Coordinated Regulation Of Anthranilate Metabolism And Quorum-sensing By MpaR In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a common opportunistic pathogen that often infects immunocompromised patients and causes a variety of diseases.Quorum sensing(QS)systems in P.aeruginosa mainly include the Las,Rhl and PQS system,which control the expression of many virulence factors and play an important role in bacterial pathogenicity and resistance.As an important metabolic intermediate in P.aeruginosa,anthranilate is intricately intertwined with QS systems.The Gnt R family regulators are widely distributed in bacteria and play critical roles in metabolic processes and bacterial pathogenicity.In this study,we explored the function of the Gnt R family regulator MpaR encoded by PA4132 in P.aeruginosa.Firstly we constructed the mpaR deletion mutant strain and its complementing strain.By testing the related virulence phenotypes,we found that MpaR can inhibit the formation of biofilm and promote the production of pyocyanin,but MpaR did not affect the motility of P.aeruginosa.Results of RNA sequencing analysis and q RT-PCR revealed that the levels of genes related to the anthranilate metabolic pathway and QS system were affected by mpaR deletion.It is known that the enzyme encoded by ant ABC can catalyze the conversion of anthranilate to catechol.We found that MpaR indirectly inhibited the expression of the ant operon at the level of transcription and translation.It was also proved by high-performance liquid chromatography(HPLC)that MpaR inhibited the production of catechol through Ant R.For the QS system,using the lux-based reporter driven by promoter to find the regulation pathway,and using electrophoretic mobility shift assay(EMSA)to detect whether DNA and protein interact directly,we found that MpaR directly activated Mvf R,a regulator of the PQS system,and then activited the PQS system and promoted the production of PQS.MpaR also inhibited Rhl R,a regulator of the Rhl system,and both Mvf R and Rhl R can regulate the expression of ant ABC.All results showed that the function of MpaR is integrated into the regulatory network of anthranilate metabolism and QS system.Moreover,MpaR significantly enhanced the pathogenicity of P.aeruginosa in a mouse model of acute infection.In summary,this study explored the function of the Gnt R family regulator MpaR in P.aeruginosa,which is involved in the regulation of anthranilate metabolism,quorum sensing system and bacterial virulence.