ABSTRACT
Objective To investigate the role and the mechanism of ppk1 gene (coding for polyphosphate kinase 1) in oxidative stress resistance in uropathogenic Escherichia coli (UPEC).MethodsMutant strains with ppk1-deletion (△pk1) and complemented strains (△pk1-C) were constructed based on the UPEC strain CFT073.A comparative analysis was conducted to analyze survival rates of CFT073, △pk1 and △pk1-C strains at different time points while they were under oxidative stress.Differences in protein expression between CFT073 and △pk1 strains were analyzed using mass spectrometric analysis.Differences between CFT073 and △pk1 strains in expression of katG and katE genes were analyzed using real-time quantitative RT-PCR.Results The survival rate of △pk1 strains was lower than that of CFT073 strains at every time point, while the survival rate of △pk1-C strains was basically the same as that of CFT073 strains.Gel image analysis and mass spectrometric analysis revealed that six proteins were down-regulated and one was up-regulated in △pk1 strains as compared with those in CFT073 strains.Expression of the catalase-coding genes katG and katE in △pk1 strains were respectively (20.5±8.2)% and (20.9±6.9)% of those in CFT073 strains (P<0.05).Conclusion The ppk1 gene plays an important role in oxidative stress resistance in UPEC by modulating the expression of catalase-coding genes katG and katE.
ABSTRACT
Objective To construct a Polyphosphate kinase 1 ( ppk1) gene deletion mutant of uro-pathogenic Escherichia coli (E.coli) CFT073, and to explore the biological properties of the mutant strain . Methods The ppk1 gene deletion strain (△pk1) was constructed based on CFT073 E.coli strain by usingλRed homologous recombination technology .A comparison analysis was conducted on adhesive and invasive abilities between CFT073 wild type strain and △pk1 strain in in vitro model of human bladder cancer epithe-lial cell 5637 .Crystal violet staining method was used to evaluate the influences of ppk1 gene deletion on biofilm formation.Results The CFT073 ppk1 deletion mutant strain was successfully constructed .Com-pared with the wild type strain ,△pk1 strain showed impaired adhesive and invasive abilities to 5637 cells. Moreover , the absorbance values of crystal violet at 570 nm at each time point of the mutant strain group were also lower than those of the wild-type strain group .Conclusion The ppk1 gene deletion mutant of uro-pathogenic E.coli CFT073 could be successfully constructed by Red homologous recombination technology and its biological properties indicates that ppk1 gene plays an important role in the pathogenesis of uropatho-genic E.coli infection through regulating the abilities of adhesion , invasion and biofilm formation .