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【目的】结核分枝杆菌可以在宿主细胞内的酸性环境中长期存活。为了探明天冬酰胺酶(AnsA)代谢通道介导的分枝杆菌在酸性环境中的适应机制,分别通过体内和体外实验,对AnsA的活性、突变体性质进行了分析。【方法】以无致病性的卡介苗分枝杆菌(M.bovis BCG)为模式菌,扩增天冬酰胺酶编码基因ansA并在大肠杆菌中进行表达和纯化,对AnsA的酶学活性进行了分析。在卡介苗分枝杆菌中将ansA敲除,对其抗酸、产氨等特性进行了研究。【结果】纯化的重组AnsA在体外可以将天冬酰胺分解并产生氨。在酸性培养基中,分枝杆菌利用天冬酰胺产生的氨,可以释放到培养基中,将酸性培养基中和。敲除ansA后,卡介苗分枝杆菌在酸性环境中的生长滞后10天左右。为了更好的理解天冬酰胺介导的抗酸机制,绘制了天冬酰胺代谢、氨产生和转运示意图。【结论】结核分枝杆菌的抗酸特性之一是通过分泌氨将周围的酸性环境中和来实现的。氨来源于分枝杆菌AnsA对底物天冬酰胺的分解。这为揭示结核分枝杆菌在宿主巨噬细胞内酸性环境中的生存机制提供了线索。
【Objective】 Mycobacterium tuberculosis can survive long-term in the acidic environment of host cells. In order to investigate the adaptive mechanism of mycobacterial metabolic pathway-mediated Mycobacterium tuberculosis in acidic environment, AnsA activity and the nature of mutants were analyzed by in vitro and in vivo experiments respectively. 【Method】 Asparaginase-encoding gene ansA was amplified by non-pathogenic Mycobacterium bovis BCG and expressed in Escherichia coli. The enzymatic activity of AnsA analysis. AnsA was knocked out in M. bovis and the characteristics of acid-fast and ammonia-producing were studied. [Results] The purified recombinant AnsA can decompose asparagine and produce ammonia in vitro. In acidic media, mycobacteria use asparagine to produce ammonia, which can be released into the medium and acidified. After knocking out ansA, M. bovis lags about 10 days behind in an acidic environment. In order to better understand the asparagine-mediated mechanism of acid-fastness, schematics of asparagine metabolism, ammonia production and transport were drawn. 【Conclusion】 One of the antacid properties of Mycobacterium tuberculosis is achieved by the neutralization of the surrounding acidic environment through the secretion of ammonia. Ammonia is derived from the decomposition of the substrate asparagine by mycobacterium AnsA. This provides a clue to reveal the survival mechanism of M. tuberculosis in the acidic environment of host macrophages.