Interdependence of Multi-Drug Efflux Pumps and Quorum Sensing Systems in Pseudomonas Aeruginosa

Background: Multi-drug resistance (MDR) efflux pumps belonging to the nodulation cell division (RND) family in Pseudomonas aeruginosa are able to extrude out a wide variety of drugs and antibiotics. These complexes of inner-membrane, periplasm, and outer-membrane protein components are not merely molecular machines that pump out drugs. The very drugs that are pumped out by these efflux pumps can also induce expression of the efflux genes/proteins. In addition, the MDR efflux pumps transport out quorum-sensing molecules, which can induce expression of several genes including those related to biofilm formation, virulence, and efflux, thereby conferring additional resistance. Therefore, a complete understanding of the function of the MDR efflux pumps requires measurement and modeling of three processes: (a) how a given drug is extruded through these pumps; (b) how the same drug induces the expression of these pumps; and (c) how the release of quorum-sensing molecules cause the expression of biofilm, virulence, and efflux genes. Methods & Materials: We measured and modeled the survival kinetics and intra- and extra-cellular concentration of ciprofloxacin in the wild-type P. aeruginosa strain PAO1 and the mutants deficient in efflux and quorum sensing. We also measured the formation of biofilm, antibiotic-induced expression of virulence, and efflux genes by real-time qPCR (Fluidigm) and modeled how the expression of these genes confers additional drug resistance via biofilm formation and enhanced virulence. Results: We showed that (i) the P. aeruginosa tripartite MexA-MexB-OprM protein complex plays an important role in the efflux of ciprofloxacin and in the induction of the quorum-sensing pathways (ii) a sharp increase in the efflux within 5–10 minutes after the treatment of ciprofloxacin after which the efflux levels off (iii) the quorum sensing mediated biofilm, virulence, and efflux genes are expressed at higher level by mature biofilm than the nascent one providing additional resistance. Conclusion: Combination of experimental and modeling studies provides an insight into how MDR efflux and quorum sensing systems work together to confer drug resistance. A similar approach can be extended to understand the structural, genetic, and cellular processes underlying the function of MDR efflux pumps.