Sun Qing, Zou Yan, Feng Qian, Gong Zongyue, Song Manlin, Li Machao, Chen Zhuang
Department of Medicine, Hunan Traditional Chinese Medical College, No.136, Lusong Road, Lusong District, Zhuzhou, 412000, Hunan Province, China.
Hunan Traditional Chinese Medical College, Zhuzhou, China.
Arch Microbiol. 2023 Sep 23;205(10):337. doi: 10.1007/s00203-023-03676-9.
EmbR, a substrate of pknH in Mycobacterium tuberculosis (Mtb), is related to the ethambutol (EMB) resistance. This study aimed to investigate the relationship between acetylation of pknH and the resistance of EMB mono-resistant Mtb. The EMB mono-resistant Mtb strain was constructed based on the MYCOTB and the Löwenstein-Jensen (LJ) proportion method. The growth kinetics was used to evaluate the bacterial growth. Escherichia coli, as the host of Mtb, was used for cloning and protein purification. Moreover, the immunoprecipitation was performed along with western blot to evaluate the EmbR phosphorylation and pknH acetylation. Each independent experiment was conducted in triplicate. EMB mono-resistant Mtb strain was successfully constructed according to the results of MIC values of 14 anti-Mtb drugs. The EMB resistant (ER) Mtb strain showed faster growth than the wild-type (WT) Mtb strain, and the difference was statistically significant. Moreover, pknH robustly phosphorylates EmbR, and pknH and acetylated pknH protein levels were downregulated in ER strain. The acetylation of pknH may reduce the phosphorylation of EmbR to inhibit the growth of Mtb strain. Enhancing the acetylation of pknH may be a promising method to inhibit the EMB resistance against Mtb.
EmbR是结核分枝杆菌（Mtb）中pknH的底物，与乙胺丁醇（EMB）耐药性有关。本研究旨在探讨pknH乙酰化与EMB单耐药Mtb耐药性之间的关系。基于MYCOTB和罗氏（LJ）比例法构建了EMB单耐药Mtb菌株。采用生长动力学评估细菌生长情况。以大肠杆菌作为Mtb的宿主进行克隆和蛋白质纯化。此外，通过免疫沉淀和蛋白质印迹法评估EmbR磷酸化和pknH乙酰化情况。每个独立实验均重复进行三次。根据14种抗结核药物的MIC值结果，成功构建了EMB单耐药Mtb菌株。EMB耐药（ER）Mtb菌株的生长速度比野生型（WT）Mtb菌株快，差异具有统计学意义。此外，pknH能强烈磷酸化EmbR，且ER菌株中pknH和乙酰化pknH的蛋白水平下调。pknH的乙酰化可能会减少EmbR的磷酸化，从而抑制Mtb菌株的生长。增强pknH的乙酰化可能是一种抑制Mtb对EMB耐药的有前景的方法。
