Quantification of Gelatinase Expression in a Biofilm Deficient Mutant of Enterococcus faecalis OG1RF
Enterococcus faecalis, a Gram-positive bacterium, has been characterized as a commensal organism in the gastrointestinal (GI) tract of humans. It has become a leading cause of nosocomial infections in the United States, treatment of which is complicated by the formation of biofilms which protect the bacteria from antimicrobial agents. Biofilm phosphatase (bph) is required for surface attachment and biofilm formation in wild-type (OG1RF) strains. Mutant strains lacking bph (Δbph) display significant growth and attachment defects, in addition to global changes in gene expression genes. Specifically, Δbph strains show markedly reduced production of gelatinase (GelE), a secreted Zn-metalloprotease implicated in a “housekeeping role,” on the surface of E. faecalis. Despite this, gelatin plate assays show a retention of gelatinase activity. Determining the regulatory role of Bph in GelE production is critical for a complete understanding of E. faecalis biology. To quantify gelatinase transcript and protein levels in OG1RF and the bph mutant, we employed a number of quantitative analyses to measure gelatinase expression and activity via RT-qPCR and Azo-protein (Azocoll and Azocasein) dye-release assays, respectively. We demonstrate that Bph regulates gelatinase production at the level of transcription. RT-qPCR shows a 10-fold decrease of gelE expression in Δbph mutants, while GelE enzymatic activity decreased by 2- to 5- fold (substrate dependent). These findings have relevance to the development of effective therapeutic strategies that utilize the regulatory role of bph in synthesis of proteins necessary for biofilm production and pathogenesis.