State Key Laboratory of Microbial Metabolism, Joint International Laboratory on Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Institute of Physics Belgrade, University of Belgrade, Belgrade 11000, Serbia.
J Antimicrob Chemother. 2023 Apr 3;78(4):1066-1075. doi: 10.1093/jac/dkad048.
Bacterial toxin-antitoxin (TA) modules respond to various stressful conditions. The Gcn5-related N-acetyltransferase-type toxin (GNAT) protein encoded by the GNAT-RHH TA locus is involved in the antibiotic tolerance of Klebsiella pneumoniae.
To investigate the transcriptional mechanism of the GNAT-RHH operon kacAT under antibiotic stress.
The transcriptional level of the kacAT operon of K. pneumoniae was measured by quantitative real-time (qRT) PCR assay. The degradation of antitoxin KacA was examined by western blot and fluorescent protein. The ratio of [KacA]:[KacT] was calculated by the fluorescence intensity of KacA-eGFP and mCherry-KacT. Mathematical modelling predicted protein and transcript synthesis dynamics.
A meropenem-induced increase in transcript levels of kacA and kacT resulted from the relief from transcriptional autoregulation of the kacAT operon. Meropenem induces the degradation of KacA through Lon protease, resulting in a reduction in the ratio of [KacA]:[KacT]. The decreased ratio causes the dissociation of the KacAT complex from its promoter region, which eliminates the repression of kacAT transcription. In addition, our dynamic model of kacAT expression regulation quantitatively reproduced the experimentally observed reduction of the [KacA]:[KacT] ratio and a large increase in kacAT transcript levels under the condition of strong promoter autorepression by the KacAT complex.
Meropenem promotes the degradation of antitoxin by enhancing the expression of Lon protease. Degradation of antitoxin reduces the ratio of intracellular [antitoxin]:[toxin], leading to detachment of the TA complex from its promoter, and releasing repression of TA operon transcription. These results may provide an important insight into the transcriptional mechanism of GNAT-RHH TA modules under antibiotic stress.
细菌毒素-抗毒素 (TA) 模块对各种应激条件作出响应。由 GNAT-RHH TA 基因座编码的 Gcn5 相关 N-乙酰转移酶型毒素 (GNAT) 蛋白参与了肺炎克雷伯菌对抗生素的耐受性。
研究抗生素应激下 GNAT-RHH 操纵子 kacAT 的转录机制。
通过实时定量 (qRT) PCR 检测肺炎克雷伯菌 kacAT 操纵子的转录水平。通过 Western blot 和荧光蛋白检测抗毒素 KacA 的降解。通过 KacA-eGFP 和 mCherry-KacT 的荧光强度计算 [KacA]:[KacT] 的比值。数学模型预测了蛋白质和转录物合成的动力学。
美罗培南诱导 kacA 和 kacT 的转录本水平增加，这是由于 kacAT 操纵子的转录自动调节得到缓解。美罗培南通过 Lon 蛋白酶诱导 KacA 降解，导致 [KacA]:[KacT] 的比值降低。降低的比值导致 KacAT 复合物与其启动子区域解离，从而消除了 kacAT 转录的抑制。此外，我们对 kacAT 表达调控的动态模型定量再现了实验观察到的 [KacA]:[KacT] 比值降低和 KacAT 转录本水平在 KacAT 复合物强烈启动子自抑制条件下大幅增加的情况。
美罗培南通过增强 Lon 蛋白酶的表达促进了抗毒素的降解。抗毒素的降解降低了细胞内 [抗毒素]:[毒素] 的比值，导致 TA 复合物从其启动子上脱离，释放了 TA 操纵子转录的抑制。这些结果可能为抗生素应激下 GNAT-RHH TA 模块的转录机制提供重要的见解。
