结核分枝杆菌硫醇乙酰基转移酶基因敲除株的构建及其生物学特性分析

为探索硫醇乙酰基转移酶(mycothiol acetyltransferase，MshD)在结核分枝杆菌中的生物学特性，本实验利用噬菌体为载体的同源重组技术，构建结核分枝杆菌mshD基因敲除株、mshD基因回补株，用实时定量聚合酶链反应(real time-quantitative polymerase chain reaction, RT-qPCR)对所构建的菌株进行验证。分别收集H37Ra野生株、mshD基因敲除株、mshD基因回补株对数生长期菌液各5 mL, 离心收集菌体并培养，以观察菌落形态、生物膜形成及生长曲线测定；用5 mmol/L H₂O₂、0.05% SDS，50 ℃热激及低氧条件下分别处理基因敲出菌株和野生菌株，将菌液进行10倍梯度稀释，培养4～6周后检测抗胁迫能力并计算存活率。结果显示， 与野生株H37Ra相比，mshD基因敲除株菌落褶皱减少且菌落偏小，生长趋势较为缓慢；生物膜形成所需时间增长且褶皱明显减少；抗逆能力下降，存活率略低于野生株和回补株。揭示了mshD基因对结核分枝杆菌的生长具有重要作用，为进一步揭示该基因的功能和作用机制奠定了基础。

A mycothiol acetyltransferase knockout in Mycobacterium tuberculosis and its biological characteristics

Mycothiol is the major low-molecular-mass thiol in M. tuberculosis, which plays an important role in maintaining redox homeostasis. Mycothiol acetyltransferase (MshD) finishes the synthesis process by transferring the acetyl group of Acetyl-CoA to the mycothiol precursor. In order to explore the role of MshD in M. tuberculosis, we constructed a MshD knockout strain with a phage vector-based homologous recombination technology. Compared with the wild strain H37Ra, the knockout had smaller colonies, slower growth rate, decreased biofilm, and was more sensitive to chemical challengers by 5 mmol/L H₂O₂, 0.05% SDS, 50 ℃ heat shock and hypoxic conditions. The above results indicated that the mshD gene played an important role in M. tuberculosis. This study laid the foundation for further revealing the function and mechanism of the gene.