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. 2010 Apr 16;285(16):12140-8.
doi: 10.1074/jbc.M109.066928. Epub 2010 Feb 17.

A novel glucosyltransferase is required for glycosylation of a serine-rich adhesin and biofilm formation by Streptococcus parasanguinis

Meixian Zhou  1 , Fan Zhu, Shengli Dong, David G Pritchard, Hui Wu
Affiliations

A novel glucosyltransferase is required for glycosylation of a serine-rich adhesin and biofilm formation by Streptococcus parasanguinis

Meixian Zhou et al. J Biol Chem. .

Abstract

Fap1-like serine-rich glycoproteins are conserved in streptococci, staphylococci, and lactobacilli, and are required for bacterial biofilm formation and pathogenesis. Glycosylation of Fap1 is mediated by a gene cluster flanking the fap1 locus. The key enzymes responsible for the first step of Fap1 glycosylation are glycosyltransferases Gtf1 and Gtf2. They form a functional enzyme complex that catalyzes the transfer of N-acetylglucosamine (GlcNAc) residues to the Fap1 polypeptide. However, until now nothing was known about the subsequent step in Fap1 glycosylation. Here, we show that the second step in Fap1 glycosylation is catalyzed by nucleotide-sugar synthetase-like (Nss) protein. The nss gene located upstream of fap1 is also highly conserved in streptococci and lactobacilli. Nss-deficient mutants failed to catalyze the second step of Fap1 glycosylation in vivo in Streptococcus parasanguinis and in a recombinant Fap1 glycosylation system. Nss catalyzed the direct transfer of the glucosyl residue to the GlcNAc-modified Fap1 substrate in vitro, demonstrating that Nss is a glucosyltransferase. Thus we renamed Nss as glucosyltransferase 3 (Gtf3). A gtf3 mutant exhibited a biofilm defect. Taken together, we conclude that this new glucosyltransferase mediates the second step of Fap1 glycosylation and is required for biofilm formation.

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Figures

FIGURE 1.
FIGURE 1.
The Nss mutant has a defect in Fap1 glycosylation. Whole cell extracts prepared from the same number of bacterial cells (1 × 108) of wild type (WT), fap1 mutant (fap1−), nss mutant (nss−), nss mutant transformed with pVPT-CHSV (nss−/pVPT), and nss mutant transformed with pVPT-Nss containing the full-length nss gene (nss−/pVPT-nss) were subjected to Western blotting analysis using Fap1-specific mAbs E42, D10, and F51.
FIGURE 2.
FIGURE 2.
Nss-mediated Fap1 glycosylation occurs after Gtf1-Gtf2-catalyzed glycosylation and prior to Gap1-mediated Fap1 biogenesis. Fap1 glycosylation and production profile by the nss mutant and two double mutants, gtf1-nss and gap1-nss. A, Fap1 glycosylation profiled by sWGA pull-down assays. 400 μg of whole cell lysates proteins prepared from the nss mutant (nss−) and a gtf1 mutant, VT508 (gtf1−) were subjected to sWGA pull-down and Western blotting analyses using Fap1 peptide-specific antibody mAb E42. B, Fap1 production profiled by Western blotting analysis of the gtf1 nss double mutant and its parent strains. The whole cell lysates prepared from the same number of bacterial cells (1 × 108) of wild type (WT), fap1 mutant (fap1−), VT508 (gtf1−), nss mutant (nss−), and VT508::Δnss (gtf1−nss−) were probed with Fap1-specific mAbs. C, a schematic representation of the proposed Fap1 processing intermediates. The 360-kDa unglycosylated Fap1 protein and the 400-kDa GlcNAc-modified Fap1 intermediate are recognized by peptide-specific Fap1 antibody mAb E42. The mature 200-kDa Fap1 protein reacts with all peptide- and glycan-specific Fap1 antibodies mAb E42, D10, and F51. D, Fap1 production profile by the gap1 nss double mutant and its parent strains. The whole cell lysates prepared from the same number of bacterial cells (1 × 108) of wild type (WT), fap1 mutant (fap1−), nss mutant (nss−), VT324 (gap1−), and VT324::Δnss (gap1−nss−) were subjected to Western blotting analysis using Fap1-specific mAbs.
FIGURE 3.
FIGURE 3.
Analysis of Fap1ΔRII expression in a recombinant E. coli glycosylation system. A, an E. coli strain carrying pHSG576/fap1ΔRII was transformed with pGEX-6p-1 (lane 1), pGEX-6p-1/gtf1–2 (lane 2), pGEX-6p-1/gtf1–2/pVPT-gly-nss-galT1-galT2 (lane 3), and pGEX-6p-1/gtf1–2/pVPT-gly-galT1-galT2 (lane 4), respectively, and subjected to Western blotting analysis. B, an E. coli strain carrying pHSG576/fap1ΔRII was transformed with pGEX-6p-1 (lane 1), pGEX-6p-1/gtf1–2 (lane 2), pGEX-6p-1/pVPT-nss (lane 3), pGEX-6p-1/gtf1–2/pVPT-gly (lane 4), pGEX-6p-1/gtf1–2/pVPT-nss (lane 5), pGEX-6p-1/gtf1–2/pVPT-galT1 (lane 6), and pGEX-6p-1/gtf1–2/pVPT-galT2 (lane 7), respectively, and subjected to Western blotting analysis.
FIGURE 4.
FIGURE 4.
GC-MS analysis of glycosyl composition of recombinant Fap1 proteins. Sugar standard GlcNAc (A), glucose (B), and recombinant Fap1 glycosylated by Gtf1 and Gtf2 (C) or Gtf1, Gtf2, and Nss (D) were subjected to GC-MS analysis for glycan composition.
FIGURE 5.
FIGURE 5.
Nss enzymatic activity determined by an in vitro glycosylation assay. A, Nss is a glucosyltransferase determined by an in vitro glycosyltransferase assay. B, UDP-glucose effectively inhibited the transfer of 3H-labeled UDP-glucose to the GlcNAc-modified Fap1 polypeptide. C, [3H]UDP-GlcNAc is not an active sugar donor for Nss. HI, heat inactivated.
FIGURE 6.
FIGURE 6.
Biofilm formation of S. parasanguinis derivatives. Biofilm formation of wild type (WT), fap1 mutant (fap1−), gtf3 mutant (gtf3−), gtf3 mutant transformed with pVPT-CHSV (gtf3−/pVPT), and gtf3 mutant transformed with pVPT-Nss harboring the full-length gtf3 gene (gtf3−/pVPT-gtf3) was determined by a microtiter plate method using crystal violet staining (A) and confocal laser scanning fluorescence microscopy analyses (B). Bar, 50 μm.
FIGURE 7.
FIGURE 7.
A model for the Fap1 glycosylation. Gtf1 and Gtf2 form an enzyme complex that catalyzes the transfer of GlcNAc to the unglycosylated Fap1 polypeptide, Gtf3 transfers the Glc residues to the GlcNAc-modified Fap1. Other glycosyltransferases, GalT1, GalT2, and Gly, are responsible for the further glycosylation.
FIGURE 8.
FIGURE 8.
Distribution of Gtf3 in streptococci and lactobacilli that possess Fap1-like serine-rich glycoproteins. Genes encoding glycosyltransferases including Gtf1, -2, and -3 and accessory secretion components of a variety of serine-rich glycoproteins were collected from sequenced bacterial genomes and aligned (A). Collected genes coding for Gtf3 homologues of streptococci and lactobacilli were subjected to phylogenetic analyses using MEGA4 (B).
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