Transcriptional Regulation Mechanism Of The Type Ⅲ Secretion System In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a human pathogen that causes serious and often life-threatening infections. Type Ⅲ secretion system (T3SS) is an important virulence factor in P. aeruginosa that is extremely important for its successful establishment of infection and evasion of phagocytosis. It is essential to understand the pathways and controlling mechanisms of the T3SS. T3SS also provides a novel target for the development of new therapeutic strategies against P. aeruginosa infections. Although dozens of genes have been identified in the past10years that are implicated in regulation of the T3SS in P. aeruginosa, the exact molecular basis of the central regulatory pathways is not completely clear. Important regulators of the T3SS are still to be discovered.In order to obtain a complete picture of the T3SS regulatory pathways, a P. aeruginosa transposon insertion library of about20000clones was constructed and screened for genes involved in T3SS regulation using the T3SS effector gene exoS as a reporter. A strain carrying the exoS-luxCDABE reporter on the chromosome was constructed and used in the screening, and transposon mutants with altered transcription of exoS were selected by the changed light production. The sites of transposon insertion in the selected mutants were determined by semi-arbitrary primed PCR and subsequent sequencing of the PCR products. Twenty four genes that affected exoS expression were identified. Among these genes, four genes were found to repress the expression of exoS and twenty genes were required for the activity of exoS. Most of these identified genes have not been reported to have a role in the regulation of T3SS although several known regulatory genes such as exsA and exsD were identified. Some of these genes including PA4595and PA1611were selected for further study and the specific functions of these genes in the regulation of T3SS were characterized.Several isolated mutants showing elevated exoS expression were found to have the gene PA4595disrupted by the transposon. In addition of exoS, the expression of other T3SS genes including exoY, exoT, exsCEBA and exsD-pscB-L was also increased significantly in the PA4595knockout mutant, and the influence of PA4595on T3SS transcription was confirmed by the levels of secreted T3SS effectors. Furthermore, this mutant exhibited increased swarming motility, increased pyocyanin production and decreased biofilm formation. Complementation by inserting an intact copy of the gene at the attB site on the chromosome restored these phenotypes to the wild-type levels. PA4595encodes a probable soluble ATP binding cassette (ABC) ATPase and has86%similar to putative transport system protein YjjK in Escherichia coli which belongs to the REG subfamily in the phylogenetic classification of ABC ATPases. Our study indicated that PA4595may not be involved in transport process. The highly conserved aspartate residues in the Walker motif B were essential for the function of PA4595. We purified the recombinant GST-PA4595protein and demonstrated that this protein displayed ATPase activity. Gel mobility shift assay indicated that the recombinant GST-PA4595protein could bind to DNA or RNA with no sequence specificity. These results add a novel player in the T3SS expression in P. aeruginosa and provide a new functional clue to the REG subfamily ATPases.The opportunistic pathogen P. aeruginosa causes a wide range of acute and chronic infections. Several two-component system (TCS) components (including RetS, LadS, and GacS/GacA) are proposed to be involved in the switch between acute and chronic infections. In this study we identified a hybrid sensor kinase PA1611that is involved in controlling the expression of genes associated with acute and chronic infections in P. aeruginosa PAO1. Overexpression of PA1611inhibited the type Ⅲ secretion, cytotoxicity and swarming motility, and promoted biofilm formation. In addition, PA1611controlled the expression of two small RNAs, rsmY and rsmZ. Its effect was suppressed by mutation in gacA but not influenced by deletion of PA3346, a component of HptB signalling pathway. These phenomena were similar to those of retS mutant. Further study indicated that the conserved phosphorelay residues were not essential for the function of PA1611. Using a bacterial two-hybrid assay, we found that PA1611could interact with RetS. In addition, as PA1611was expressed in low levels in the wild-type PAO1under general conditions, we carried out another round of whole genome mutagenesis to identify genes that control PA1611expression. We found that mutation of PA1775caused dramatical activation of PA1611expression. PA1775is predicted to encode a conserved cytoplasmic membrane protein which has the characteristic of the mechanosensitive ion channel. According to these results, we speculate that PA1775monitors some kinds of external stimuli and converts these stimuli into signals which in turn control the expression of PA1611. These results indicate that when the expression of PA1611is actitived, it can affect acute and chronic infection through direct interaction with RetS instead of via the phosphorelay pathways. A new interaction mechanism for hybrid sensor kinases in P. aeruginosa is proposed.