Meixian Zhou

Glycosylation and biogenesis of a family of serine-rich bacterial adhesins.

Glycosylation of bacterial proteins is an important process for bacterial physiology and pathophysiology. Both O- and N-linked glycan moieties have been identified in bacterial glycoproteins. The N-linked glycosylation pathways are well established in Gram-negative bacteria. However, the O-linked glycosylation pathways are not well defined due to the complex nature of known O-linked glycoproteins in bacteria. In this review, we examine a new family of serine-rich O-linked glycoproteins which are represented by fimbriae-associated adhesin Fap1 of Streptococcus parasanguinis and human platelet-binding protein GspB of Streptococcus gordonii. This family of glycoproteins is conserved in streptococcal and staphylococcal species. A gene cluster coding for glycosyltransferases and accessory Sec proteins has been implicated in the protein glycosylation. A two-step glycosylation model is proposed. Two glycosyltransferases interact with each other and catalyse the first step of the protein glycosylation in the cytoplasm; the cross-talk between glycosylation-associated proteins and accessory Sec components mediates the second step of the protein glycosylation, an emerging mechanism for bacterial O-linked protein glycosylation. Dissecting the molecular mechanism of this conserved biosynthetic pathway offers opportunities to develop new therapeutic strategies targeting this previously unrecognized pathway, as serine-rich glycoproteins have been shown to play a role in bacterial pathogenesis.

Interaction between Two Putative Glycosyltransferases Is Required for Glycosylation of a Serine-Rich Streptococcal Adhesin

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.

A Novel Glucosyltransferase Is Required for Glycosylation of a Serine-rich Adhesin and Biofilm Formation by Streptococcus parasanguinis

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.

Role of gap3 in Fap1 glycosylation, stability, in vitro adhesion, and fimbrial and biofilm formation of Streptococcus parasanguinis

BACKGROUND/AIMS Streptococcus parasanguinis is a primary colonizer of the tooth surface. Its adhesion is mediated by the long fimbriae, which are composed of multiple subunits of a serine-rich glycoprotein, Fap1. Previous studies revealed that a chromosomal region located downstream of fap1 is involved in the secretion and glycosylation of Fap1. In this study, we investigated the role of a glycosylation-associated gene, gap3, in Fap1 biogenesis. METHODS A gap3 non-polar mutant was constructed by insertional inactivation. The phenotype of the mutant and the subcellular distribution of its products were investigated. The binding ability of the mutant was tested with saliva-coated hydroxyapatite (SHA). Electron microscopy was used to observe the morphological changes on the mutant cell surface. Confocal microscopy was utilized to determine biofilm formation ability. RESULTS The gap3 mutant produced a partially glycosylated Fap1 precursor, that was less stable than mature Fap1. The Fap1 precursor was distributed in all subcellular fractions including the cell surface and culture medium although in decreased amounts. These data suggest a role for Gap3 in Fap1 glycosylation as well as a link between glycosylation and secretion of Fap1. The gap3 mutant had reduced binding to saliva-coated hydroxyapatite. Electron microscopy revealed that the gap3 mutant had lost its long fimbriae. Biofilm formation was also inhibited by the gap3 mutation. Fewer gap3 mutant cells adhered to the biofilm surface and microcolony formation was decreased. CONCLUSION Gap3 is required for the complete glycosylation and secretion of Fap1, which is important for fimbrial assembly, bacterial adhesion, and in vitro biofilm formation.

The highly conserved domain of unknown function 1792 has a distinct glycosyltransferase fold

More than 33,000 glycosyltransferases have been identified. Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B. Here we report a 1.34 Å resolution X-ray crystallographic structure of a previously uncharacterized “domain of unknown function” 1792 (DUF1792) and show that the domain adopts a new fold and is required for glycosylation of a family of serine-rich repeat streptococcal adhesins. Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyzes the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis. DUF1792 homologs from both Gram-positive and Gram-negative bacteria also exhibit the activity. Thus DUF1792 represents a new family of glycosyltransferases, so we designate it as a GT-D glycosyltransferase fold. As the domain is highly conserved in bacteria and not found in eukaryotes, it can be explored as a new antibacterial target.

Structural and Functional Analysis of a New Subfamily of Glycosyltransferases Required for Glycosylation of Serine-rich Streptococcal Adhesins.

Serine-rich repeat glycoproteins (SRRPs) are a growing family of bacterial adhesins found in many streptococci and staphylococci; they play important roles in bacterial biofilm formation and pathogenesis. Glycosylation of this family of adhesins is essential for their biogenesis. A glucosyltransferase (Gtf3) catalyzes the second step of glycosylation of a SRRP (Fap1) from an oral streptococcus, Streptococcus parasanguinis. Although Gtf3 homologs are highly conserved in SRRP-containing streptococci, they share minimal homology with functionally known glycosyltransferases. We report here the 2.3 Å crystal structure of Gtf3. The structural analysis indicates that Gtf3 forms a tetramer and shares significant structural homology with glycosyltransferases from GT4, GT5, and GT20 subfamilies. Combining crystal structural analysis with site-directed mutagenesis and in vitro glycosyltransferase assays, we identified residues that are required for UDP- or UDP-glucose binding and for oligomerization of Gtf3 and determined their contribution to the enzymatic activity of Gtf3. Further in vivo studies revealed that the critical amino acid residues identified by the structural analysis are crucial for Fap1 glycosylation in S. parasanguinis in vivo. Moreover, Gtf3 homologs from other streptococci were able to rescue the gtf3 knock-out mutant of S. parasanguinis in vivo and catalyze the sugar transfer to the modified SRRP substrate in vitro, demonstrating the importance and conservation of the Gtf3 homologs in glycosylation of SRRPs. As the Gtf3 homologs only exist in SRRP-containing streptococci, we conclude that the Gtf3 homologs represent a unique subfamily of glycosyltransferases.

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

Fap1‐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.

A Conserved C-Terminal 13-Amino-Acid Motif of Gap1 Is Required for Gap1 Function and Necessary for the Biogenesis of a Serine-Rich Glycoprotein of Streptococcus parasanguinis

ABSTRACT Adhesion of Streptococcus parasanguinis to saliva-coated hydroxyapatite (SHA), an in vitro tooth model, is mediated by long peritrichous fimbriae. Fap1, a fimbria-associated serine-rich glycoprotein, is required for fimbrial assembly. Biogenesis of Fap1 is controlled by an 11-gene cluster that contains gly, nss, galT1 and -2, secY2, gap1 to -3, secA2, and gtf1 and -2. We had previously isolated a collection of nine nonadherent mutants using random chemical mutagenesis approaches. These mutants fail to adhere to the in vitro tooth model and to form fimbriae. In this report, we further characterized these randomly selected nonadherent mutants and classified them into three distinct groups. Two groups of genes were previously implicated in Fap1 biogenesis. One group has a mutation in a glycosyltransferase gene, gtf1, that is essential for the first step of Fap1 glycosylation, whereas the other group has defects in the fap1 structural gene. The third group mutant produces an incompletely glycosylated Fap1 and exhibits a mutant phenotype similar to that of a glycosylation-associated protein 1 (Gap1) mutant. Analysis of this new mutant revealed that a conserved C-terminal 13-amino-acid motif was missing in Gap1. Site-directed mutagenesis of a highly conserved amino acid tryptophan within this motif recapitulated the deletion phenotype, demonstrating the importance of the Gap1 C-terminal motif for Fap1 biogenesis. Furthermore, the C-terminal mutation does not affect Gap1-Gap3 protein-protein interaction, which has been shown to mediate Fap1 glycosylation, suggesting the C-terminal motif has a distinct function related to Fap1 biogenesis.

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

Fap1, a serine-rich repeat glycoprotein (SRRP), is required for bacterial biofilm formation of Streptococcus parasanguinis. Fap1-like SRRPs are found in many gram-positive bacteria and have been implicated in bacterial fitness and virulence. A conserved five-gene cluster, secY2-gap1-gap2-gap3-secA2, located immediately downstream of fap1, is required for Fap1 biogenesis. secA2, gap1, and gap3 encode three putative accessory Sec proteins. SecA2 mediates export of mature Fap1, and Gap1 and Gap3 are required for Fap1 biogenesis. Interestingly, gap1 and gap3 mutants exhibited the same phenotype as a secA2 mutant, implying that Gap1 and Gap3 may interact with SecA2 to mediate Fap1 biogenesis. Glutathione S-transferase pulldown experiments revealed a direct interaction between SecA2, Gap1, and Gap3 in vitro. Coimmunoprecipitation analysis demonstrated the formation of a SecA2-Gap1-Gap3 complex. Homologues of SecA2, Gap1, and Gap3 are conserved in many streptococci and staphylococci. The corresponding homologues from Streptococcus agalactiae also interacted with each other and formed a protein complex. Furthermore, the Gap1 homologues from S. agalactiae and Streptococcus sanguinis rescued the Fap1 defect in the Gap1 mutant, indicating the functional conservation of the accessory Sec complex. Importantly, canonical SecA interacted with the accessory Sec protein complex, suggesting that the biogenesis of SRRPs mediated by the accessory Sec system is linked to the canonical Sec system.

Gap1 functions as a molecular chaperone to stabilize its interactive partner Gap3 during biogenesis of serine‐rich repeat bacterial adhesin

Serine‐rich repeat glycoproteins (SRRPs) are important bacterial adhesins that are conserved in streptococci and staphylococci. Fimbriae‐associated protein (Fap1) from Streptococcus parasanguinis, was the first SRRP identified; it plays an important role in bacterial biofilm formation. A gene cluster encoding glycosyltransferases and accessory secretion components is required for Fap1 biogenesis. Two glycosylation‐associated proteins, Gap1 and Gap3 within the cluster, interact with each other and function in concert in Fap1 biogenesis. Here we report the new molecular events underlying contribution of the interaction to Fap1 biogenesis. The Gap1‐deficient mutant rendered Gap3 unstable and degraded in vitro and in vivo. Inactivation of a gene encoding protease ClpP reversed the phenotype of the gap1 mutant, suggesting that ClpP is responsible for degradation of Gap3. Molecular chaperone GroEL was co‐purified with Gap3 only when Gap1 was absent and also reacted with Gap1 monoclonal antibody, suggesting that Gap1 functions as a specific chaperone for Gap3. The N‐terminal interacting domains of Gap1 mediated the Gap3 stability and Fap1 biogenesis. Gap1 homologues from Streptococcus agalactiae and Staphylococcus aureus also interacted with and stabilized corresponding Gap3 homologues, suggesting that the chaperone activity of the Gap1 homologues is common in biogenesis of SRRPs.