环二核苷酸磷酸二酯酶调控牛支原体生长特性的分子机制研究

Mycoplasma bovis (M.bovis) causes serious diseases in cattle such as pneumonia and mastitis. However, no specific and highly effective control and treatment measures are available against the diseases. The factors mostly associated with these problems are the unclear metabolism and virulence mechanism. The current applicant recently identified a novel protein designated as cyclic dinucleotide phosphodiesterase (shorten as CDNPase) essential for M.bovis growth and able to degrade cyclic dinucleotides (CDNs) including c-di-AMP, c-di-GMP and cGAMP, the second messengers. This study would be aimed to determine the molecular mechanism of growth regulatory functions of this CDNPase. The main activities include as follows: the effect of CDNPase on intra-bacterial CDNs concentrations will be determined by using LC-MS/MS to detect intra-bacterial CDNs of wild type and CDNPase mutant; the mutants whose growth are affected by CDNs accumulation will be screened from mutant library already constructed in this laboratory by adding over dose CDNs into the normal media, the genes and their functions are identified by sequencing the mutants and bioinformatics analysis; the CDNs receptors are further identified with pull-down techniques; With RNA-seq techniques, the differential effects on genome-wide gene expression in wild type and CDNPase mutant will be revealed and the potential regulatory mechanisms are predicted; the proteinomic study on wild type and CDNPase mutant is further performed to assay the potential regulatory effects of CDNPase at protein level and potential regulatory mechanisms are predicted. By integrating all the above findings on potential regulatory mechanisms of CDNPase, 1-2 critical pathways are selected to be confirmed in M.bovis strains of wild type, CDNPase mutant and its revertant, and other mutants) on M.bovis medium culture and cell infection models. The results would make breakthrough contribution to elucidate metabolism and virulence mechanism of M.bovis and discover potential targets for prevention and treatment of M.bovis caused diseases.

牛支原体致牛肺炎、乳腺炎等重要疾病，无特效防治措施，其生长代谢和毒力机制不清是重要制约因素。申请者新鉴定了一种牛支原体生长必需蛋白，能降解环二核苷酸（CDNs），命名为环二核苷酸磷酸二酯酶（CDNPase）。本研究旨在证明该酶在牛支原体的生长调节机制。内容包括：确定CDNPase对菌内CDNs浓度的影响；培养基中人工添加CDNs，从已构建的突变库中筛选鉴定CDNs积累影响生长的突变体并鉴定相关基因；利用pull down技术等鉴定CDNs受体，预测CDNs调节功能；利用RNA-seq和蛋白组学技术(2-DE/LC-MS/MS），揭示CDNPase基于全基因组的可能的生长调控机制；在整合分析基础上，选择1-2个关键调节通路，利用野生株、突变体和回补株在牛支原体无细胞培养条件下和细胞感染模型上进行生长特性验证。结果将为阐明牛支原体生长代谢机制和毒力相关机制、确定支原体防治潜在靶标提供理论依据。

牛支原体是牛的重要病原体，主要导致肺炎、乳腺炎和关节炎等。由于支原体密码子偏爱性、大部分基因为未知基因、遗传操作工具少、小动物模型缺乏等原因，其代谢和致病机制知之甚少，且进展缓慢。因此，本项目在前期研究基础上，旨在研究和发现一种牛支原体第二信使降解酶—环二核苷酸磷酸二酯酶调控牛支原体代谢的分子机制，为阐明其代谢机制和致病机制、研发高效防控措施提供理论基础。.项目主要取得如下结果：.（1）利用细胞与牛支原体共培养模型，鉴定了13个共培养条件下生长缺陷的突变菌株，发现MbovGdpP除具有磷酸二酯酶和ATP酶活性外，还具有nanoRNase和ADP酶活性。.（2）成功构建了CDNPase（Mbov_0328）回补菌株，确定了CDNPase对牛支原体菌内CDNs的降解功能，并进一步证实H219和204-209aa是催化c-di-AMP降解的关键位点。.（3）利用转录组学和蛋白组学技术检测CDNPase突变株及野生株，确定差异基因主要富集在牛支原体新陈代谢等途径，并从中验证到一个受CDNs调节的基因Mbov_0701，研究表明其可以降解胞外DNA以支持支原体生长。.（4）利用Pulldown和质谱分析等蛋白互作技术，筛选到一批CDNs潜在受体，筛选出9个最重要的侯选蛋白，并初步验证了MbovP0421（KtrC）蛋白（一种钾离子通道蛋白）与c-di-AMP 间存在特异性结合。.项目实施期间发表高水平SCI收录论文4篇；申报国家发明专利8项，获得授权4项；制定发布行业标准1项；培养研究生5名，其中博士1名、硕士4名。全面完成了计划目标。研究结果推进了对牛支原体代谢调节机制的认知，对深入阐明牛支原体致病机制和研发新型防控手段均具有重要意义。