Functional characterization of ABC-MFP membrane transport systems and a TolC homologue in Brucella suis

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

Atlanta, USA

Congreso; American Society for Microbiology, 105 th ASM General Meeting; 2005

American Society for Microbiology

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 export system (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. Brucella is a Gram-negative intracellular pathogen able to replicate and survive inside phagocytic and non phagocytic cells. Therefore, protein secretion and efflux of toxic compounds may be crucial in the infection process. Two ABC-MFP putative systems and only one protein of the TolC family have been identified in the B. suis genome. Phylogenetic analysis of the MFP components placed them close to those involved in drug efflux. Both loci encoding for the ABC-MFP systems and the tolCb gene were cloned under the inducible ara promoter. Complementation studies on heterologous mutants affected in protein secretion or drug efflux were performed. Protein secretion was assayed by different plate assays and SDS-PAGE. Drug efflux was analyzed by measuring the minimum inhibitory concentration (MIC) of different compounds such as detergents, drugs and antibiotics. The cloned ABC-MFP systems were unable to complement the secretion phenotype of the R. leguminosarum prsD-prsE mutant, which encodes for an ABC-MFP protein secretion system. However, one of these systems restored resistance to nalidixic acid and rhodamine 6-G in the acrAB hypersensitive mutant of E. coli. No evidence for complementation of the resistance phenotype of the tolC::Tn5 mutant with tolCb was obtained and only partial complementation of the hemolysin secretion was observed. A mutant in a tolC homologue had been isolated in Rhizobium. Interestingly, this mutant showed a defect in growth that was restored by tolCb of Brucella. These results suggest that in B. suis at least one of the ABCMFP systems may be involved in efflux of toxic compounds. In addition, proteins of the TolC family may play in Rhizobiaceae some other physiological role that has not yet been described.