Association Between Pnc A Gene Mutations, Pyrazinamidase Activity, And Pyrazinamide Susceptibility In Mycobacterium Tuberculosis

Objective: This study analyzes the effect of different mutations of pnc A gene on PZase activity.Method: 142 clinical isolates of Mycobacterium tuberculosis were tested the susceptibility of PZA with BACTECTM MGITTM 960 PZA Kit(Becton, Dickinson and Company)and sequenced of the pnc A gene.We selected wild-type pnc A gene and mutations on active site or mutations in PZA sensitive strains, cloning, expression,purification protein and measured its activity.Results: Among 142 clinical isolates 57.7%(82/142) of MDR strains were resistant to PZA while another 71.1%(32/45) of extensively drug resistant(XDR) strains and 63.4%(26/41) of pre-XDR strains were also resistant to PZA.XDR and pre-XDR strains had higher PZA resistance rate than that of MDR strains which were still sensitive to fluoroquinolone and aminoglycoside(42.9%, 24/56)(c2=8.922, P=0.012).When including the promoter region, totally 85.4%(70/82) PZA resistant MDR strains had pnc A mutations. The value of sensitivity,specificity,positive predict value(PPV) and negative predict value(NPV) was 85.4%,91.7%,93.3% and 82.1%.Among 9 recombinant PZAses,the activity of wild-type pnc A gene protein was 37.2μmol·min-1·mg-1,two mutants(His51Pro 和 His51Gln)showed no enzyme activity,four mutants(Ala146Thr、His57Asp、Thr76Pro 、His71Tyr)showed low activity comparable to the wild-type protein,and one(78deletion GCTGGC)was insoluble.Conclusion: Mutations of pnc A gene had good correlation with Pyrazinamide resistsnce and pnc A mutations caused the level of protein activity reduced or lost.