The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

Abstract

fThe WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in ␤-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species. Enterococcus faecium is one of the "no ESKAPE" pathogens responsible for a considerable percentage of nosocomial infections; its ability to cause life-threatening infections and resistance to antibiotics cause considerable difficulties for patients and physicians (1). To date, many of the potential virulence determinants that have been described in E. faecium are MSCRAMMs (microbial surface components recognizing adhesive matrix molecules); these include adhesins or pili anchored to the cell surface by an LPxTG-like motif and contain IgG-like folds (2). Our group previously performed a bioinformatic search for novel surface proteins that might be virulence factors in the E. faecium endocarditis strain TX16 (also known as DO) (3). During this search, we identified a locus which included predicted proteins possessing the previously described WxL domain colocated with a predicted LPxTG-like protein (Fms6) (3, 4).The WxL domain is comprised of several conserved residues along with a highly conserved YXXX(L/I/V)TWXLXXXP motif and a second WxL proximal motif in the last ϳ120 to 190 C-terminal residues of a protein (4,5). Genes encoding proteins with the WxL domain were first described in Lactobacillus plantarum as part of a novel gene cluster found in a subset of low-GϩC-content Gram-positive bacterial species, one of which is E. faecium (6, 7). Bioinformatics and transcriptome data in L. plantarum predicted these loci form cell surface protein complexes involved in ca...

Vancomycin-Resistant Enterococci

“…In addition, WxL proteins from locus A were able to bind collagen and fibronectin and deletion of all 3 loci-attenuated virulence in a mixed infection rat endocarditis model. 43 Glycolipids and polysaccharides are also important components of the enterococcal cell envelope. Lipoteichoic acid is an antigenic component of gram-positive cell walls, and antibodies directed against this epitope were shown to enhance opsonization and killing of enterococci by complement and leukocytes.…”

How does temperature influences the development of lactococcosis? Transcriptomic and immunoproteomic in vitro approaches

“…2a and Table 2B. Of particular significance is the protein gi|515938667 containing a WxL domain, which is characteristic of proteins that are non-covalently anchored to peptidoglycan of the cell wall (Galloway-Peña et al 2015) and play a role in carbon source acquisition or adhesion to the host cells (Siezen et al 2006). Also, the protein gi|518123066 seems to be a lytic enzyme and is codified by a gene (gene 25 of contig 19) previously identified among those upregulated at 18°C in comparison with 13 and 37°C (Table 1).…”

The Fibronectin-Binding Protein Fnm Contributes to Adherence to Extracellular Matrix Components and Virulence of Enterococcus faecium

“…Previous studies have shown that E. faecium cells express a variety of surface proteins that can contribute to adhesion (41). These include an endocarditis-and biofilm-associated pilus protein (Ebp fm ) (42), an enterococcal surface protein (Esp) (43), serine-glutamate repeat-containing protein A (SgrA) (44), and the WxL proteins (26). In addition, we identified a significant association between collagen adherence and the presence of a functional acm gene, encoding an adhesin of collagen, in clinical but not community isolates (45,46).…”

“…E. faecium genomic DNA was isolated from a single colony after overnight growth in BHI broth, as described previously (26). Plasmids were isolated from E. coli using Wizard Plus SV MiniPreps system columns (Promega Corporation).…”