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 gram-positive 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.

FIG. 1.

Multiple-amino-acid sequence alignment of the 27 proteins of E. faecalis strain V583 using the ClustalW program. WxL domains are indicated by bold type and shading. The distances between the two WxL domains are indicated in parentheses. Amino acids that are identical or similar are indicated by shading. The groups of amino acids considered similar are I, L, M, and V; A, S, G, and T; H, K, and R; D, E, N, and Q; and F, W, and Y.

FIG. 2.

Localization of Nuc fusions by fractionation. Proteins from E. faecalis strains expressing P_usp45::sp_Usp45::nucB (A), P_usp45::sp_Usp45::nucB::cwa_M6(277-415) (B), P_usp45::sp_Usp45::nucB::EF2686_WxL2 (C), and P_usp45::sp_Usp45::nucB::EF0392_WxL2 (D) were fractionated in three compartments, protoplasts (P), cell walls (CW), and the supernatant (SN). Equivalent amounts of proteins were subjected to SDS-12% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, and immunodetection was performed using Nuc monoclonal antibodies. The arrows indicate bands migrating at the expected positions for the proteins indicated. The lower bands in panels A and B migrated at the position of the protein from which the N-terminal propeptide was also cleaved (26). The upper band in panel B migrated at the position of the full-length protein containing the signal peptide (38).

FIG. 3.

Interaction of Nuc fusions with JH2-2 and VE14258 whole cells. Whole cells of strain VE14258 were incubated with supernatants of strains expressing secreted Nuc and Nuc-EF2686_WxL2. Strain JH2-2 was incubated with supernatants of strains expressing secreted Nuc and Nuc-EF0392_WxL2. Proteins were fractionated in two compartments, cells (C) and the supernatant (SN), and were analyzed by Western blotting.

FIG. 4.

(A) Schematic diagrams of Nuc fusion proteins and derivatives used in this study. SP, signal peptide. The open bars represent Nuc; the cross-hatched bars represent the C-terminal domains of EF2686 and EF0392. (B) Western blot analysis of protein extracts of P, CW, and SN fractions from E. faecalis JH2-2 strains expressing Nuc fused to proteins with complete or deleted WxL regions, as indicated above the lanes. (C) Reassociation of Nuc fusions with E. faecalis cells. VE14258 cells were incubated with culture supernatants of strains expressing Nuc-EF2686_WxL2, Nuc-EF2686_WxL1, and Nuc-EF2686_WxL0. Strain JH2-2 cells were incubated with culture supernatants of strains expressing Nuc-EF0392_WxL2, EF0392_WxL1, and EF0392_WxL0. Cells (C) and supernatants (SN) were separated and analyzed by Western blotting. Immunoblotting was done using Nuc monoclonal antibodies.

FIG. 5.

Detection of Nuc fusion proteins on the E. faecalis cell surface by immunofluorescence. Strains expressing the different fusions were treated with lysozyme, labeled with anti-Nuc monoclonal antibodies and Alexa 555 secondary antibodies, and visualized by fluorescence.

FIG. 6.

Interaction of Nuc fusion proteins with gram-positive bacterial cells (A) and peptidoglycan of B. subtilis (B). (A) Strain VE14258 and B. subtilis, S. agalactiae, L. johnsonii, S. aureus, and L. innocua strains were incubated with supernatants of cultures of strains expressing Nuc-EF2686_WxL2 and Nuc-EF2686_WxL0 or expressing Nuc-EF0392_WxL2 and Nuc-EF0392_WxL0. Cell proteins were analyzed by Western blotting, using anti-Nuc antibodies. (B) Interaction of Nuc fusions proteins. One hundred fifty micrograms of insoluble peptidoglycan of B. subtilis was incubated with supernatants of strains expressing Nuc-EF2686_WxL2, Nuc-EF2686_WxL1 and Nuc-EF2686_WxL0, or Nuc-EF0392_WxL2, Nuc-EF0392_WxL1, and Nuc-EF0392_WxL0. Suspensions were then centrifuged, and the pellets were subjected to SDS-polyacrylamide gel electrophoresis analysis and Western blot analysis using anti-Nuc monoclonal antibody.