The Molecular Regulation Mechanism Of The Two-component Regulating System RrhRS

Pseudomonas aeruginosa is a kind of Gram-negative bacteria, which is estimated that about10%of hospital infection. The innate resistance to drug of Pseudomonas aeruginosa is very strong. The acquired infection rates and resistance rates of the pathogen in hospital and community are increasing year by year, which has attracted great attention of governments and infection control experts.Two-components signaling system is a bacterial signal transduction system which responds to the stimulation of the external environment. A typical two-component system is composited of histidine kinase and response regulator. When the external signal is transited to extracellular ligand binding domain of the histidine kinase, histidine autophosphorylation occurs, which triggered the transferation of phosphate group to the regulon of reaction, thereby generating a series of regulation. Hitherto,64genes encoding two-component system sensor kinase. Most of the known functions of the two-component system in Pseudomonas aeruginosa are closely related to the resistance and pathogenicity. RrhS (Regulation of rhamnolipid Sensor) is a sensor kinase of two-component system, and RrhR (Regulation of rhamnolipid Regulator) is another corresponding response regulator protein in the Pseudomonas aeruginosa MPAO1. Two-component system RrhR/S of Pseudomonas aeruginosa has been reported to play an very important role in biofilm formation, development and other aspects.In previous work, we found that Pseudomonas aeruginosa lost the ability of secretion the key virulence factors rhamnolipid and pyocyanin, when deletion of rrhS gene. Furthermore, rrhS gene deletion mutant has reduced pathogenicity. In this paper, we intend to study the mechanism of RrhS regulation rhamnolipid, pyocyaninin in Pseudomonas aeruginosa.First, rrhR-luciferase reporter assay (rrhR-lvx) revealed that rrhR gene was highly expressed in rrhS gene deletion mutant. Furthermore, Western-blot verified the high enrichment of RrhR protein in rrhS gene deletion mutant. Through electrophoretic mobility shift assay and DNase I footprinting comfirmed that RrhR could specifically bind its own promoter. However, reporter gene showed that expression of rrhS in rrhRS double gene deletion mutant was similar to that of wild-type. These results indicate that the mutation of rrhS result in loss of inhibition for the expression of the rrhR, which is nevertheless self-regulating.Expression profiles of rrhS gene deletion mutant and wild-type were then analysed by Microarray,which revealed that the expression profiles found the expression of genes encoding rhamnolipid synthase-related protein rhlA, rhlB, and rhlC were significantly reduced in rrhS mutant. Subsequently, RrhR was determined to specifically bind the promoter region of the rhlR using electrophoretic mobility and DNaseI Footprinting assays. Furthermore, the of expression of the rhlR was suppressed in the mutant. Using report strain of N-butanoyl-1-homoserine lactone (C4-HSL), we found that the amount of C4-HSL in the wild-type is10folds to that of rrhS mutant. These results suggest that two-component system RrhRS regulates the production of rhamnolipid, and gene deletion mutant. These results suggest that two-component system RrhRS regulates the production of rhamnolipid and pyocyanin generation through RhlR-RhlI quorum sensing system.Additionaly, we found that RrhR binding the promoter region of the genes exsA, a key regulatory of type Ⅲ secretion system. Futher analysis, showed that the expression of exsA gene and its downstream genes exoT and exoS was activated in rrhS deletion mutants, suggesting that rrhS negatively regulates the expression of the genes in type Ⅲ secretion pathway. These results indicate that the rrhS gene negatively regulates the expression of the type Ⅲ secretion pathway genes.In addition, through a series of Footprinting analysis, we found that the DNA binding sites of the RrhR contain a conserved sequence GATACA.This study reveals two previously unexplored signal transduction pathways, RrhS/RrhR/RhlR and RrhS/RrhR/ExsA, for the expression of multiple virulence determinants in P. aeruginosa and may inform theoretical basis and strategies designed to limit infections caused by P. aeruginosa.