泛素样蛋白蛋白酶体系统对单耐异烟肼结核杆菌耐药性的影响研究

Study on the effect of ubiquitin like protein-proteasome system reacts to the drug-resistance of isoniazid monoresistant Mycobacterium tuberculosis

为探讨泛素样蛋白蛋白酶体系统对单耐异烟肼结核杆菌耐药性的影响及其机制。采用刃天青显色法,比较单耐异烟肼结核杆菌(对照组)与4种基因(Pup基因、Dop基因、PafA基因、Mpa基因)过表达的单耐异烟肼结核杆菌菌株和4种相同基因缺失突变的单耐异烟肼结核杆菌菌株异烟肼最低抑菌浓度(MIC)值的差异;比较在应用外排泵抑制剂后,单耐异烟肼结核杆菌(对照组)与4种基因(Pup基因、Dop基因、PafA基因、Mpa基因)过表达的单耐异烟肼结核杆菌菌株和4种相同基因缺失突变的单耐异烟肼结核杆菌菌株异烟肼MIC差值间的差异。结果显示:(1)与单耐异烟肼结核杆菌相比,过表达PafA基因的该菌株异烟肼的MIC值增加了1.03μg/m L,过表达Dop、Mpa基因的该菌株异烟肼的MIC值分别降低了1.03μg/m L、0.68μg/m L,差异均无统计学意义(P>0.05);与单耐异烟肼结核杆菌相比,过表达Pup基因的该菌株异烟肼的MIC值增加了8μg/m L,缺失Pup、Dop、PafA、Mpa基因的该菌株异烟肼的MIC值分别降低了4.82、4.98、4.99、4.9μg/m L,差异均有统计学意义(P<0.05)。(2)与在应用外排泵抑制剂后单耐异烟肼结核杆菌相比,过表达Pup基因的该菌株异烟肼的MIC差值增加了8μg/m L,缺失Pup、Dop、PafA、Mpa基因的该菌株异烟肼的MIC差值分别降低4.65、4.81、4.83、4.75μg/m L,差异均有统计学意义(P<0.05);与在应用外排泵抑制剂后单耐异烟肼结核杆菌相比,过表达Dop、PafA、Mpa基因的该菌株异烟肼的MIC差值分别降低了1、0.99、0.65μg/m L,差异均无统计学意义(P>0.05)。由此可知,结核杆菌泛素样蛋白蛋白酶体系统对单耐异烟肼结核杆菌耐药性的产生有调控作用,其作用机制可能是通过调控单耐异烟肼结核杆菌外排泵的功能来调控其耐药性的产生。

To explore the effect and mechanism of ubiquitin like protein-proteasome system reacts to the drug-resistance of isoniazid monoresistant Mycobacteffum tuberculosis. Using the method of resazurin chromogenic, the difference of minimum inhibitory concentration （MIC） of isoniazid were examined among the isoniazid monoresistant Mycobacteffum tuberculosis （INH-MTB, as a control group） strain, four over-expression genes（ Pup, Dop, PafA and Mpa ） INH-MTB strains and four same deleted genes （△Pup, △Dop, △PafA and △Mpa） INH-MTB strains.The difference value of MIC of isoniazid were analyzed among INH-MTB strain, four over-expression genes （ Pup, Dop , PafA and Mpa ） INH-MTB strains and four same deleted genes （△Pup, △Dop, △PafA and △Mpa） INH-MTB strains. The results showed that the MIC value of isoniazid was 1.03 μ g/mL higher in PafA over-expression INH-MTB strain than that in INH-MTB; the MIC value of isoniazid in Dop, Mpa over-expression strains were 1.03 μ g/mL and 0.68 μ g/mL lower than that in INH-MTB, respectively. The differences had no statistical significance （P〉0.05）. The MIC value of isoniazid was 8 μ g/mL higher in Pup over-expression INH-MTB strain than that in INH-MTB; the MIC value of isoniazid were 4.82ug/ml, 4.98 μg/mL, 4.99ug/ml and 4.9 μg/mL lower in △Pup, △Dop, △PafA and △Mpa INH-MTB strains than that in INH-MTB, respectively. The differences had statistical significance （P〈0.05）. After using efflux pump inhibitors, the difference Value of MIC of isoniazid in Pup over-expression INH-MTB strain was 8 μg/mL higher than that in INH-MTB; the difference Value of MIC of isoniazid were 4.65 μg/mL, 4.81 μg/mL, 4.83 μg/mL, 4.75 μg/mL lower in APup, △Dop, △PafA and △Mpa INH-MTB strains than that in INH-MTB, respectively. The differences had statistical significance （P〈0.05）. The difference value of MIC of isoniazid in Dop, PafA, Mpa over-expression INH-MTB strain were 1 μ g/mL, 0.99 μ g/mL, 0.65μ g/mL lower than that in INH-MTB, respectively. The differences had no statistical significance （1〉0.05）. It can be concluded that Mycobactenum tuberculosis ubiquitin like protein-proteasome system regulate the development of drug-resistance of INH-MTB strain and may affect its resistance by regulating the function of efflux pumps.