Studies on multidrug efflux systems and triclosan resistance in Pseudomonas aeruginosa

Abstract

In this dissertation, the role of RND multidrug efflux system in triclosan resistance was studied in Pseudomonas aeruginosa. Infections with P. aeruginosa are notoriously known for being very difficult to treat because of its high intrinsic drug resistance and its ability to develop resistance to a wide range of antimicrobial agents. This high resistance primarily results from synergy of its low outer membrane permeability and the expression of multidrug efflux pumps. Genomic DNA sequence analysis revealed that there are as many as 37 efflux systems representing all known efflux pump families. Twelve of these belong to the resistance nodulation division (RND) family and these have emerged as clinically significant in Gram-negative bacteria, including P. aeruginosa. Four RND efflux systems - MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY - had been described in wild-type strain PAO1 when this dissertation research was started. Two additional ones were found since - MexGHI-OpmD and MexVW - and this research identified a seventh, MexJK. Only MexAB-OprM is constitutively expressed at low levels in PAO1 and the other RND efflux systems are only expressed in strains containing regulatory mutations. The characterization of new efflux pumps therefore awaits identification of regulatory mutations expressing these pumps and these can usually only be obtained after identification of the respective pump substrates or the fortuitous discovery of clinical isolates expressing the pumps after antibiotic exposure. P. aeruginosa is well known for its high intrinsic resistance to triclosan, a broad spectrum bisphenolic biocide that is the active ingredient in many consumer products with antimicrobial properties. Although wild-type P. aeruginosa strains express the triclosan target enoyl-acyl carrier protein reductase (FabI), a crucial enzyme of the bacterial fatty acid biosynthetic cycle, they are triclosan-resistant due to constitutive expression of the MexAB-OprM efflux system. In this study, triclosan was proven to be a good substrate for most of characterized multidrug efflux systems including MexAB-OprM, MexCD-OprJ and MexEF-OprN, but not MexXY. In fact, experiments presented in this dissertation showed that RND pumps are solely responsible for high-level triclosan resistance of P. aeruginosa, which enables this bacterium to survive in the presence of triclosan concentrations in excess of 1000 μg/ml. Since triclosan is a good and perhaps universal RND efflux pump substrate, it was reasoned that it could be used as a tool to isolate regulatory mutants expressing hitherto unidentified efflux pumps and to use these mutants to study efflux pump function, molecular architecture and regulation. Proof-of-concept was obtained by exposure of a triclosan-susceptible Δ(mexAB-oprM) P. aeruginosa strain to triclosan. This procedure selected triclosan-resistant bacteria at high frequencies and these bacteria became simultaneously multidrug resistant (M DR) due constitutive overexpression of MexCD-OprJ caused by mutations in its regulatory gene, nfxB. The types of mutations obtained were similar to those previously obtained after exposure to fluoroquinolones in laboratory and clinical settings. These experiments supported the notion that triclosan and antibiotics can cause drug resistance via similar mechanisms and that a link between antiseptic, triclosan, and antibiotic resistance does indeed exist. The next step was as to prove that triclosan is also an excellent tool for selection of regulatory mutants expressing normally silent multidrug efflux pumps. By exposing a susceptible (mexAB-oprM) (mexCD-oprJ) strain to triclosan, a new RND efflux pump, MexJK, was discovered and characterized. It was found that expression of the mexJK operon is negatively governed by the product of a regulatory gene, mexL, located upstream of and transcribed divergently from the mexJK operon. The regulatory mutants obtained by tricosan exposure constitutively expressed MexJK due to an alanine to aspartate change in the putative helix-turn-helix motif of MexL. Gene fusion analysis verified the negative effect of mexL on mexJK expression, indicated that the degree of mexJK repression/derepression is dependent on the growth medium and showed that MexL autoregulates its own expression. To determine modes of regulation of mexL and mexJK expression at the molecular level, MexL was overexpressed and purified as a fusion protein containing a carboxy-terminal hexahistidine peptide tag. The purified protein showed an apparent molecular weight of 25,000 daltons. Biochemical and genetic experiments showed that MexL oligomerizes and exists in solution as a tetrameric protein. Gel mobility shift and footprinting assays demonstrated that MexL is a specific DNA binding protein and that MexL binds to both DNA strands of the 94 bp mexL-mexJ intergenic region. The protected region encompassed two inverted repeats of the GTATTT hexamer sequence, which may be recognized by MexL as part of its operator site. The mexL and mexJK promoter regions were also localized to the MexL protected region. These two promoter regions overlap and share a common -10 region. The mexL promoter was verified by mapping the mexL transcript start site by RNase protection assays and the mexJ promoter was localized using mexJ-lacZ gene fusions. In summary, the studies presented in this dissertation verified that triclosan is a substrate for most P. aeruginosa RND efflux pumps, can be used to isolate regulatory mutants expressing known and unknown RND efflux pumps, and is therefore an excellent tool for efflux pump discovery and characterization. The materials and tools developed during these studies will be useful for further studies, especially those aimed at understanding efflux pump function - e.g., pump assembly and substrate recognition - and efflux pump inhibitor discovery.