Genetic Analysis Of The Genes Involved In The Regulation Of Type Ⅲ Secretion System In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium capable of causing serious and often life-threatening infections in cystic fibrosis patients and immunocompromised humans.The bacterium is notorious for its ability to resistant to many conventional antibiotics,and drug treatment has become increasingly difficult to control its infection.The Type Ⅲ Secretion System(T3SS)of P.aeruginosa is one of the major virulence determinant associated with the bacterial acute infection.T3 SS is similar to a needle-like structure consisting of four separate but synergistic protein complexes that including secretion apparatus,translocation apparatus,effector proteins and cognate chaperones.At present,the specific regulatory network of T3 SS is not fully understood,but a range of regulators are known to be associated with T3 SS regulation.Internal adjustment factor such as two-component system,Crc,Vfr,RNA helicase Dea D and Fis,which regulate T3 SS by influencing the expression or function of the T3 SS master regulator Exs A.Expression of P.aeruginosa T3 SS is also influenced by various environmental conditions,such as calcium depletion and direct contact with host cells or serum.Other regulators also known to affect the expression of T3 SS genes are small chemical molecules,such as quorum sensing signals(for example,PQS),acetyl-Co A,histidine,spermidine,etc.Therefore,genetic analyses of the genes involved in the regulation of T3 SS and related regulatory networks in P.aeruginosa hold a great significance in basic research and practical applications.This study focuses on identification and characterization of the genes related to T3 SS regulation.The main findings are as follows:Guided by the results of previous study,I knocked out 4 genes,including PA2768,PA3598,PA3218,and PA0317,which are potentially associated with T3 SS regulation,and generated corresponding deletion mutants in the wild-type strain PAO1 and T3 SS report strain PCZ.In order to study whether these genes could affect the production of those quorum sensing-dependent virulence factors,such as growth curve,elastase,pyocyanin,motility,and biofilm,P.aeruginosa wild type strain and the constructed mutants were used for phenotype analyses.My data showed that deletion of PA2768,PA3598,PA3218,and PA0317,respectively,did not affect the growth,and did not affect the production of elastase,biofilm and pyocyanin,as well bacterial motility,compared with the wild type strain.In order to study whether the above four genes have a regulatory effect on T3 SS,I conducted the the β-galactosidase analysis using the T3 SS reporter strain PCZ and its deletion mutants.The results showed that the β-galactosidase activity the PA2768 knocked out mutant was decreased by 50% compared with its wild type PCZ.The β-galactosidase activity of the PA2768 mutant was fully restored by in trans expression of the wild type PA2768.To further validate the above findings,the expression levels of the T3 SS major regulator gene exs A and its effector genes exo S,exo Y and exo T were studied at the transcriptional level using wildtype strain PAO1,its PA2768 deletion mutant and complemented strain.The results showed that deletion of PA2768 led to significant reduction in expression of exs A,exo S,exo Y and exo T.After complementation with the wild type gene PA2768,the expression level of exs A,exo S,exo Y and exo T were returned to wild-type level.At the translational level,deletion of PA2768 also caused a significant decrease in Exs A expression.The cytotoxicity experiment showed the PA2768 mutant was attenuated in virulence to the host cells compared with wild type strain PAO1.These results established that the regulatory role of PA2768,which encodes a hypothetical membrane protein,on P.aeruginosa T3 SS,and shed a new light on understanding the complicated T3 SS regulatory systems and for future development of practical treatment against P.aeruginosa infections.