C-Terminal WxL Domain Mediates Cell Wall Binding in Enterococcus faecalis and Other Gram-Positive Bacteria

Abstract

Analysis of the genome sequence of Enterococcus faecalis clinical isolate V583 revealed novel genes encoding surface proteins. Twenty-seven of these proteins, annotated as having unknown functions, possess a putative N-terminal signal peptide and a conserved C-terminal region characterized by a novel conserved domain designated WxL. Proteins having similar characteristics were also detected in other low-G؉C-content grampositive bacteria. We hypothesized that the WxL region might be a determinant of bacterial cell location. This hypothesis was tested by generating protein fusions between the C-terminal regions of two WxL proteins in E. faecalis and a nuclease reporter protein. We demonstrated that the C-terminal regions of both proteins conferred a cell surface localization to the reporter fusions in E. faecalis. This localization was eliminated by introducing specific deletions into the domains. Interestingly, exogenously added protein fusions displayed binding to whole cells of various gram-positive bacteria. We also showed that the peptidoglycan was a binding ligand for WxL domain attachment to the cell surface and that neither proteins nor carbohydrates were necessary for binding. Based on our findings, we propose that the WxL region is a novel cell wall binding domain in E. faecalis and other gram-positive bacteria.Gram-positive bacteria contain a variety of extracellular proteins, some of which are crucial for adaptation and survival in the environment. These proteins participate in various important functions, such as nutrient catabolism, adhesion for niche colonization, translocation, and defense against the host immune system in the case of invading pathogens (35). At this point, cell surface-exposed proteins secreted via the Sec pathway can be classified into the following seven categories according to their means of associating with the cell surface (1, 4, 6, 8): (i) membrane-spanning proteins; (ii) lipoproteins (52, 55); (iii) cell wall-anchored proteins, which are covalently linked to the peptidoglycan at the carboxyl terminus via the LPXTG motif (34, 48); (iv) proteins containing LysM domains that bind to peptidoglycan, most of which are cell wall hydrolases (23, 44, 51); (v) GW proteins, which interact with lipoteichoic acids on the bacterial cell surface and also with mammalian cells (as shown for InlB and Ami of Listeria monocytogenes [2,21,22,33]); (vi) choline-binding proteins, which bind to phosphorylcholine in the cell wall (12); and (vii) proteins containing surface layer homology domains, which interact with peptidoglycan or cell wall-associated polymers, forming a crystalline surface layer (41). The latter class is restricted to bacilli and lactobacilli so far (32). A new C-terminal domain designated WxL was recently predicted based on in silico global genome analyses of Lactobacillus plantarum (25), Lactobacillus coryniformis (47), and Lactobacillus sakei (5). In these studies, neither biological properties nor cell localization was examined.With the ever-increasing availability of bacter...

Listeria monocytogenesSurface Proteins: from Genome Predictions to Function

“…This hypothesis is supported by the demonstration that GW repeats bind to cellular proteoglycans and that these interactions are required for efficient entry (87). Other internalin-like proteins, such as E. faecalis EF2686 (ElrA), S. pyogenes Slr, and S. agalactiae BrlD are also buried in the cell wall (28,190). The accessibility of these proteins at the bacterial surface …”

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

“…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).…”

Exploring Surface Display Technology for Enhancement of Delivering Viable Lactic Acid Bacteria to Gastrointestinal Tract

“…These results indicated that the fusion proteins had noncovalent interactions with the cell wall of E. faecalis. The fusion proteins were able to attach to the cell surface of E. faecalis and L. johnsonii when they were added exogenously (Brinster et al, 2007).…”