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.
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.
Sillanpää
Jouko
J
Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, TX, USA.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School, Houston, TX, USA.
Nallapareddy
Sreedhar R
SR
Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, TX, USA.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School, Houston, TX, USA.
Prakash
Vittal P
VP
Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, TX, USA.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School, Houston, TX, USA.
Qin
Xiang
X
Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA.
Höök
Magnus
M
Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA.
Weinstock
George M
GM
Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA.
Murray
Barbara E
BE
Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, TX, USA.|Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, TX, USA.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School, Houston, TX, USA.
D018829:Adhesins, Bacterial / Q000235:genetics* / Q000378:metabolism; D001422:Bacterial Adhesion; D002942:Circular Dichroism; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D016984:Enterococcus faecium / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D010861:Fimbriae, Bacterial / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D005810:Multigene Family; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D018829:Adhesins, Bacterial; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D003094:Collagen