Transcriptional regulation of RND drug efflux pumps in Brucella suis”

MARTÍN, F A; POSADAS, D. M.; RUIZ, V.; CARRICA, MC; CRAVERO S; O'CALLAGHAN, D; ZORREGUIETA A

Rosario

Congreso; V Congreso Argentino de Microbiología General SAMIGE; 2008

SAMIGE

The resistance nodulation cell division-type (RND) efflux pumps are responsible for the multi-drug resistance phenotype observed in many clinically relevant species. Besides, RND pumps were implicated in physiological processes with a role in the virulence mechanisms of several pathogenic bacteria. Our previous results have shown the functionality of two RND pumps named BepDE and BepFG, involved in the resistance to structurally unrelated compounds in the intracellular pathogen Brucella suis. In other bacterial species the RND efflux pumps has been demonstrated to be transcriptionally regulated by proteins from the TetR family. The context of bepDE showed the presence of a gene encoding a putative repressor from the TetR family named BepR, while the regions around bepFG did not suggest the presence of any putative regulator. The aim of the present work was to determine the bepDE and bepFG promoter activities both in vitro and in the intracellular environment and analyse the function of BepR in the transcriptional regulation of bepDE. We constructed transcriptional fusions of PbepDE and PbepFG predicted promoters to the GFP-promoter-less plasmid pKGFP. The fluorescence activity was evaluated in rich (TS, triptic soy broth) and modified minimal medium E (MME) using a multi-plate reader (Berthold Technologies). Although a significant level of PbepDE-GFP expression was observed in both media, the presence of stechiometric dose of bepR strongly repressed PbepDE-GFP expression, suggesting that BepR is a transcriptional repressor of bepDE. Interestingly, The presence of sodium deoxicholate (DOC), which is a substrate of both pumps released the repression mediated by BepR.   We also evaluated the expression of BepR promoter (PbepR) using the same reporter system. A significant increase in reporter expression from  PbepR when DOC was added to the culture media was also observed. Although bepFG expression (from PbepFG-GFP) showed undetectable level of expression in TS or MME media, PbepFG activity showed 5-fold induction in a BepDE-defective mutant. HeLa cells infected with B. suis harbouring the construction with the PbepDE-GFP (and the same number of bepR copies), or the PbepFG-GFP construction showed reporter activity at early stages of infection (5 h) that sustained for 48 h.  Similarly, PbepR was clearly expressed in HeLa cells. Taken together, these results suggest that BepR acts as a local repressor of bepDE, which can be released by the presence of DOC. Moreover a regulatory interplay betweem bepDE and bepFG was observed suggesting that  a global regulatory system fine tunes the expression of efflux systems in Brucella suis.