Sortase A utilizes an ancillary protein anchor for efficient cell wall anchoring of pili in Streptococcus agalactiae

Abstract

Pili are putative virulence factors and promising vaccine candidates in Streptococcus agalactiae (group B Streptococcus [GBS]) infection, a leading cause of neonatal sepsis and meningitis. The genes necessary for pilus synthesis and assembly are clustered in pilus islands (PI). Each gene encodes three structural subunits (a backbone and two ancillary proteins) bearing a C-terminal LPXTG motif and two subfamily C sortases (SrtC) involved in covalent polymerization of the subunits. GBS strains also possess the conserved "housekeeping" sortase A (SrtA), but its role in pilus assembly is unclear. To address this issue, pilus expression and cell wall anchoring were analyzed in srtA deletion mutants. Loss of SrtA did not affect pilus polymerization. However, pilus expression on the cell surface was reduced, and pili accumulated in the culture supernatant. Furthermore, cell-associated pili could be readily released by detergent treatment, indicating that SrtA is involved in covalent anchoring of pili to the cell wall. When each of the genes comprising PI-2a was systematically deleted, only the absence of ancillary subunit GBS150 or the SrtC required for incorporation of GBS150 into pili mimicked the srtA mutant phenotype. Thus, from these data a model for GBS pilus assembly can be proposed in which PI sortases are responsible for polymerization of the pilus structure, while SrtA is required to covalently attach it to the cell wall, utilizing ancillary pilus subunit GBS150 as the anchor protein.

FIG. 1.

Schematic representation of GBS PIs. Genes encoding the three LPXTG proteins that comprise the pilus structure are represented by black (backbone subunit) and white (ancillary subunits) arrows. Subfamily SrtC transpeptidases that polymerize the protein subunits are shown in gray. Gene designations correspond to GBS strain 2603V/R, accession number AE009948 (PI-1 and PI-2a) or strain COH1, accession number AAJR00000000 (PI-2b).

FIG. 2.

Confirmation of S. agalactiae srtA mutant generation. (A) Transcription of srtA and flanking genes (gyrA, SAG0962, and SAG0963) in wild-type and srtA mutant strains. RNA was extracted from wild-type (wt), srtA deletion (ΔsrtA) and complemented (ΔsrtA+) strains, cDNA was synthesized, and the presence of transcripts was detected by PCR. Wild-type genomic DNA (50 ng) was used as a positive control, as indicated. DNA markers (bp) are given on the left-hand side. Schematic indicates positioning of primer sites. (B) Comparative growth of wild-type (filled squares) and srtA deletion (empty squares) and complemented strains (empty triangles), along with strain 515 (pAMp) as a complementation vector control (filled triangles). Bacteria were grown for 7 h at 37°C in 5% CO₂ in THB medium, and the optical density at 600 nm was measured at the indicated times. (C) Western immunoblot analyses of wild-type and srtA mutant strains with antiserum against LPXTG family surface proteins. Total protein extracts (20 μg) were collected as described in Materials and Methods, blotted onto nitrocellulose, and probed with antisera directed against SAG0392, SAG0771, and SAG1462 (right panel). Extracts were also stained with Coomassie blue as a protein loading control (left panel). Molecular size markers (kDa) are indicated.

FIG. 3.

Relative abundance of pilus proteins associated with wild-type 515 and srtA mutant strains. (A) Proteins were collected from FMC culture supernatants or harvested cell pellets of wild-type 515 (wt) and srtA deletion (ΔsrtA) and complemented (ΔsrtA+) strains, blotted onto nitrocellulose, and probed with antiserum directed against each of the PI-2a pilus proteins (GBS59, GBS67, and GBS150). (B) Total protein extracts were also stained with Coomassie blue as protein loading controls. Molecular size markers (kDa) are indicated. (C) Transcription of PI-2a pilus protein subunits in wild-type and srtA mutant strains. RNA was extracted from wild-type (wt) and srtA deletion (ΔsrtA) and complemented (ΔsrtA+) strains; cDNA was synthesized, and the presence of backbone (GBS59) and ancillary (GBS67/GBS150) protein transcripts was detected by PCR. Wild-type genomic DNA (50 ng) was used as a positive control, as indicated. α, anti.

FIG. 4.

Effects of mild detergent treatment on pilus anchoring by wild-type (wt) and srtA mutant strains. (A) Bacterial cells were incubated with (+) or without (−) 0.5% SDS for 1.5 h, and the supernatants were harvested. Proteins were then extracted as described in Materials and Methods, blotted onto nitrocellulose, and probed with antiserum directed against the pilus backbone protein (GBS59). (B) Flow cytometry analysis of bacterial cells. Cells were incubated with 0.5% SDS for 1.5 h, paraformaldehyde fixed, and then stained with antiserum against each of the three pilus proteins followed by an R-phycoerythrin secondary antibody. Filled histograms correspond to staining of bacteria with preimmune serum, while black histograms indicate staining with specific antiserum. The change in mean fluorescence is indicated in the top right-hand corner of each plot. wt, wild-type; ΔsrtA, srtA deletion mutant; ΔsrtA+, srtA complemented mutant; α, anti.

FIG. 5.

Relative abundance of pilus proteins associated with sortase mutants (A) or pilus protein mutants (B) of PI-2a. Proteins were collected from FMC culture supernatants (lower panels) or harvested cell pellets (upper panels), as described in Materials and Methods, blotted onto nitrocellulose, and probed with antiserum directed against each of the pilus proteins (GBS59, GBS67, and GBS150). wt, wild-type; ΔsrtA, srtA deletion mutant; Δ1405 and Δ1406, pilus-associated sortase C family deletion mutants; Δ59, -67, and -150, pilus protein deletion mutants; α, anti.

FIG. 6.

Effects of mild detergent treatment on sortase mutants (A) or pilus protein mutants (B) of PI-2a. Bacterial cells were incubated with (+) or without (−) 0.5% SDS for 1.5 h, and the supernatants were harvested. Proteins were then extracted as described in Materials and Methods, blotted onto nitrocellulose, and probed with antiserum directed against the pilus backbone protein (GBS59). wt, wild-type; ΔsrtA, srtA deletion mutant; Δ1405 and Δ1406, pilus-associated sortase C family deletion mutants; Δ59, -67, and -150, pilus protein deletion mutants.

FIG. 7.

Fate of ancillary protein GBS150 in the absence of SrtA or the pilus-associated SrtC transpeptidases. Bacterial cells were incubated with 0.5% SDS for 1.5 h, and the suspensions were subsequently separated into cell-associated (A) and extracellular (B) fractions. Proteins were extracted as described in Materials and Methods, blotted onto nitrocellulose, and probed with antiserum directed against ancillary protein GBS150. wt, wild-type; ΔsrtA, srtA deletion mutant; Δ1405 and Δ1406, pilus-associated sortase C family deletion mutants.