IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Transcriptional regulation of RND drug efflux pumps in Brucella suis.
Autor/es:
MARTÍN F.A.; POSADAS DM; RUÍZ V; CARRICA MC; CRAVERO SL; O'CALLAGHAN D; ZORREGUIETA A.
Lugar:
Rosario, Santa Fé
Reunión:
Congreso; 5ta Reunión de la Sociedad Argentina de Microbiología General SAMIGE; 2008
Institución organizadora:
samige
Resumen:
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 and
bepR itself, which can be released by the presence of DOC.
Moreover a regulatory interplay between bepDE and bepFG was
observed suggesting that a global regulatory system fine tunes the expression of
efflux systems in Brucella suis.