Enterococcus faecalis biofilms: Analysis of the roles of enterococcal surface protein, ESP, and a newly identified genetic locus

Biofilms are bacterial communities attached to a surface, and encased in an extracellular polymeric matrix. Enterococci have emerged as one of the leading causes of nosocomial infections, and are frequently associated with biofilms isolated from various indwelling medical devices. Enterococcal surface protein, Esp, has been found to contribute significantly to biofilm formation in E. faecalis. However, certain strains have been shown to form biofilms independent of Esp. The first aim of this study was to precisely define the role Esp plays in E. faecalis biofilm formation. Esp was expressed at the cell surface of natively Esp-deficient strains. Isogenic Esp-positive and Esp-deficient strains were then compared for their biofilm forming abilities. The results show that Esp expression leads to a significant enhancement in biofilm formation, and this process is regulated in a glucose-dependent manner. Since mature Esp consists of multiple domains including a nonrepeat N-terminal domain, a central domain consisting of series of tandem repeats followed by a non-repeat C-terminal domain, the second aim was to localize the domain of Esp critical to biofilm enhancement. In-frame deletion mutants expressing truncated forms of Esp in an isogenic background were assessed for their biofilm forming abilities. We find that the strain expressing Esp lacking the N-terminal domain, formed significantly less biofilms than the strain expressing wild type Esp. Furthermore, an E. faecalis strain expressing the N-terminal domain of Esp alone, fused to a heterologous protein anchor formed biofilms that were quantitatively similar to those formed by a strain expressing full-length Esp. These results imply that the N-terminal domain of Esp is sufficient for Esp-mediated biofilm enhancement in E. faecalis. The third aim was to identify factors other than Esp that may be involved in the process of enterococcal biofilm development. A library of transposon insertion mutants was generated in a high-biofilm forming E. faecalis strain, E99, and screened for mutants with a reduced ability to form biofilms. Using this strategy, we have identified a novel locus encoding surface proteins, which appears to contribute to the high-biofilm phenotype. Moreover, this locus is apparently carried on a mobile genetic element that mediates high-frequency conjugal transfer.