A bistable genetic switch controls antibiotic resistance transfer in Enterococcus faecalis.

Abstract

The recent rise in microbial drug resistance is a growing challenge for future therapy of bacterial infections. Increased prevalence of antibiotic resistance in bacteria is an outcome of evolution via natural selection. However, the built-in design feature of bacteria to transfer DNA containing antibiotic resistance both within the same species and across species is the main culprit for the spread of drug resistance. One of the main factors driving the rise of drug resistant microbes is the transfer of antibiotic resistance genes present on mobile plasmids between donor and recipient cells via the mechanism of conjugation. In order to combat microbial drug resistance, novel strategies need to be developed to block such transmission of antibiotic resistance. In this work, the gene regulatory components involved in transfer of tetracycline resistance confers plasmid pCF10 between plasmid-carrying donor cells and plasmid-deficient recipient cells in bacterium Enterococcus faecalis is investigated. In the native state the donor cell exists in an OFF or conjugation-incompetent state. A pheromone released by the recipient cells serves as the chemical trigger for switching the donor cell from OFF to an ON or conjugation-competent state. The onset of conjugation is tightly regulated via multi-layered regulation offered by two-key genes prgQ and prgX present on pCF10 in response to the pheromone secreted by recipient cells. Using mathematical modeling and experimentation, we describe a novel mechanism of gene-regulation due to transcriptional interference and sense-antisense RNA interaction as a result of convergent transcription in the prgX/prgQ operon. We demonstrate that such a multi-layered gene-regulatory mechanism confers the system a bistable genetic switch controlling conjugative gene transfer between donor and recipient cells. A similar regulatory advantage offered by convergent transcription in attributing a bistable switch-like behavior in the scbA-scbR operon controlling antibiotic production in S.coelicolor is also investigated. Both mathematical model and experiments demonstrate that donor cells also control response to pheromone by changing the number of copies of pCF10 plasmid inside the cell. Cells with higher copies show increased robustness of the bistable switch and lower sensitivity to pheromone. Once bistable genetic-switch is ON, expression of genes encoding various proteins involved in the transfer of the plasmid are induced, however, this also causes production of an inhibitor of conjugation, thus giving rise to negative feedback loop which causes the donor to return to OFF state. Modeling and experimental analysis of dynamic response to induction indicate that this negative feed-back loop causes a brief surge of expression of the entire operon. We show that the inhibitor signaling peptide for pCF10 based system, acts as quorum-sensing signal with the role of turning-OFF conjugation at a population-wide scale. An interplay of positive and negative feedback loops allows the donor cell to quickly transition between ON and OFF states and is critical both for the transfer of plasmid and survival of the cell. Studying both the turning-ON and turning-OFF mechanisms of the switch allows identification of potential drug targets for blocking transmission of antibiotic resistance for use in future therapy.