| Pseudomonas aeruginosa, a Gram-negative bacterium, is an opportunistic human pathogen exhibiting innate resistance to multiple antimicrobial agents. Intrinsic multidrug resistance of P. aeruginosa is caused synergistically by its low-permeability outer membrane and the existance of a large number of multidrug efflux systems. There are five known multidrug efflux pumps families in bacteria and the RND (resistance/nodulation/cell division) efflux system is the most important multidrug efflux family which mainly contributes to intrinsic multidrug resistance. It is important to study RND pumps in order to understand the mechanism of antibiotic resistance, and the screening for inhibitors of RND pumps in P. aeruginosa is one the promising approaches for controlling antibiotic resistance in pathogens.In this study, the functions of the twelve RND pump gene clusters in P. aeruginosa were systematically investigated using knockout mutants of the 12 RND pumps. The expression of the RND punps was also examined using the promoter-reporter fusions which were constructed by cloning the promoter regions in front of the promoterless luxCDABE. The results and conclusions are presented in four parts:Part one, the expression profiles of the RND pumps with and without zinc in the medium in P. aeruginosa PAO1 were measured. The results showed that mexJK and muxABC-opmB were constitutively expressed in laboratory conditions in addition to the reported mexAB-oprM, mexGHI-opmD, mexVW and mexXY. Antibiotic susceptibility of the RND efflux pump mutants was tested, and the results indicate that two RND pump mutants, MexGHI-OpmD and MuxABC-OpmB, showed increased antibiotic resistance. These results suggest that MexAB-OprM is the main member in RND family; it is the first line for effluxing chloramphenicol, ciprofloxacin, tetracycline, oxytetracycline HCl and streptomycin. MexAB-OprM and MexMN-OprN show the main defence to ampicillin in RND family in wild type PAO1. For the zinc resistance, CzcCBA, MexGHI-OpmB and MexXY-OprM are the first defence line in RND family in wild type PAO1.Zinc induced the expression of mexAB-oprM, mexJK, mexXY and czcCBA. MexGHI-OpmD and MexXY-OprM were involed in efflux of zinc. It was also observed that antibiotic susceptibility was decreased in the zinc efflux pump CzcCBA mutant, PAO(ΔczcC), revealing a relationship between antibiotic resistance and metal ion efflux. The results also show the zinc affect the antibiotic resistance of P. aeruginosa PAO1.Part two, the function of MuxABC-OpmB in antibiotic efflux and bacterial virulence was tested in a MuxABC-OpmB mutant constructed by insertion of LacZ and gentamycin resistance (lacZ-GENr) cassette in muxA. The drug susceptibility and virulence were measured in the muxA mutant PAO(ΔmuxA) and compared with that of wild type PAO1. Elevated resistance toβ-lactams including ampicillin and carbenicillin was observed in this mutant. The mutant also showed decreased twitching motility and attenuated virulence both in Brassica rapa pekinensis and Drosophila melanogaster infection models. These results indicate that the MuxABC-OpmB RND pump is associated with ampicillin and carbenicillin resistance and also involved in pathogenesis in P. aeruginosa.Part three, a series of controllable expression vectors (pLY expression system) were constructed based on the zinc regulated promoter of the czcCBA in P. aeruginosa. These vectors allow controlled expression of proteins or RNAs in P. aeruginosa. As proof of principle, the luxABCDE and sacB were cloned into the vectors and the zinc controlled expression was tested. The results demonstrated that this expression system was functional in P. aeruginosa. It provides a useful tool for controlled expresson in P. aeruginosa.Part four, six metal transport pumps were identified in the geneome of PAO1 by bioinformatics analysis. Experiments were carried out to test the funcrtions of these pumps. Promoter reporter and knockout mutants were constructed for each of the six pumps. The expression profiles were measured and their substrate spectra tested. The involvement of these metal pumps in metal resistance as well as antibiotic resistance in P. aeruginosa PAO1 was examined. The expression of the eleven RND was induced to different various extents by different metal ions and antibiotics. The results of drug susceptibility tests indicated that ZntAlwas involved in cadmium and zinc resistance, ZntA2 was associated with cadmium resistance, and CzcD2 could efflux cobalt. However, these mutants did not show any change of antibiotic susceptibilities.In summary, we systematically investigated the expression and function of the twelve RND pumps and six metal iron transport complexes in P. aeruginosa. These data should facilitate our understanding of the intrinsic resistance to antibiotics and other agents in P. aeruginosa...
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