The Regulatory Mechanisms Of PA4857 Involved In Type Ⅲ Secretion System In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious acute or chronic infections in humans.Acute infections typically involve the type III secretion system(T3SS)and bacterial motility,whereas chronic infections are often associated with the type VI secretion system and biofilm formation.The ability of P.aeruginosa to avoid phagocytic clearance is a major virulence determinant that primarily depends on the T3SS.The T3SS of P.aeruginosa has been shown to contribute to epithelial cell and macrophage damage,disrupt intracellular signaling and cause cell death.P.aeruginosa uses its T3SS to produce and directly inject four virulence effectors:ExoS,ExoT,ExoU and ExoY.But so far the regulatory pathways of T3SS are unknown.In this study,we constructed a transposon mutagenesis library in a wildtype PAO1 strain containing an exoS-lux transcriptional reporter integrated into the chromosome at the vacant phage CTX attachment site and screened for mutants with altered exoS-lux expression.According to the changed lux activity,15 mutants were selected,including the known genes:exsA,pcrD,vqsM and so on,and the unknown genes such as PA5264,and PA4857.This study mainly focus on studying the function of PA4857(We designated PA4857 as tspR).Deletion of tspR affects ExoS protein levels,bacterial motility,and biofilm formation.Meanwhile,tspR mutant reduced the virulence in a mouse acute lung infection model and diminished cytotoxicity.We also found that TspR negativly regulates the two small RNAs,RsmY and RsmZ,which control RsmA expression.The expression of ExoS and the biofilm formation in the two double mutants ΔtspRΔrsmY,ΔtspRΔrsmZ restored to wild type levels.In addition,our data demonstrated that defects in T3SS expression and biofilm formation in retS mutant could be partially restored by overexpression of tspR.These results indicate that RetS positively regulate tspR,whereas the TspR regulates the T3SS through the two small RNA,RsmYand RsmZ.Taken together,our results demonstrated that the newly identified retS-tspR pathway is coordinated with the retS-gacS system,which regulates the genes associated with acute and chronic infections.This study can help us understand the complex regulatory network of T3SS in P.aeruginosa.