reactive lectin sWGA (B). MW, molecular mass.

FIG. 4.

Interaction between Gtf1 and Gtf2 determined by yeast two-hybrid and in vitro GST pull-down assays. (A) Yeast two-hybrid analysis of the interaction between Gtf1 and Gtf2. The full-length gtf1 and gtf2 genes were cloned into yeast two-hybrid reporter vectors pBD and pAD to construct pAD-gtf1, pBD-gtf1, pAD-gtf2, and pBD-gtf2, respectively. AH109 yeast was transformed with different combinations of bait and prey plasmids. The resulting growth of cotransformants was used to score protein-protein interactions. (B) In vitro GST pull-down assays for interactions between purified GST fusion proteins and in vitro-translated Gtf1 or Gtf2 products. The purified GST, GST-Gtf2, or GST-Gtf1 glutathione Sepharose beads were incubated with in vitro-translated c-Myc-Gtf1 (A) and c-Myc-Gtf2 (B) to pull down the interacting partners, and the captured protein complexes were subjected to Western blot analyses with c-Myc antibody. Inputs represented in vitro-translated c-Myc-Gtf1 (A) and c-Myc-Gtf2 (B) and serve as positive controls.

FIG. 5.

Gtf1 and Gtf2 interact with each other in the E. coli glycosylation system. (A) In vivo GST pull down. The recombinant Fap1 substrate plasmid pAL81 was cotransformed with various pGEX6p1 derivatives into E. coli to generate a series of recombinant strains, AL400/pGEX6p1 (lane 1), AL402/pGEX6p1-gtf1(Δ21-485)-2 (lane 2), AL403/pGEX6p1-gtf1-2(Δ15-417) (lane 3), and AL401/pGEX6p1-gtf1-2 (lane 4). GST fusion proteins were purified from these recombinant strains and stained with Coomassie blue. MW, molecular mass. (B) MS analysis of Gtf2. A 50-kDa protein was copurified with GST-Gtf1, excised, and subjected to LC/MS/MS (B). Six tryptic peptides identified by MS matched with predicted peptides from Gtf2. Numbers indicate the amino acid positions in Gtf2.

FIG. 6.

The Gtf1 N-terminal region is critical for Gtf1 and Gtf2 interaction and Fap1 glycosylation. A variety of plasmids generated by using the EZ-Tn5 transposon system (Table 4) were cotransformed with pAL81 into E. coli to construct a positive control strain, AL406/pGEX6p1-gtf1-2T7 (lane 1); a negative control, AL407/pGEX6p1-gtf2T7 (lane 2); and several in-frame insertional mutants, AL408 gtf1::Tn31-gtf2 (lane 3), AL409 gtf1::Tn65-gtf2 (lane 4), AL410 gtf1::Tn159-gtf2 (lane 5), and AL411 gtf1::Tn416-gtf2 (lane 6). Cotransformants were subjected to immune blotting with lectin sWGA (A) and MAb E42 (B). GST fusion proteins were purified from those cotransformants and analyzed by Western blotting assays with anti-T7 antibody (C). MW, molecular mass.