Regulatory Mechanism Of Pseudomonas Aeruginosa Cold Shock Protein CspC On The Expression Of Type Ⅲ Secretory System And Quorum Sensing System

Pseudomonas aeruginosa is a widely distributed human opportunistic pathogen,which can adapt to a variety of environments,including the host in vivo environment.The ability to effectively sense and fine tune global gene expression in respond to the host environment is critical for the virulence of pathogenic bacteria.The host temperature is exploited by the bacteria as a cue for triggering virulence gene expression and enhancing the infection process.During infection,bacteria need to efficiently aquire and utilize the nutrients from the host to achieve long-term colonization.However,little is known about the response mechanisms of P.aeruginosa to host temperature and nutrients in vivo.The cold shock protein family is a group of conserved RNA binding proteins that regulate gene expression in bacteria.The cold shock proteins of P.aeruginosa and their functions remain largely unexplored.In this study,we first identified and explored the functions of P.aeruginosa cold shock proteins,and found that Csp C(PA0456)controls the bacterial virulence.Combining transcriptomic analyses,RNA-immunoprecipitation and high-throughput sequencing(RIP-Seq),we demonstrated that Csp C represses the type Ⅲ secretion system(T3SS)by binding to the 5’ untranslated region of the m RNA of exs A,which encodes the T3 SS master regulatory protein.In addition,the acetylation modification of cold shock protein was confirmed for the first time and we further demonstrated that acetylation at K41 of the Csp C reduces its affinity to nucleic acids.Shifting the culture temperature from 25 ℃ to 37 ℃ or infection of mouse lung increases the Csp C acetylation,which derepresses the expression of the T3 SS genes,resulting in elevated virulence.In addition,we found that Csp C regulates the quorum sensing(QS)systems by repressing the translation of a QS negative regulatory gene rsa L.Through RNA immunoprecipitation coupled with real time quantitative reverse transcription PCR(RIP-q RT-PCR)and electrophoretic mobility shift assays(EMSAs),we found that Csp C binds to the 5’ untranslated region of the rsa L m RNA to achieve its function.Acetylation modification also affects this regulation process.Comparing to glucose,itaconate(a metabolite generated by macrophages during infection)reduces the acetylation of Csp C,which increases the affinity between Csp C and the rsa L m RNA,leading to upregulation of the QS systems.Overall,this study identifies the regulatory targets of Csp C and reveal a regulatory mechanism of the T3 SS in response to temperature shift and host in vivo environment and a novel regulatory mechanism of the QS systems in response to a host generated metabolite.