Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium - PubMed (original) (raw)

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium

Jouko Sillanpää et al. Microbiology (Reading). 2008 Oct.

Abstract

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

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Figures

Fig. 1

Domain organization of E. faecium CWA proteins with Ig-like folds. (a) S, signal peptide; W, cell-wall-spanning region; M, membrane anchor region; C, cytoplasmic tail with charged residues; Shaded A region, non-repetitive sequences; hatched B region, repetitive sequences; lollypop denotes presence of a stop codon due to a nucleotide substitution/deletion in Fms15, Fms16 and Fms19 of TX0016; solid black bars, His6-tag expressed regions of Scm (Fms10). Arrows above Scm show regions with predicted Ig-like folding. (b) Contiguously arranged ORFs encoding CWA proteins in E. faecium genome; srt, sortase. *Fms14 lacks an identifiable signal peptide, but has all the other characteristics of CWA MSCRAMMs.

Fig. 2

Binding of recombinant Scm proteins to ECM proteins in a solid phase ELISA-type ligand binding assay. (a) Binding of 20 μM rScm65 (aa 27-624) and rScm36 (aa 27-333) to a collection of immobilized ECM proteins. CI, type I collagen, Ln, laminin; Fn, fibronectin; Fg, fibrinogen; BSA, bovine serum albumin. Data points for rScm36 represent the means of _A_405nm values of 18 wells representing six independent experiments and including proteins from two different purifications, and for rScm65, 15 wells from five independent assays. (b) Binding of rScm36 to CI, CV and fibrinogen with increasing rScm36 concentrations. Data points represent the means of _A_405 nm values from three wells. The experiment was repeated up to six times using protein from two purifications. The reported KD value is the average ± standard error from the six assays. BSA values were subtracted from the respective collagen-binding values after which affinity calculations were performed with the one-ligand binding site model. The resulting curves are depicted in the figure.

Fig. 3

Secondary structure analysis by far-UV circular dichroism spectroscopy. (a) Collected CD-spectra of Scm36, Ace19 and Acm37. Mean residue weight ellipticity is reported in deg cm2 dmol-1. (b) Summary of secondary structure compositions. *Scm36 contains the predicted A-domain of Scm (aa 27-333). Ace19 and Acm37 contain the corresponding minimal ligand binding regions of the collagen binding MSCRAMMs Ace (E. faecalis) and Acm (E. faecium), respectively.

Fig. 4

Quantitation of Scm surface expression by FACS analysis. Reactivity of both preimmune Igs (PI) and anti-Scm Igs for each strain was shown. Although some E. faecium isolates exhibit slight binding to goat PI, significant difference was observed between PI and anti-Scm Igs. Bacteria were analyzed using side scatter as the threshold for detection. Specific binding by anti-Scm antibodies is indicated as log fluorescence intensity on the X-axis. Each histogram represents 50 000 events of bacterium-sized particles.

Fig. 5

Western blot analysis of cell wall anchored proteins from E. faecium strains TX0082 and TX0016. The mutanolysin cell wall extracts (10 μg total protein/lane) were separated on 4-15% SDS-PAGE gels, transferred to PVDF membranes, and probed using affinity-purified anti-rEbpCfm62 antibodies. Purified rScm36 and rEbpCfm62 proteins (20 ng/lane) were included as controls.

Fig. 6

Transcriptional analysis of the ebpAfm to bpsfm gene cluster. (a) Northern hybridization of total RNA (30 μg/lane) from TX0082 probed with intragenic fragments of ebpAfm, ebpBfm, ebpCfm, and bpsfm. The acm probe was used as a control. RNA bands with expected sizes are indicated with arrows. (b) RT-PCR analysis of the ebpAfm to bpsfm gene cluster. Location of each primer pair is shown with arrows in the schematic representation of the gene cluster. A lollypop between ebpCfm and bps depicts a predicted transcriptional terminator. Top gel, RT-PCR with DNase-treated total RNA (10 ng) as template; middle gel, control reaction of the same total RNA (100ng) preparation amplified without reverse transcriptase; bottom gel, control reaction amplified with genomic TX0082 DNA as template. An intragenic region of gyrase A was used as a control. M, molecular weight marker.

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Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium - PubMed (original) (raw)