The Functions And Mechanisms Of Pseudomonas Aeruginosa Two-component Regulatory System NioS/NioR In Skin Infection

Pseudomonas aeruginosa?P.aeruginosa?is a clinically significant opportunistic pathogen that causes a wide variety of infections.Most infections with this organism occur in compromised hosts,including disrupted skin barriers and dysfunctional immune mechanisms.Two-component regulatory system?TCS?is the dominant signal transduction pathway for prokaryotic cells,and has an important role in the process of bacteria to adapt to different environments.However,the molecular mechanism involved in P.aeruginosa skin infection is less clear.In this study,Tn-seq was used to screen the potential pathogenic TCSs of P.aeruginosa PA14 which may play important roles during skin infection.The sequencing reads of 684 genes of PA14 were significantly decreased in skin infection after 1-,3-and 5-day post infection compared with LB-growth input,including 17 TCS genes.Among these TCSs,PA14₃2570/PA14₃2580?named NioS/NioR?with the most profound change were chose to further investigate their functions during skin infection.Firstly,all NioS/NioR deletions and their complement strains were constructed.All mutant strains showed decreased survival ability after 3-day post infection in skin,while the complement strains restored the surivival ability.These results verfied the Tnseq results and showed that NioS/NioR play important roles during skin infection.Next,we investigated whether NioS/NioR could regulate the biofilm formation of P.aeruginosa PA14,which usually has been regarded as an important characteristic of virulence.The results showed that the deletion of NioS/NioR genes decreased the expression of genes related to flagellum synthesis,thus decreasing the swarming motility and biofilm formation of P.aeruginosa PA14.Since phagocytic clearance by neutrophils and macrophages is important to endogenous control of P.aeruginosa infection,we next investigated whether NioS/NioR TCS would impair phagocytic function of macrophages during skin infection.The results demonstrated that NioS/NioR TCS increased phagocytic resistance via upregulating the expression of fliC and algD genes which also could increase biofilm formation.To further identify genes regulated by NioS/NioR TCS,RNA-seq analysis was performed and showed that NioS/NioR TCS regulated multiple bacterial systems including denitrification pathway,pyochelin,porin and protease.Further investigations proved NioS/NioR promote the degradation of nitric oxide via regulating the mRNA expression of nitric oxide reductase systhetic genes norB and norC to against the macrophages' killing.Taken together,our data demonstrates that the NioS/NioR TCS can regulate the expression of various virulence factors of P.aeruginosa PA14 to increase pathogenicity during skin infection.These findings provide new insights into the pathogenic mechanism of P.aeruginosa in skin infection,and provide a potential therapeutic target for treatment of skin infection by P.aeruginosa.