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

Abstract

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.

Fig. 1. Production of Gap3 was inhibited in the gap1 mutant

(A) Cell lysates of wild type (WT), gap3 mutant (gap3⁻), gap1 mutant (gap1⁻) and the gap1 mutant transformed with pVT1666 (gap1⁻/pVT1666) or with pVPT-Gap1-GFP (gap1⁻/pVPT-Gap1-GFP) were subjected to Western blot analysis using Gap3 (upper panel) and FimA (lower panel) antibodies, respectively. (B) Cell lysates of wild type (WT), gap1 mutant (gap1⁻) and gap3 mutant (gap3⁻) of S. parasanguinis were subjected to Western blot analysis using anti-Gap1 (upper panel) and anti-FimA (lower panel) antibodies, respectively. (C) The Gap1 deficiency did not affect expression of gap3 by RT-PCR. Total RNA prepared from wild type and the gap1 mutant was reverse-transcribed with M-MLV reverse transcriptase, and used for PCR with gap3-specific primers for gap3 (550bp) and fimA-specific primers for fimA (930bp). Genomic DNA samples were used as PCR controls.

Fig. 2. Gap1 stabilized Gap3

Top10 E. coli cells were transformed with pVPT-Gap3-CHSV (A), or pVPT-Gap1-3-CHSV (B). The gap1 mutant cells were transformed with pVPT-Gap3-CHSV (C) or with pVPT-Gap1-3-CHSV (D). The recombinant bacteria were grown to exponential phase (OD₆₀₀ = 0.6) and treated with chloramphenicol at 200 µg/ml for 0, 10, 20, 40 and 60 min. The cell lysates were prepared and then subjected to Western blot analysis using anti-Gap3, and anti-HSV antibody, respectively.

Fig. 3. Gap3 alone was more susceptible to proteolytic digestion in vitro

Proteolytic assays for degradation of Gap1, Gap3 and Gap1/3 complex. Purified Gap1, Gap3 and Gap1/3 complex were treated with trypsin (A) and (B) endoproteinase Glu-C, and subjected to SDS-PAGE analysis and stained with Coomassie brilliant blue.

Fig. 4. The Clp deficiency restored production of Gap3 in the gap1 mutant

Cell lysates prepared from wild type (WT), clpP mutant (clpP⁻), VT324 (gap1Δ513–525), double mutant (VT324/clpP⁻), gap1 mutant (gap1⁻) and gap3 mutant (gap3⁻) were subjected to Western blot analysis using Gap3 (upper panel) and FimA (bottom panel) antibody, respectively.

Fig. 5. The N-terminus of Gap1 was required for the Gap3 stability, for the interaction with Gap3 and for Fap1 maturation

(A) Interaction between Gap1 mutant variants and Gap3. Equal amounts of purified GST, GST-Gap1 and GST-tagged Gap1 mutant variants (Gap1Δ1–10, 11–20, 21–28 and 29–45) bound to glutathione Sepharose 4B beads were incubated with in vitro translated c-Myc-Gap3. The pull-down protein complexes were analyzed by Western blotting using c-Myc monoclonal antibody. Input represents the in vitro translated protein product. (B) Effect of the Gap1 deletions on production of Gap3. Cell lysates of gap1 mutant transformed with the full-length Gap1 and with various Gap1 deletion constructs (Δ1–10, Δ11–20, Δ21–28, Δ29–45) were subjected to Western blot analysis using anti-Gap3 (upper panel), anti-Gap1 (middle panel) and anti-FimA (bottom panel) antibody respectively. (C) Effect of the Gap1 deletions on production of Fap1. Complementation of the gap1 mutant was carried out using the gap1 deletion mutants. Cell lysates of the gap1 mutant transformed with the full-length Gap1 and with various Gap1 deletion mutants were subjected to Western blot analysis using mature Fap1-specific antibody F51.

Fig. 6. Gap1 monoclonal antibody recognized GroEL co-purified with Gap3

His-SUMO-tagged Gap3 (Lane 1), Gap1Δ21–28 (Lane 2) and Gap1/Gap3 (Lane 3) proteins were purified with Ni-NTA resin, eluted by cleavage of the SUMO tag and subjected to SDS-PAGE (A) and Western blot analysis using anti-Gap1 (B). A protein band co-purified with Gap3 (Lane 1, top band) was excised, digested with trypsin and subjected to mass spectrometry (LC/MS/MS) to determine the protein identity.

Fig. 7. Asp1 interacted with Asp3 from S. agalactiae J48 and S. aureus COL

(A) GST-Asp3 interacted with c-Myc-Asp1. Equal amounts of purified GST and GST-Asp3 (J48) bound to glutathione Sepharose 4B beads were each incubated with in vitro translated c-Myc-Asp1 (J48). (B) GST-Asp1 interacted with c-Myc-Asp3. Equal amounts of purified GST and GST-Asp1 (J48) bound to glutathione Sepharose 4B beads were each incubated with in vitro translated c-Myc-Asp3 (J48). The pull-down protein complexes were analyzed by Western blotting using c-Myc monoclonal antibody. Inputs represent the in vitro translated protein products. (C) Asp1 of S. aureus interacted with Asp3. pGEX-Asp1 (COL) and pVPT-Asp3-CHSV (COL) were co-transformed into E. coli Top10, and recombinant GST-Asp1 was purified using glutathione Sepharose 4B beads and subjected to Western blot analysis with anti-HSV monoclonal antibody. The recombinant E. coli strain harboring pGEX-5X-1 (GST) and pVPT-Asp3-CHSV (COL) was used as a control.

Fig. 8. Gap1 homologues stabilized Gap3 homologues

Top10 E. coli cells transformed with pVPT-Asp3-CHSV (J48) (A), pVPT-Asp3-CHSV/pGEX-Asp1 (J48) (B), pVPT-Asp3-CHSV(COL) (C) or pVPT-Asp3-CHSV/pGEX-Asp1 (COL) (D) respectively were grown to exponential phase (OD₆₀₀ = 0.6), treated with 200 µg/ml of chloramphenicol for 0, 10, 20, 40, and 60 min, harvested and subjected to Western blot analysis using anti-HSV antibody.