Interplay between two RND systems mediating antimicrobial resistance in Brucella suis.
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 DeltabepE 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 DeltabepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the DeltabepE DeltabepG double mutant showed a more severe phenotype than the DeltabepE 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.
Martin
Fernando A
FA
Fundación Instituto Leloir, IIBBA CONICET and FCEyN, Universidad de Buenos Aires, Patricias Argentinas 435, (C1405BWE) Buenos Aires, Argentina.
Posadas
Diana M
DM
Carrica
Mariela C
MC
Cravero
Silvio L
SL
O'Callaghan
David
D
Zorreguieta
Angeles
A
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D041021:Brucella suis / Q000187:drug effects* / Q000472:pathogenicity / Q000502:physiology; D003840:Deoxycholic Acid / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D004847:Epithelial Cells / Q000382:microbiology; D004996:Ethidium / Q000494:pharmacology; D017353:Gene Deletion; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005840:Gentian Violet / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014774:Virulence
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D012097:Repressor Proteins; D003840:Deoxycholic Acid; D004996:Ethidium; D005840:Gentian Violet