Transcriptome Analysis Of Pseudomonas Aeruginosa Deletion Mutant PAO1(Δ4429-31)

Pseudomonas aeruginosa(PA)is a prevalent gram-negative bacteria in nature,which can cause serious nosocomial infection and induce pulmonary cystic fibrosis(CF).Therefore,Pseudomonas aeruginosa which is belong to Pseudomonas is listed one of the world’s three major pathogenic bacteria by the World Health Organization.Previously,we not only proved experimentally that the PA4429-31 gene of Pseudomonas aeruginosa PAO1 encodes respiratory chain complex III(cytobc1),but also found that it played important roles in antibiotic resistance and pathogenicity.It resulted in a remarkable decrease in pathogenicity,and increase in the aminoglycoside antibiotic resistance.Therefore,the study want to explore the mechanism which is PA4429-31 regulating pathogenicity,aminoglycoside antibiotic resistance of PAO1,RNA-seq analysis was performed on the mutant PAO1(Δ4429-31)and wild-type PAO1.The experimental results which is statistical analysis of RNA-seq fragments and the transcription levels of alg A,fur,las A,pqs A,ton B1 in the wild-type strain PAO1 and the mutant PAO1(Δ4429-31)were illustrated that the quality of RNA-seq was credibility.RNA-seq results showed that 293 genes which was included 145 down-regulated genes and 148 up-regulated genes were significantly changed in the PAO1(Δ4429-31).The results of DEGs GO/KEGG pathway analysis were illustrated that 293 DEGs were widely enriched in the two-component system,iron ion absorption system,oxidative phosphorylation pathway,aromatic compound metabolism and carbon metabolism on the Pseudomonas aeruginosa PAO1.In order to explore what PA4429–31 deletion can regulate the pathogenicity and aminoglycoside antibiotic resistance in the PAO1(Δ4429-31)by affected the expression levels of DEGs enrichment pathways.Furthermore,heat map enrichment analysis was applied in the DEGs enrichment pathways,and the results were as follows.The result about heat map analysis of Fatty acid synthesis and respiratory pathway showed that transcription level expression of fatty acid synthesis and anaerobic respiration were increased in PAO1(Δ4429-31),and nitric oxide determination and biofilm results showed that anaerobic respiration rate increased and biofilm thickened in PAO1(Δ4429-31).Hence,this experimental results were developed the speculate which is the knockouted of PA4429-31 could made the biofilm content increase to resist the antibiotic through inducing the synthesis of fatty acids and the nitric oxide in PAO1(Δ4429-31).The results of quorum-sensing system heat map analysis and PQS system signal molecule detection showed that the expression level of QS system was decreased in the PAO1(Δ4429-31).And degradation rate of the anthranilate which is made the precursor for the PQS signal molecule synthesis was more higher than the synthesis rate at the transcription level.These results suggest that PAO1(Δ4429-31)may regulate the secretion of PQS signal molecules and pqs system expression by reducing the production of anthranilate.In order to further explore the reasons about decrease of anthranilate in PAO1(Δ4429-31),the study detected the content of iron ions and the expression of two small RNA(s RNAs),Prr F1 and Prr F2 in PAO1(Δ4429-31).The experimntal result show that the content of iron ion and the expression of Prr F1 and Prr F2 were significantly reduced.Therefore,we speculate the fact which is the the low expression of Prr F1 and Prr F2 releases the inhibitory effect on the degradation process of anthranilate,resulting in decline of production of PQS signal molecules,the expression of pqs system and quorum-sensing system in the PAO1(Δ4429-31).The quorum sensing system and the level of iron ions were directly related to PA virulence and pathogenicity.Therefore,the above results suggest that pathogenicity was decreased in the mutant PAO1(Δ4429-31)by reducing the intracellular iron content and the level of QS system.In the end,in order to explore the causes which is iron content decline in the PAO1(Δ4429-31).The study find the fact which is iron content decline has nothing to do with pyoverdine in the PAO1(Δ4429-31)through the detection of pyoverdine production.Therefore,the reason for the decrease of iron ion content in PAO1(Δ4429-31)needs explore.In addition,PA4429-31 deletion affected DNA repair,metal ion transport and homeostasis,carbon metabolism and synthesis,aromatic amino acid metabolism pathway in the PAO1(Δ4429-31)through regulating the expression level of five s RNAs which is included NC-002516-pred RNA1,NC-002516-pred RNA40,NC-002516-pred RNA274,NC-002516-pred RNA97 and NC-002516-pred RNA89.In summary,The study explored the mechanism which is PA4429-31 regulating the pathogenicity and adaptive resistance in the PAO1 through RNA-seq and experiment analyse,and provided research ideas and directions for further exploring the composition and function of respiratory chain and new methods to treat P.aeruginosa infection.