Gelatinase Is Important for Translocation of Enterococcus faecalis across Polarized Human Enterocyte-Like T84 Cells

Abstract

ABSTRACT Previously, in our laboratory, we established a two-chamber system to study translocation of Enterococcus faecalis across monolayers of polarized human colon carcinoma-derived T84 cells. By using the same system in the present study, we now show that disruption of gelE of strain OG1RF, which also has a polar effect on the cotranscribed sprE , as well as disruption of its regulatory system ( fsrA , fsrB , and fsrC ) resulted in a loss of detectable translocation by E. faecalis OG1RF; these mutants lost gelatinase (GelE) and serine protease (SprE) production by standard assay. A gelE deletion mutant of OG1RF (GelE − SprE + ) also showed that significantly reduced translocation and complementation with the gelE gene (pTEX5438) in trans restored gelatinase and translocation, demonstrating that gelatinase is important for E. faecalis translocation. Complementation of fsrA , fsrB , and fsrC mutants with all three fsr genes also resulted in production of gelatinase and translocation. Furthermore, introduction of fsr genes into two non-gelatinase-producing E. faecalis isolates, the well-characterized laboratory strain JH2-2 and a human-derived fecal isolate, TX1322 (both of which have gelE but not fsrA or fsrB , are gelatinase negative, and do not translocate), resulted in gelatinase production by these strains and restored translocation across T84 monolayers, while transformation with pTEX5438 ( gelE ) showed little or no translocation and no detectable gelatinase, confirming the importance of both fsr and gelatinase for E. faecalis translocation. The importance of gelatinase production was also corroborated among 20 E. faecalis human isolates (7 fecal, 7 endocarditis, and 6 urine isolates), which showed translocation by all gelatinase-positive isolates but little to no translocation for gelatinase nonproducers. These results indicate that gelatinase is important for the successful in vitro translocation of E. faecalis across human enterocyte-like T84 cells.