Functional characterization of a Brucella suis ABC-MFP translocase

MARTÍN, F A; POSADAS, D. M.; DELPINO M V; BALDI P C; CAMPOS E; ZORREGUIETA, A

Merida, Yucatan, Mexico

Congreso; Brucellosis 2005, International Research Conference Including the 58th Brucellosis Research Conference,; 2005

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Gram-negative bacteria have evolved transport complexes that export macromolecules and toxic substances across both inner and outer membranes in a single energy coupled step. The process requires (1) a cytoplasmic membrane protein (ABC or RND), (2) an accessory protein (MFP), and (3) an outer membrane factor (OMF) of the TolC family. The roles of these systems in Brucella spp. remain unexplored. Both protein export and efflux of toxic compounds may be crucial in survival and pathogenicity in Brucella spp.. Three putative ABC-MFP systems have been identified in the B. suis genome. Phylogenetic analysis of the MFP components placed them close to those involved in drug efflux. In order to study the possible roles of the ABC-MFP systems, heterologous complementation studies were performed on  an E. coli acrAB hypersensitive mutant, which is affected in drug efflux. Drug efflux was analyzed by measuring the minimum inhibitory concentration (MIC) of different compounds such as detergents, drugs and antibiotics. One of the transport loci (eflA1-eflB1), cloned under the inducible ara promoter, restored resistance to unrelated drugs such as nalidixic acid, rhodamine 6G and erithromycin in the acrAB mutant of E. coli. Furthermore, a fluorometric assay showed that eflA1-eflB1 conferred to the E. coli acrAB mutant, a clear reduction of rhodamine 6G accumulation. In addition, EflA1-EflB1 was unable to restore drug resistance in an acrAB- tolC double mutant of E. coli indicating that efflux by EflA1-EflB1 in E. coli is OMF dependent. These results show that the EflA1-EflB1 complex is capable of promoting efflux of toxic compounds. However, disruption of eflA1 in B. suis did not decrease the level of resistance of Brucella to several drugs, suggesting that there may be additional systems which could also contribute to drug efflux. Key words: efflux, ABC , MFP, transport. This work was supported by a grant from University of Buenos Aires (X-245)