Cell wall location and antigenicity of WapE in Streptococcus mutans

Experiments with isolated S. mutans cell walls have implicated wall-associated proteins as the important components of caries vaccines and 5 wall-associated proteins of S. mutans with the sortase-targeted motif LPXTG have been characterized (WapA, SpaP, GbpC, DexA, FruA). In addition, WapE was identified by bioinformatic analysis of the S. mutans UA159 genome. The wapE gene encodes a mature protein with a predicted molecular mass of approximately 55 kDa and a C-terminal LPXTG motif indicating a cell wall location. Objectives: The aim of this study was to explore the cellular location and immunogenicity of WapE. Methods: The wapE gene was cloned into pQE30 and rWapE expressed as a His-tagged protein, purified from E. coli and used for production of specific antiserum. Knockout mutants of wapE and srtA were constructed by insertional mutagenesis. Results: rWapE expressed in E. coli was recognized by antibody to whole cells and cells walls of S. mutans and also by specific antibody to rWapE. This antibody caused agglutination of S. mutans cells, indicating that WapE was exposed at the cell surface. However, WapE could not be detected in SDS-extracts or culture supernatants of S. mutans. Inactivation of the sortase gene (srtA) resulted in release of WapE into the supernatant, detectable by western blots. Blots with specific antibody were used to confirm the lack of expression and release of WapE in a wapE/srtA double mutant. Assays of whole cell hydrophobicity and biofilm formation in the presence and absence of sucrose failed to show any effect of the wapE knockout. Conclusion: These results indicate that WapE is expressed and exposed at the cell surface but not released into the supernatant.