CdaR-DAC信号系统调控钩端螺旋体合成第二信使分子c-di-AMP  

CdaR-DAC signaling systems regulating the synthesis of a second messenger c-di-AMP in Leptospira interrogans

作　　者：方葆 李阳 严杰[3] 胡玮琳[3] Fang Bao;Li Yang;Yan Jie;Hu Weilin(Department of Microbiology Kaihua Center For Clinical Laboratory,Quzhou 324300,China;Clinical Laboratory Children's Hospital of Soochow University,Suzhou 215025,China;Department of Medical Microbiology and Parasitology,Medical School of Zhejiang University,Hangzhou 310058, China)

机构地区：[1]开化县临床检验检测中心微生物室,衢州324300 [2]苏州大学附属儿童医院检验科,215025 [3]浙江大学医学院病原生物学系,杭州310058

出　　处：《中华微生物学和免疫学杂志》2018年第12期881-885,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(81501713);浙江省自然科学基金(LY18H190001).

摘　　要：目的确定致病性问号钩端螺旋体(简称钩体)LA3304基因产物DAC合成c-di-AMP的活性,LA3303基因产物CdaR增强DAC酶活。方法采用PCR扩增问号钩体黄疸出血群赖型赖株LA3304基因,并构建其原核表达系统。Ni-NTA亲和层析法提纯表达的目的重组蛋白rDAC。采用高效液相色谱法(HPLC)检测rDAC环化ATP底物为c-di-AMP的活性。采用细菌双杂交法检测CdaR与DAC相互作用情况。构建细菌共表达系统,结合HPLC法检测CdaR增强DAC酶活情况。结果所构建的问号钩体赖株LA3304基因原核表达系统在IPTG诱导下能高效表达rDAC,Ni-NTA亲和层析法提纯的rDAC经SDS-PAGE后显示为单一蛋白条带。rDAC具有体外合成ATP底物为c-di-AMP的功能。CdaR与DAC存在相互作用,CdaR可显著增强DAC酶活(P<0.05)。结论LA3304基因产物DAC具有c-di-AMP合成酶活性,CdaR可增强DAC活性,并与DAC构成CdaR-DAC信号系统,共同参与调节钩体c-di-AMP合成。Objective To analyze the activity of diadenylate cyclase (DAC) encoded by LA3304 gene of Leptospira interrogans (L.interrogans) and to investigate the influence of CdaR encoded by LA3303 gene on DAC activity. Methods The LA3304 gene in L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR and inserted into a prokaryotic expression system for expressing DAC. The expressed recombinant protein, rDAC, was purified by Ni-NTA affinity chromatography. High Performance Liquid Chromatography (HPLC) was used to measure the synthesis of c-di-AMP from ATP by rDAC in vitro. Bacterial two-hybrid analysis was used to detect the interaction between CdaR and DAC. Prokaryotic co-expression system was constructed and used in combination with HPLC to analyze the role of CdaR in activating DAC. Results The constructed prokaryotic expression system for LA3304 gene of L. interrogans strain Lai could highly express the rDAC upon the induction of IPTG. The purified rDAC showed high purity with a single protein band in gel as indicated by SDS-PAGE. rDAC could synthesize c-di-AMP from ATP in vitro. CdaR interacted with DAC and enhanced the activity of DAC (P<0.05). Conclusion DAC encoded by LA3304 gene was a diadenylate cyclase that could convert ATP into c-di-AMP. CdaR promoted the activation of DAC and formed a CdaR-DAC system with DAC. The system was involved in the synthesis of c-di-AMP in L. interrogans.

关 键 词：钩端螺旋体 c—di—AMP DAC CDAR 

分 类 号：R377.5[医药卫生—病原生物学]