Background: Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies.

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 (Aand B-domains) and Scm 36 (A-domain) bound to collagen type V efficiently in a concentrationdependent 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 bsheets, 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 pilusencoding 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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