转录因子IscR调控睾丸酮丛毛单胞菌对重金属锑和头孢氨苄的共抗性机制

Antimonials, as the common used drugs for parasitic diseases, have a high frequency of cross-contamination with antibiotics. The cross-contamination leads environmental bacteria to evolve synergistic resistance to antimony (Sb) and antibiotics, which poses serious challenges to the living environment and the healthcare industry. Until now, mechanism of bacterial resistance to Sb is poorly understood, to say nothing of the mechanism of microbial synergistic resistance to Sb and antibiotics. In the prophase of this project, we have constructed a mutant library of a Comamonas testosterone strain with the ability to resist and oxidize Sb(III) using Tn5 transposon insertion mutagenesis. The whole-genome of the bacterium was sequenced. By phenotypic analyses of wild-type and mutant strain of C. testosterone under duress of Sb(III) and/or cephalexin, we performed the initial screening of potential synergistic genes coupling Sb and cephalexin resistance. Based on these preliminary results，we subsequently plan to study the bacterial Sb(III)/cephalexin resistant genes and related regulatory genes, and to explore bacterial-Sb(III)/cephalexin interaction and regulation mechanism by means of the comparative genomics, gene knockout and complementation, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), real-time quantitative RT-PCR, LacZ fusion protein expression and other molecular biology techniques. The results of this project will not only contribute to propose a model of synergistic induction regulation for bacterial co-resistance to Sb(III) and antibiotic, but also lay a certain foundation for new drug targets discovery and drug development.

重金属锑作为治疗寄生虫疾病的药物和抗生素的交叉污染的频率较高。重金属和抗生素的交叉污染迫使细菌产生协同抗性，给人类生存环境及医疗行业带来严峻挑战。目前，关于细菌对锑的抗性机制知之甚少，对锑和抗生素的协同抗性机理更是鲜有报道。本项目前期借助Tn5转座子插入突变技术构建了一株锑(III)抗性/氧化性睾丸酮丛毛单胞菌突变文库，并对该菌进行了全基因组测序，通过分析锑(III)或/和头孢氨苄胁迫下野生型和突变株的表型，初步筛选潜在的偶联锑和头孢氨苄协同抗性相关基因。拟借助比较基因组学，基因敲除和互补，凝胶阻滞，染色质免疫共沉淀，实时定量RT-PCR和LacZ融合蛋白表达等分子生物学技术研究细菌与锑(III)和头孢氨苄抗性相关的基因和调控基因，探索互作机制和调控机理。本项目研究结果将为建立细菌对锑(III)和抗生素共抗性的协同感应调控模型奠定基础，对药物新靶标的发现和新药开发也具有一定理论意义。

本项目发现Comamonas testosterone S44除了对锑(III)具有较高抗性外，还有较好头孢菌素类抗性能力。借助Tn5转座子插入突变构建随机插入突变体库，筛选出对锑(III)抗性降低表型最明显的突变株iscR-280，同时也表现出对头孢氨苄抗性显著降低，借助质粒拯救确定突变基因位点为铁硫簇合成isc操纵子(iscRSUA-hscBA-fdx) 的调控基因iscR。通过分析锑(III)或/和头孢氨苄胁迫下野生株，突变株和互补株的生长表型，确定iscR基因突变导致菌株S44对锑(III)和头孢氨苄抗性同时降低，iscR基因回补使突变株抗性表型回复。研究发现iscRSUA共转录，共用同一个启动子，IscR可以和该启动子区结合。有研究表明，在重金属胁迫下可能会诱导细胞产生氧化压力，足以对铁硫簇的组装和硫醇代谢造成损伤，项目组发现IscR突变会导致菌株S44细胞内的谷胱甘肽含量和谷胱甘肽合成限速酶活性显著下降。因此，项目组推测IscR通过调控铁硫簇合成和硫醇化合物代谢，间接赋予菌株S44对锑(III)的氧化胁迫应激反应。.项目组在S44基因组上发现2个反向紧邻的β-内酰胺酶编码基因及调控基因，并命名为CzoA和CzoR。研究表明CzoR和IscR可以结合在czoA-czoR间隔区的不同位点，并确定了精确结合位点。 CzoA异源表达赋予E. coli DH5α菌株对头孢氨苄等多重抗生素抗性。体外酶活实验表明CzoA可以水解头孢氨苄。敲除iscR或czoR均会导致菌株S44对头孢氨苄抗性的显著下降，相应iscR/czoR互补株对头孢氨苄的抗性回复。头孢氨苄可诱导iscR, czoA和 czoR的转录表达，CzoR对于czoA的转录表达是必须的，并且CzoR正调控czoA。 IscR正调控czoR，CzoR对iscR无调控作用。研究表明IscR通过调控czoR和czoA介导菌株S44对头孢氨苄和头孢唑啉等抗生素的抗性。.综上所述，IscR介导的共调控抗性可能是Sb(III)和头孢氨苄的共选择抗性机制，这种共调控抗性机制中各种调控体系在转录上是相互关联的，因此，细菌暴露于一种有毒物质可导致对另一种未知的或未被识别的有毒物质产生抗性，即IscR协同调控isc和czo操纵子的转录表达进而表现出对头孢氨的抗性和对Sb(III)胁迫产生的氧化胁迫应激。