The Activation Of PhzA2 Of Subinhibitory Concentrations Of Spectinomycin And The Regulatory Function Of Gene PA3114 In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is one of the major nosocomial pathogens causing a series of infections ranging from mild skin infection to organ failure and death. The pathogenicity of P. aeruginosa relies on the variety of pathogenic factors it possesses and the complex regulatory network that controls virulence factors in response to host environment.Phenazine compound pyocyanin is one of the secondary metabolites of P. aeruginosa, whichserves as an antibiotic inhibiting the growth of other bacteria. Pyocyanin is also a virulence factor and can act as a signaling molecule to regulate gene. The synthesis of phenazine compounds in P. aeruginosa is mainly catalyzed by the products encoded by the two gene clusters phzl (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2). Previously, studies in our lab revealed that subinhibitory concentrations of spectinomycin activated the expression of phzA2, and obtained a PA3114 transponson insertion mutant in which the activation effect of spectinomycin on phzA2 was significantly reduced. In this study, the regulatory function of the gene PA3114 has been investigated, and the relationship between PA3114 and the phzA2 gene cluster examined. The potential regulatory pathways through which PA31114 functions have been explored together.A PA3114 knockout mutant PAO1 (?PA3114) was first constructed. The results show that the expression of phenazine synthesis gene clusters phzA1, phzA2 and phzS decreased significantly in mutation PAO1(?PA3114) compared with the wild type PAO1. In agreement with the decreased expression, pyocyanin production was decreased significantly in the mutant. Since phenazine production is under the control of the PQS quorum sensing system in P. aeruginosa, we compared the expression of gene pqsA,pqsR and pqsH bet ween the mutant and the wild type. The results obtained indicate the expression the PQS genes was significantly lower in the mutant, and so is the production of PQS signal molecules. These results suggest that the effect of PA3114 on phenazine synthesis may be through the PQS pathway.Further study found, in addition to phenazine production, PA3114 also affected the expression of genes in the type three secretion system, i.e. exoT, exoS, exoY. Twitching motility of PAO1 (APA3114) mutant strain was also abolished, while the mutant showed enhanced swarming ability compared with PAO1. Electron microscopy showed the flagella number did not change but the number of fimbriae of PAO1(APA3114) strain appeared to be reduced. In search for the potential mechanism, it was found that the expression of rsmA, a global regulator was reduced in the mutant while the expression of rsm Y, rsmZ was increased significantly. These findings may explain the changes in the mutant and suggest PA3114 is a potential regulator of the Rsm system.Further study found that the activation effect of spectinomycin on phzA2 was stronger under higher concentrations of spectinomycin. While the MIC of spectinomycin for the mutant was not different from that of the wild type PAO1, the activation effect by spectinomycin was decreased significantly in the mutant strain. In addition, several genes in Pseudomonas aeruginosa such as phzS, phzA1, and pilG etc were activated by subinhibitory concentration of spectinomycin. Clearly, the mechanism of the spectinomycin activation effect on phzA2 requires further study.