Identification and characterization of novel genetic determinants of biofilm formation in Enteroccous faecals.

Abstract

The nosocomial pathogen Enterococcus faecalis is a normal resident of the intestinal tracts of many vertebrates and invertebrates, including humans, and is readily isolated from many other environments. It has been well documented that E. faecalis can form biofilms on biotic and abiotic surfaces, and enterococcal biofilms likely play a role in virulence, persistence, and horizontal gene transfer of this organism. In my thesis research I used a large genetic screen to identify over 68 potential determinants of enterococcal biofilm formation. This, in conjunction with another genetic screen from our lab, constitute the first comprehensive examination of the core genome of E. faecalis for genetic determinants of biofilm formation. I characterized the role in biofilm formation of a novel transcription factor, Enterococcal Biofilm Regulator (EbrA), identified in a Recombinase Based In Vivo Expression Techonology (RIVET) screen. This transcription factor was differentially expressed between biofilm and planktonic cells at 24 hours, and a mutant strain in which the open reading frame was deleted was defective in its ability to form biofilms as compared to wild-type. To determine the regulon of EbrA, I utilized methods that examined the proteome and the transcriptome and determined that EbrA is responsible for modulating the metabolic rate to promote survival of the biofilm population when it undergoes nutrient stress. As a function of this survival role, my experiments suggest that EbrA is also a negative regulator of the cell lysis mechanism, that is thought to mediate eDNA release for biofilm matrix production. The current model of eDNA release did not include a negative regulator. Biofilm development is a dynamic process in which the population undergoes changes in both gene expression and metabolic activity. Previous characterizations have focused mostly on biofilms grown for at least 24 h. There is little information about the cellular processes associated with this transition from planktonic to biofilm growth in non-motile species, such as E. faecalis. The low amounts of biomass present in these initial stages of biofilm development preclude the use of standard expression profiling analyses of mRNA and protein. My thesis research also describes comparative analysis of the increase in biomass and adherent bacterial populations in the early stages of biofilm formation by the laboratory strain in relation to several clinical isolates. I then combined this analysis with high-resolution Field Emission Scanning Electron Microscopy (FESEM) analysis of the cell surface and the extracellular matrix of the developing biofilms. These studies revealed a dramatic temporal change in the appearance and biochemical composition of the extracellular matrix that has not been previously reported. The data also suggest that the biochemical composition of the biofilm matrix changes over time. One of those phenotypes has not been previously described. This work highlights the importance of careful kinetics studies, including very early time points, to identify novel phenotypes in complex biofilm growth systems.