Characterization Of The Regulation Of RND Pump MexGHI-OpmD By Pyocyanin In Pseudomonas Aeruginosa

Pseudomonas aeruginosa(PA) is a Gram-negative pathogen capable of causing serious diseases which can cause acute or chronic infections in hospitals. It is the major cause of mortality in patients with cystic fibrosis. PA can produce many virulence factors which enable the pathogen to cause extensive tissue infections and blood invasion.Phenazine compounds are secondary metabolites which are involved in PA pathogenecity. They also can act as signal molecule regulating the expression of many genes. Previous work in our laboratory has found that pyocyanin, one of the phenazine compounds produced by PA, can activate the expression of the RND pump gene cluster mexGHI-opmD. the mexGHI-opmD RND pump is involved in resistance to several antibiotics such as Tetracycline, Ticarcillin and Clavulanate Potassium netilmicin. However, the molecular mechanism of the mexGHI-opmD regulation by pyocyanin remains unknown.In the present study, we aim to characterize the regulation pathways that are involved in the regulation of mexGHI-opmD by PYO. Transposon mutagenesis was carried out to screen for mutants that have altered regulation of mexGHI-opmD by PYO. After three rounds of screening,17transposon mutans were selected. By locating the transposon element on the chromosome through semi-arbitrary PCR and the sequencing analysis, we identified the genes disrupted by transposon insertion. In order to characterize the function and effect of the mutated genes, we tested of the phenotypes of the transposon mutants, including motility, antibiotic susceptibility, and lipopolysaccharide production. The mutant No.37was then investigated further. It was found that the transposon inserted into gene PA2621which firms an operon with PA2620. Gene complementation indicates that both PA2621and PA2620affected mexGHI-opmD expression regardless the presence or absence of PYO. Comparing with the wide type PAO1, PA2621and PA2620deletion mutants showed increased pyocyanin production, decreased susceptibility to antibiotic Carbenicillin and increased susceptibility to Rifampin. No obvious change was observed with motility, colony morphology, and protease production in the mutants.Phenazine synthesis is strictly controlled by the quorum-sensing (QS) system in P. aeruginosa and on the other hand, phenazine pyocyanin is the terminal signal molecule of the homoserine lactone based quorum sensing system. Therefore, we tested the effect of PA2621and PA2620on quorum sensing system. Both gene affected the QS system and T3SS(Type â…¢ secretion system). Interestingly, the expression of genes pqsA, lasR, rhlR, exoS and exoY was increased in mutant PA2620but the expression of pqsH, pqsR, rhlI and exoT was depressed.Taken together, these results indicate that PA2621and PA2620play important roles in antibiotic resistance, QS system and T3SS. They are probably components of the regulation pathways through which phenazine (pyocyanin) activates the RND pump mexGHI-opmD. These results provide a starting point for revealing the molecular mechanism underlying phenazine regulation in P. aeruginosa.