吡嗪酰胺是重要的一线抗结核药物之一，当其与异烟肼和利福平联合应用后可明显缩短化疗时间。结核分枝杆菌对吡嗪酰胺耐药给结核病治疗带来困难。目前已知的吡嗪酰胺耐药机制为编码吡嗪酰胺酶的pncA基因发生突变，但存在一些结核分枝杆菌的pncA基因无突变依然表型耐药、机制不清。本课题在前期发现的基础上围绕这一现象提出假说：可能存在蛋白质与pncA基因相结合从而抑制其转录和翻译而无吡嗪酰胺酶合成导致耐药。拟利用Reverse ChIP和细菌单杂交技术分别"垂钓"与pncA基因相结合的蛋白，进而用蛋白质谱确定蛋白特性；而后利用基因敲除编码该蛋白的基因，分析该蛋白对pncA基因表达的影响；并利用电泳迁移位移试验确定蛋白质与pncA基因结合的区域。本课题从DNA与蛋白质相互作用的角度探讨结核分枝杆菌对吡嗪酰胺耐药的分子机理，对进一步阐明吡嗪酰胺耐药机制、完善耐药临床检测方法、设计合理的治疗药物等具有重要意义。

Pyrazinamide is one of the most important first-line drugs for M.tuberculosis infection.Its inclusion in isoniazid and rifampin-containing regimens has enabled the shortening of the treatment period. However, the mechanism of M.tuberculosis resistance to pyrazinamide is not so clear.Mutations within the pncA, which encodes the pyrazinamidase, had been described as the major mechanism of PZA-resistance in M.tuberculosis. But, some PZA-resistant isolates without mutation in the gene of pncA. In this project, other related mechanism in M.tuberculosis to PZA will be investigated. For this purpose, we put forward the hypotheses: it may exist some proteins that combine with pncA so that the transcription and translation of pncA was prohibited. The proteins will be captured by the Reverse ChIP and bacterial one-hybrid reporter system from two directions and characterised by the protein mass spectrum technique. After this, the gene of encoding protein will be knocked out to analize its function acted on pncA.And the binding sites between pncA and protein will be deteced by electrophoretic mobility shift assay to reveal the mechanism of M.tuberculosis resistance to PZA. This study is important for fully understanding the mechanism of PZA-resistant tuberculosis and is beneficial to develope PZA drug susceptibility test and design more effective anti-tuberculosis drugs.