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翟小倩, 鲍朗, 罗涛等. 结核杆菌CRISPR-associated Csm4(Rv2820c)诱导iNOS表达对耻垢杆菌胞内存活的影响[J]. 四川大学学报(医学版), 2018, 49(3): 319-324.
引用本文: 翟小倩, 鲍朗, 罗涛等. 结核杆菌CRISPR-associated Csm4(Rv2820c)诱导iNOS表达对耻垢杆菌胞内存活的影响[J]. 四川大学学报(医学版), 2018, 49(3): 319-324.
ZHAI Xiao-qian, BAO Lang, LUO Tao. et al. Effect of the Expression of iNOS Induced by Mycobacterium tuberculosis CRISPR-associated Csm4(Rv2820c) on Intracellular Viability of Mycobacterium smegmatis[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(3): 319-324.
Citation: ZHAI Xiao-qian, BAO Lang, LUO Tao. et al. Effect of the Expression of iNOS Induced by Mycobacterium tuberculosis CRISPR-associated Csm4(Rv2820c) on Intracellular Viability of Mycobacterium smegmatis[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(3): 319-324.

结核杆菌CRISPR-associated Csm4(Rv2820c)诱导iNOS表达对耻垢杆菌胞内存活的影响

Effect of the Expression of iNOS Induced by Mycobacterium tuberculosis CRISPR-associated Csm4(Rv2820c) on Intracellular Viability of Mycobacterium smegmatis

  • 摘要: 目的 研究Csm4蛋白在重组耻垢分枝杆菌(Mycobacterium smegmatis, MS)中的表达及其影响胞内存活的机制。方法 PCR扩增Csm4基因,构建重组pMV261-Csm4穿梭表达质粒,将重组质粒和空白质粒电穿孔进MS生成重组菌MS_Csm4和对照重组菌MS_V,采用Western blot对重组菌Csm4蛋白表达进行检测。体外培养观察MS_Csm4和MS_V生长曲线,并检测活性氮、活性氧环境下的集落形成单位(CFU)变化。MS_Csm4和MS_V重组菌感染人源巨噬细胞THP-1,计数CFU反映胞内存活,实时荧光定量PCR检测诱导性一氧化氮合酶基因(inducible nitric oxide synthase, iNOS) 表达。硝酸还原酶法检测一氧化氮(nitric oxide, NO)释放指标。结果 Csm4蛋白在MS_Csm4中成功表达,且其表达不影响MS_Csm4的生长情况;MS_Csm4在体外活性氮、氧环境中CFU下降,与MS_V相比,差异有统计学意义(PCsm4作用于THP-1细胞后诱导iNOS表达上调,促进NO的释放以及减少胞内生存,与MS_V相比,差异有统计学意义(PCsm4不能耐受体外活性氮、氧压力环境,可诱导宿主细胞iNOS表达上调,促进NO释放,从而影响其胞内生存能力。

     

    Abstract: Objective To determine how Csm4 protein expression affects intracellular survival of Mycobacterium smegmatis(MS). Methods Csm 4 gene was amplified by PCR to construct pMV261-Csm4 shuttle expression plasmid. The Csm4 protein expression in MS_Csm4 was detected by Western blot after electroporation of the recombinant plasmid into MS. The growth kinetics of MS_Csm4 in vitro and the influence of reactive N, O species on the growth of MS_Csm4were observed. The intracellular survival of MS_Csm4 and expressions of inducible nitric oxide synthase gene (iNOS) and nitric oxide production (NO) were detected after infection with THP-1 macrophages. Results Csm4 protein was successfully expressed in MS_Csm4, which did not affect the growth of the recombinant MS. Reactive N, O species decreased MS_Csm4 colony forming unit (CFU) in vitro. THP-1 increased the expression of iNOS and NO production and decreased intracellular survival of MS_Csm4. Conclusion Recombinant MS_Csm4 is susceptible to reactive N, O species in vitro. THP-1 promotes NO release and thus discourages intracellular survival of MS.

     

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