Study On Correlation Between Regulator PA0610 Of Pyocin And Trimethoprim Resistance In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a widely distributed opportunistic pathogen that causes many infections in humans,especially those,cystic fibrosis and people with burns and low immunity.Treatment of infections caused by P.aeruginosa has become increasingly difficult due to the intrinsic and acquired resistance in this pathogen against antibiotics.Hence,it is important to study the drug resistance mechanism in P.aeruginosa.Pyocin is a unique bacteriocin synthesized by P.aeruginosa.PAO1 is able to synthesize five pyocins: R2(PA0614-PA0627),F2(PA0632-PA0648),S2(PA1150),S4(PA3866)and S5(PA0985).TMP is a broad-spectrum antibiotic clinically used for treatment of P.aeruginosa infections.And it is an inhibitor of bacterial dihydrofolate reductase,TMP can interfere with the folate metabolism of bacteria,thereby inhibiting the growth of bacteria.In previous research in our lab,it was discovered that the double mutants of genes prt N(PA0610)and top A showed resistance to trimethoprim,it was confirmed that the resistance phenotype was derived from the mutation of prt N.In this study,the connection of prt N mutant and TMP resistance has been investigated.A knockout mutant PAO1(?prt N)was constructed that had an MIC to TMP about 4-fold higher than the wild type PAO1.The gene encoding hydrofolate reductase in P.aeruginosa is fol A(PA0350).We examined the expression of fol A in the mutant and compared the expression level with that in the wild type.Surprisingly the results showed that the expression level of fol A decreased in PAO(?prt N)compared with that in PAO1.To test whether intrinsic resistance is involved,the yield of biofilms and the expression of efflux pumps were measured with the mutant.The yields of biofilm was similar to those of the wild type.And the sensitivity to three antibiotics that are known to be affected by efflux pumps was also measured,and no difference was observed.Prt N is a transcriptional regulator of pyocin synthesis.Recently,it has been reported that the synthesis of R-type and F-type pyocin may affect antibiotic resistance in P.aeruginosa.Therefore,we constructed six pyocin synthesis mutants,?R2,?F2,?RF,?S2,?S4 and ?S5,tested their resistance to TMP.However,these mutants did not show any change in TMP susceptibility.As the interaction between pyocin and its binding site may also affect bacterial antibiotic resistance,we constructed a mutant PAO1(?wbp L)of P.aeruginosa.The LPS of this mutant is deficient in A-band and B-band structure,so the binding site of R-type pyocin is missing,and the results showed that PAO1(?wbp L)showed partial TMP resistance.The results reflect that the pyocin binding site may play a role in the resistance of TMP.It is possible that TMP triggers ROS outbreaks inside bacterial cells,and eventually leads to cell lysis and death.By measuring the transcription levels of ROS-related genes ahp C,kat A,and oxy R,we found that the expression of kat A in PAO(?prt N)is much higher than that of wild type.This suggests that ROS response could be the potential pathway through which the deletion of prt N leads to TMP resistance.Such information should help our understanding of antibiotic resistance in P.aeruginosa.