A collagen-binding adhesin, Acb, and ten other putative MSCRAMM and pilus family proteins of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis Group, biotype I)

Abstract

Members of the Streptococcus bovis group are important causes of endocarditis. However, factors associated with their pathogenicity, such as adhesins, remain uncharacterized. We recently demonstrated that endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates frequently adhere to extracellular matrix (ECM) proteins. Here, we generated a draft genome sequence of an ECM protein-adherent S. gallolyticus subsp. gallolyticus strain and found, by genome-wide analyses, 11 predicted LPXTG-type cell wall-anchored proteins with characteristics of MSCRAMMs, including a modular architecture of domains predicted to adopt immunoglobulin (Ig)-like folding. A recombinant segment of one of these, Acb, showed high-affinity binding to immobilized collagen, and cell surface expression of Acb correlated with the presence of acb and collagen adherence of isolates. Three of the 11 proteins have similarities to major pilus subunits and are organized in separate clusters, each including a second Ig-fold-containing MSCRAMM and a class C sortase, suggesting that the sequenced strain encodes three distinct types of pili. Reverse transcription-PCR demonstrated that all three genes of one cluster, acb-sbs7-srtC1, are cotranscribed, consistent with pilus operons of other gram-positive bacteria. Further analysis detected expression of all 11 genes in cells grown to mid to late exponential growth phases. Wide distribution of 9 of the 11 genes was observed among S. gallolyticus subsp. gallolyticus isolates with fewer genes present in other S. bovis group species/subspecies. The high prevalence of genes encoding putative MSCRAMMs and pili, including a collagen-binding MSCRAMM, among S. gallolyticus subsp. gallolyticus isolates may play an important role in the predominance of this subspecies in S. bovis endocarditis.

FIG. 1.

(A) Schematic representation of the domain organization of the predicted MSCRAMM and pilus family proteins. S, signal peptide; CWA, LPXTG-motif CWA domain; gray, nonrepetitive sequences; hatched, amino acid sequence repeats. A CWA domain is not shown for Sbs16; the location of sbs16 at the end of an unassembled contig results in missing sequence data. (B) Representation of three clusters of putative pilus encoding genes in the S. gallolyticus subsp. gallolyticus TX20005 genome. Gray ORFs, predicted pilus major subunit proteins; hatched ORFs, possible minor pilus subunit proteins; dotted ORF, other Ig-folded CWA protein; black ORFs, predicted class C sortases.

FIG. 2.

(A) Coomassie-stained 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified recombinant His₆-rAcb₃₅ protein. M, molecular mass marker. (B) Binding of rAcb₃₅ to collagen types I, IV, and V in a solid-phase ELISA-type ligand binding assay. Squares, collagen type I; triangles, collagen type IV; inverted triangles, collagen type V. Data points represent the means ± the standard deviation of A₄₀₅ values from nine wells representing three independent assays. Bovine serum albumin-binding values (ranging between 0.001 and 0.057, with the highest value observed at 20 μM of rAcb₃₅) were subtracted from the respective collagen-binding values before affinity calculations were carried out with the one-ligand binding site model. The resulting curves are depicted in the figure. The reported K_D value is the mean ± the standard error of the mean from three assays.

FIG. 3.

(A) Quantification of Acb surface expression by S. gallolyticus subsp. gallolyticus isolates by flow cytometry. Reactivity to both preimmune antiserum (PI) and anti-Acb antiserum for each isolate is shown. Bacteria were analyzed using side scatter as the threshold for detection. Labeling of cells by anti-Acb antiserum is indicated as log fluorescence intensity on the x axis. Each histogram represents ≥25,000 events of bacterium-sized particles. (B) Association between cell surface expression of Acb, presence of the acb gene in the genome, and adherence to collagen of eight S. gallolyticus subsp. gallolyticus isolates. ^a, summary of Acb cell surface expression detected by whole-cell ELISA; these results are shown in detail (see Fig. S1 in the supplemental material). Cut-off level for positive expression was set at an A₅₉₀ of 0.01. ^b, presence of the acb gene determined by colony hybridizations with gene-specific probes under high-stringency conditions. ^c, summary of adherence of the eight isolates to collagen based on results from our previous study (63).

FIG. 4.

Transcriptional analysis of the acb-sbs7-srtC1 gene cluster by RT-PCR. Arrows in the schematic representation of the gene cluster show the location of each primer pair. A lollypop downstream of srtC1 denotes the predicted transcriptional terminator. Three sets of PCRs are shown as follows: 1, mRNA, RT-PCR with DNase-treated RNA (10 ng) as a template; 2, mRNA without a template with a control reaction of the same RNA preparation amplified without reverse transcriptase; 3, genomic DNA with a control reaction amplified with genomic TX20005 DNA as a template. M, molecular weight marker.