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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

Jouko Sillanpää^1,2,

Sreedhar R. Nallapareddy^1,2,

¹ Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School, Houston, TX, USA
² Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, TX, USA
³ Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA
⁴ Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA
⁵ Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, TX, USA

Correspondence
Barbara E. Murray
bem.asst{at}uth.tmc.edu

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 Scm₆₅ (A- and B-domains) and Scm₃₆ (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 Scm₃₆ and of Acm₃₇ indicated that these proteins were rich in β-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.

These authors contributed equally to this work.

Supplementary methods, describing bacterial isolates, identification and structural analysis of CWA proteins, and sources of extracellular matrix proteins; supplementary results, describing sequence variation in fms15, fms16 and fms19, and structural motifs of the putative E. faecium MSCRAMMs; supplementary references; three supplementary figures, showing Coomassie-stained SDS-PAGE results for purified recombinant His6-Scm proteins, analysis of Scm expression on the cell surface of E. faecium isolates, and conserved lysine and glutamic acid residues in the pilin motif and E-box of predicted E. faecium major pilus proteins; and a supplementary table, listing oligonucleotides used in this study, are available with the online version of this paper.