Characterization Of The Putative Two-component System PA2571-2572 In Pseudomonas Aeruginosa

Pseudomonas aeruginosa has a very complex regulatory network,produces a variety of secreted virulence factors,and many P.aeruginosa isolates have become multi-drug resistance in recent years,resulting in difficult clinically-icomplications.Multi-drug resistant P.aeruginosa is now one of the most dangerous pathogens.Therefore,studying the drug resistance and gene regulation mechanism in P.aeruginosa not only has important theoretical value but also carries clinical significance.PA2571-PA2572 encodes a predicted two-component response regulator in P.aeruginosa.Preliminary studies had shown that the MIC of PA2571 mutant against piperacillin is 32 times was higher than that of wild type.PA2571 was shown to play an important role in the regulation of T4 P.PA2572 and PA2571 mutants also have weaker twitching than the wild type.Therefore,based on these preliminary studies,it is speculated that T4 P may be connected with antibiotic resistance,and the two-component system PA2571-2572 might be the "bridge".In this study,a PA2571-2572 deletion mutant was constructed in this study.By measuring the expression of the T4 P associated genes in the mutant PA2571-2572,we found that the expression of pil B in the mutant was about 2 times higher than that in the wild type.No significant changes were detected in other related genes.The results of RNA-Seq study indicate that besides the up-regulation of pil B in PA2571-2572,pil A,fim V,pil T,and pil G were also significantly up-regulated while the expression of pil X was down-regulated.However,the mutant strain did not differ from PAO1 in the twitching of macroscopic movement.Although PA2571-2572 significantly regulated T4 P structural genes but did not alter T4P-mediated twitching.Disc fusion assays and MIC measurements revealed that PA2571-2572 and PA2572 were slightly more sensitive to antibiotic Cip compared to PAO1,but the susceptibility of the three strains to the other five antimicrobial agents remained almost unchanged.These results indicate that the two-component system does not have a significant regulatory effect on antibiotic resistance.The appearance of the mutants was observed by TEM,and it was found that PA2571-2572 had rougher surface.Test of biofilms and other related virulence factors revealed that the biofilm production of PA2571-2572 was significantly reduced.The main reason for the significant decrease in biofilm production was probably that the expression of las I and rhl I of QS in PA2571-2572 was significantly reduced(4 times and 2 times,respectively).RNA-Seq results indicate PA2571-2572 significantly regulated some T4 P genes.Comparison between PA2571-2572 and PAO1 identified differentially expressed genes,of which 189 genes were significantly different.These genes include not only T4 P partial structural genes and QS related genes,but also many genes that are mainly involved in the metabolism of the strains,transcriptional regulation,performance of catalytic functions,and signal transduction.These results will help us to better understand the complex molecular and intercellular activities of PA2571-2572 in P.aeruginosa,and provide a basis for future studies.