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Journal of Bacteriology, April 2009, p. 2530-2540, Vol. 191, No. 8
0021-9193/09/$08.00+0     doi:10.1128/JB.01198-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Interplay between Two RND Systems Mediating Antimicrobial Resistance in Brucella suis{triangledown}

Fernando A. Martin,1 Diana M. Posadas,1 Mariela C. Carrica,2 Silvio L. Cravero,2 David O'Callaghan,3 and Angeles Zorreguieta1*

Fundación Instituto Leloir, IIBBA CONICET and FCEyN, Universidad de Buenos Aires, Patricias Argentinas 435, (C1405BWE) Buenos Aires, Argentina,1 Instituto de Biotecnología, CICVyA, INTA-Castelar, Las Cabañas y Los Reseros s/n (B1712WAA) Castelar, Buenos Aires, Argentina,2 Institut National de la Santé et de la Recherche Médicale, ESPRI 26, Université de Montpellier 1, ERA4204, UFR de Médecine, CS 83021, Avenue Kennedy, 30908 NIMES Cedex 02, France3

Received 26 August 2008/ Accepted 23 January 2009

The RND-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. Also, RND pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. We have previously shown that the BepC outer membrane factor of Brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. In the present work, we characterize two membrane fusion protein-RND translocases of B. suis encoded by the bepDE and bepFG loci. MIC assays showed that the B. suis {Delta}bepE mutant was more sensitive to deoxycholate (DOC), ethidium bromide, and crystal violet. Furthermore, multicopy bepDE increased resistance to DOC and crystal violet and also to other drugs, including ampicillin, norfloxacin, ciprofloxacin, tetracycline, and doxycycline. In contrast to the {Delta}bepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the {Delta}bepE {Delta}bepG double mutant showed a more severe phenotype than the {Delta}bepE mutant, indicating that BepFG also contributes to drug resistance. An open reading frame (bepR) coding for a putative regulatory protein of the TetR family was found upstream of the bepDE locus. BepR strongly repressed the activity of the bepDE promoter, but DOC released the repression mediated by BepR. A clear induction of the bepFG promoter activity was observed only in the BepDE-defective mutant, indicating a regulatory interplay between the two RND efflux pumps. Although only the BepFG-defective mutant showed a moderate attenuation in model cells, the activities of both bepDE and bepFG promoters were induced in the intracellular environment of HeLa cells. Our results show that B. suis harbors two functional RND efflux pumps that may contribute to virulence.

* Corresponding author. Mailing address: Fundación Instituto Leloir, IIBBA CONICET and FCEyN, Universidad de Buenos Aires, Patricias Argentinas 435, (C1405BWE) Buenos Aires, Argentina. Phone: 54-11-52387500, ext. 3204. Fax: 54-11-52387501. E-mail: azorreguieta{at}leloir.org.ar

{triangledown} Published ahead of print on 6 February 2009.

Journal of Bacteriology, April 2009, p. 2530-2540, Vol. 191, No. 8
0021-9193/09/$08.00+0     doi:10.1128/JB.01198-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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