The Effect And Mechanism Of MTB PPS On The Drug-resistance Of Isoniazid Monoresistant Mycobacterium Tuberculosis

Object: To explore the effect of ubiquitin like protein-proteasome system on the drug-resistance of isoniazid monoresistant Mycobacterium tuberculosis(INH-MTB);the mechanism of its occurrence is further discussed to lay a theoretical foundation for the drug resistance of tuberculosis.Methods: Using RT-qPCR,identify INH-MTB with over-expression of one of the four genes(Pup,Dop,PafA or Mpa)strains;Using PCR,identify INH-MTB with deletion of one of the four genes(Pup,Dop,PafA or Mpa)strains.Using Resazurin microtitre assay,the difference of minimum inhibitory concentration(MIC)of isoniazid were compared between INH-MTB and INH-MTB with over-expression of one of the four genes strains and INH-MTB with deletion of one of the four genes strains.After using efflux pump inhibitors,the difference value of MIC of isoniazid were also compared between INH-MTB and INH-MTB over-expression strains and INH-MTB deletion mutant strains.After using agent for changing cell wall permeability,the difference value of MIC of isoniazid were also compared between INH-MTB and INH-MTB over-expression strains and INH-MTB deletion mutant strains.Results:(1)We successfully constructed INH-MTB with over-expression of one of the four genes strains and INH-MTB with deletion of one of the four genes strains.(2)The MIC value of isoniazid was 8 ug/ml higher in INH-MTB with Pup over-expression strain than in INH-MTB(P<0.05).The MIC value of isoniazid was 1.03 ug/ml higher in INH-MTB with PafA over-expression strain than in INH-MTB;the MIC value of isoniazid were 1.03ug/ml and 0.68 ug/ml lower in IN H-MTB with Dop,Mpa over-expression strains than in INH-MTB,respectively.The differences had no statistical significance(P>0.05).The MIC value of isoniazid were 4.82ug/ml,4.98 ug/ml,4.99ug/ml and 4.9 ug/ml lower in INH-MTB with deletion of Pup,Dop,PafA or Mpa strains than in INH-MTB,respectively.The differences had statistical significance(P<0.05).(3)After using efflux pump inhibitors,the difference value of MIC of isoniazid was 8 ug/ml higher in INH-MTB with Pup over-expression strain than in INH-MTB;the difference value of MIC of isoniazid were 4.65ug/ml,4.81ug/ml,4.83ug/ml,4.75ug/ml lower in INH-MTB with deletion of Pup,Dop,PafA or Mpa strains than in INH-MTB,respectively.The differences had statistical significance(P<0.05).After using efflux pump inhibitors,the difference value of MIC of isoniazid were 1ug/ml,0.65ug/ml lower in INH-MTB with Dop,Mpa over-expression strains than in INH-MTB and was 0.99ug/ml higher in INH-MTB with PafA over-expression strain than in INH-MTB,respectively.The differences had no statistical significance(P>0.05).(4)After using agent for changing cell wall permeability,the difference value of MIC of isoniazid was 7.78 ug/ml higher in INH-MTB with Pup over-expression strain than in INH-MTB;the difference value of MIC of isoniazid were 4.58ug/ml,4.73ug/ml,4.75ug/ml,4.68ug/ml lower in INH-MTB with deletion of Pup,Dop,PafA or Mpa strains than in INH-MTB,respectively.The differences had statistical significance(P<0.05).After using agent for changing cell wall permeability,the difference value of MIC of isoniazid were 1ug/ml,0.7ug/ml lower in INH-MTB with Dop,Mpa over-expression strains than in INH-MTB and was 0.97ug/ml higher in INH-MTB with PafA over-expression strain than in INH-MTB,respectively.The differences had no statistical significance(P>0.05).Conclusion: Ubiquitin like protein-proteasome system of Mycobacterium tuberculosis regulates the development of drug-resistance of INH-MTB strain and may affect its resistance to isoniazid by regulating the function of efflux pumps or the permeability of cell wall.