天冬酰胺酶介导的分枝杆菌酸适应机制

Asparaginase mediated acid adaptation of mycobacteria

摘要:

摘要:【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。

Abstract:

Abstract:［Objective］Mycobacterium tuberculosis can survive inside host cells for a long time． To elucidate the acid adaptation mechanism mediated by the asparagine (Asn) metabolic pathway，the activity of asparaginase (AnsA) and growth of mycobacteria were characterized． ［Methods］The nonpathogenic M.bovis BCG was used as a model strain to characterize AnsA function. The recombinant AnsA was further expressed in E.coli，purified by Co2 + charged resin，and analyzed by enzymatic method．M．bovis BCG mutant (DansA) was constructed by homologous recombination．The growth and ammonia generation by M．bovis BCG wild type and mutant were evaluated in acidic medium．［Results］Ammonia was detected in the presence of AnsA and Asn．Deletion of ansA significantly delayed the growth of M．bovis BCG for 10 days in acidic medium． Asn was used by M．bovis BCG to produce ammonia，increasing pH．［Conclusion］The acid adaptation of M．bovis BCG was mediated by ammonia，the metabolic product of asparagine．This provides new clue to uncover the survival mechanism of M．tuberculosis inside the acidic niche environment of host macrophage．