c-di-AMP信号通路在炭疽杆菌致病机制中的功能研究

Anthrax, caused by the spore-forming pathogenic bacterium Bacillus anthracis, is a zoonosis that causes serious disease. There is a significant concern worldwide due to its high mortality rate and difficult to cure. However, no effective drug can treat anthrax after the early asymptomatic stages of infection. c-di-AMP is a recently identified secondary messenger in bacterium. It plays multiple roles in physiology and virulence of Gram-positive bacterium. We hypothesized that c-di-AMP is an important molecule regulating the virulence of B. anthracis. In our preliminary data, we found three c-di-AMP diadenylate cyclases and two c-di-AMP phosphodiesterases through bioinformatics analysis. We further expressed and purified DisA and CdaS diadenylate cyclases in vitro. At the same time we created the ΔcdaS mutant strain and compared the sporulation with the wild type strain. The results showed that the sporulation is significantly enhanced in the ΔcdaS strain. In this study, we would like to purify the rest three proteins and measure the enzymes activity. Then we would create diadenylate cyclase and phosphodiesterase mutants to alter the c-di-AMP concentration in B. anthracis. The role of c-di-AMP signaling involved in physiology of B. anthracis would be analyzed compared with Sterne strain. The in vitro invasion to macrophage and in vivo infection with mice would also be determined. Our long-term goal is to provide fundamental new knowledge regarding c-di-AMP signaling in the pathogenesis of B. anthracis and develop potential drug target to anthrax.

炭疽杆菌是致病性极强的产孢病原菌。由于其致死率高和难治愈性，该菌在世界各国受到广泛重视。迄今缺乏有效药物在感染后期控制炭疽病。c-di-AMP是新发现的细菌第二信使分子，在革兰氏阳性病菌中参与调控重要生理活动和致病力。我们认为c-di-AMP是调节炭疽杆菌致病性的重要信号分子。通过对炭疽杆菌基因组序列分析，发现该菌中存在三个c-di-AMP合成酶和两个降解酶。前期实验通过体外表达和纯化了DisA和CdaS两个合成酶，同时构建了ΔcdaS菌株。与野生型相比，该突变体产孢显著升高。我们将对剩余三个蛋白进行体外表达纯化，检测酶活；构建合成酶和降解酶基因缺失菌株，并进一步分析c-di-AMP对炭疽杆菌生理功能的影响；同时研究基因缺失菌株在体外对巨噬细胞的侵入以及体内试验中对小鼠侵染力的影响。本项目旨在揭示c-di-AMP信号通路在炭疽杆菌致病机制中的功能，同时为研发潜在的药物靶位点提供基础。

炭疽病是一种由炭疽杆菌引发的古老的致死性疾病。细菌繁殖体在感染哺乳动物宿主的过程中产生毒素因子及S蛋白。环二腺嘌呤核苷酸是新近发现的细菌第二信使分子参与调控细菌的多种生理活动。我们研究发现敲除两个c-di-AMP降解酶GdpP和PgpH导致与炭疽杆菌疫苗株Sterne相比，细菌体内的c-di-AMP的含量显著升高同时细菌的生长受到抑制。环二腺嘌呤核苷酸的富集进而抑制细菌毒素因子的表达，增强其对渗透压和去污剂的敏感性，降低对C56BL/6J及A/J小鼠的致病力。将PDE缺失菌株反式回补携带Pspac启动子的gdpP/pgpH基因可以回复细菌的生长、毒力因子的表达以及对去污剂的抗性。我们的研究结果表明c-di-AMP是一个多效性分子，对于炭疽杆菌和宿主互作起着重要的作用。我们还研究了atxA和abrB在∆∆PDE菌株毒素因子表达中起的作用，为今后解析这些因子如何参与c-di-AMP信号通路提供了良好的基础。