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.

FIG. 1.

Characterization of nonadherent gtf1 mutants. (A) Fap1 expression profile of the first group of mutants. The Fap1 expression profiles of cell lysates of the wild type (WT) (lane 1), fap1 mutant (lane 2), VT343 (lane 3), VT508 (lane 4), gtf1 mutant (lane 5), and gtf2 mutant (lane 6) were determined by Western blotting analysis with three Fap1-specific antibodies, mAbE42 (top panel), mAbD10 (middle panel), and mAbF51 (bottom panel). (B) Genetic complementation of VT343 and VT508 with genes coding for glycosyltransferases Gtf1 and Gtf2 and analyzed by BactELISA using mAbF51. (C) Western blotting analyses of the wild type (lane 1), fap1 mutant (lane 2), VT343 (lane 3), and VT508 (lane 5) and their complemented strains (lanes 4 and 6) using Fap1-specific antibodies. (D) Diagrammatic depiction of gtf1 alleles from VT343 and VT508. (E) Adhesion of S. parasanguinis FW213 and its derivatives. Wild type FW213, the fap1 mutant, VT343, and VT508 and their complemented strains were assayed for their ability to bind to SHA.

FIG. 2.

Characterization of nonadherent fap1 mutants. (A and B) Fap1 expression profiles of culture supernatants (A) and cell lysates (B) of the wild type (WT) (lane 1), fap1 mutant (lane 2), VT321 (lane 3), VT325 (lane 4), VT361 (lane 5), VT377 (lane6), VT379 (lane 7), and VT380 (lane 8), as determined by Western blotting analysis with three Fap1-specific antibodies. Arrows point to the positions corresponding to the Fap1 protein. (C and D) Western blotting analyses of culture media (C) and cell lysates (D) of the wild type (lane 1), fap1 mutant (lane 2), and fap1 chemically mutagenized strains and their complemented counterparts VT321 (lane 3), VT321/fap1⁺ (lane 4), VT325 (lane 5), VT325/fap1⁺ (lane 6), VT361 (lane 7), VT361/fap1⁺ (lane 8), VT377 (lane 9), VT377/fap1⁺ (lane 10), VT379 (lane 11), VT379/fap1⁺ (lane 12), VT380 (lane 13), and VT380/fap1⁺ (lane 14) with Fap1-specific peptide antibodies. (E) Diagrammatic depiction of fap1 alleles from VT321, VT325, and FW213. (F) Adhesion of S. parasanguinis FW213 and its fap1 mutant derivatives. Wild-type strain FW213, the fap1 mutant, and VT321, VT325, VT361, VT377, VT379, and VT380 and their complemented strains were evaluated for their ability to bind to SHA.

FIG. 3.

Characterization of a nonadherent gap1 mutant. (A) The Fap1 expression profiles of cell lysates of the wild type (lane 1), fap1 mutant (lane 2), VT324 (lane 3), secY2 mutant (lane 4), gap1 mutant (lane 5), gap3 mutant (lane 6), and secA2 mutant (lane 7) were determined by Western blotting analysis with three Fap1-specific antibodies. (B) Western blotting analyses of the wild type (lane 1), fap1 mutant (lane 2), VT324 (lane 3), and VT324 complemented with the full-length gap1 (lane 4), secY2 (lane 5), gap3 (lane 6), and secA2 (lane 7) genes. (C) Adhesion of S. parasanguinis FW213 and gap1 mutant derivatives to SHA.

FIG. 4.

The Gap1 C-terminal motif is important for Gap1 function and Fap1 biogenesis. (A) Diagrammatic depiction of a gap1 allele from VT324. (B) Homology comparison of the C-terminal domain of Gap1 of S. parasanguinis and its homologs from S. agalactiae (AAZ95528), S. gordonii (AAK16998), S. sanguinis (ABN44261), S. pneumoniae (AAK75837), Staphylococcus haemolyticus (BAE03637), and Lactobacillus johnsonii (AAS08375). The conserved amino acid residues are highlighted. (C) A key amino acid residue in the C-terminal motif is important for Gap1 function. Cell lysates prepared from the wild type (lane 1), fap1 mutant (lane 2), and VT324 (lane 3) as well as Gap1 site-directed mutants W518A (lane 4) and W518Y (lane 5) were analyzed by Western blotting with three Fap1-specific antibodies, mAbE42 (I), mAbD10 (II), and mAbF51 (III), or anti-FimA antibody (IV). Arrows point to the positions corresponding to Fap1 or FimA.

FIG. 5.

The C-terminal deletion does not affect interaction between Gap1 and Gap3. Gap1(Δ513-525) and Gap1 interactions with Gap3 were determined by in vitro GST pull-down assays. (A) SDS-polyacrylamide gel electrophoresis analyses of GST (lane 1), GST-Gap1 (lane 2), and GST-Gap1(Δ513-525) (lane 3) fusion proteins purified using glutathione Sepharose beads from respective bacterial strains. The gel was stained with Coomassie blue. M, protein marker. (B) In vitro GST pull-down assays. The purified GST (lane 1), GST-Gap1 (lane 2), or Gap1(Δ513-525) (lane 3) glutathione Sepharose beads were incubated with in vitro-translated c-Myc-Gap3. The captured protein complexes were subjected to Western blot analyses with c-Myc antibody. “Input” represents in vitro-translated c-Myc-Gap3.