分枝杆菌CpsA蛋白的功能及其致病的分子机制研究

Mycobacterial CpsA protein belongs to LCP family proteins and is related to cell wall synthesis as well as pathogenesis. Foreign scholars and us found that Mycobacterial CpsA protein bears an activity of phosphate transferase and is able to inhibit cell bactericidal activity to promote the proliferation of bacteria in the cell. But the interactional target proteins of CpsA and pathogenic molecular mechanisms are not illustrated completely. A recent discovery of CpsA by foreign investigators can interact with LAP pathway of host autophagy mechanism. on the other hand, our previous study found that CpsA can influence TNFα signal pathway and NO killing pathway as well. Therefore, we hypothesis that in addition to interfering with LAP pathway in the cell, regulating NO killing pathway and TNFa signaling pathway through the interaction between Mycobacterial CpsA protein and host target proteins is also a significant pathogenic mechanism interacts. By means of molecular biology, cell biology, immunology and proteomics research methods with macrophages and zebrafish infection model, we propose to identify the host protein and the key amino acid sites interacting with CpsA, to illuminate the molecular mechanism of immunoreaction inhibition by CpsA in Mycobacterium marinum. The research findings have significance to clarify the pathogenic role of CpsA in Mycobacterium marinum as an immunoregulator protein and reveal new interaction mechanism of bacteria and host.

分枝杆菌CpsA蛋白属LCP(LytR-CpsA-Psr)家族蛋白，与细菌的细胞壁合成和致病性相关。我们及国外学者均发现分枝杆菌CpsA具有磷酸转移酶活性，且CpsA直接抑制细胞杀菌活性，促进细菌增殖。但其与宿主相互作用的蛋白及致病分子机制尚未完全明晰。国外近期发现CpsA可干扰宿主自噬通路中的LAP途径，我们发现其还可影响细胞的TNFα及NO杀伤通路。因此，我们提出分枝杆菌CpsA与宿主靶蛋白相互作用，除干扰细胞LAP的杀菌途径外，调节NO杀伤途径及TNFα信号通路也是其重要的致病机制的科学假设。本项目拟以海分枝杆菌为对象，利用分子生物学、细胞生物学、免疫学和蛋白组学等方法，及细胞和斑马鱼感染模型鉴定与CpsA相互作用的宿主蛋白及关键位点，阐明CpsA抑制宿主免疫反应的分子机制。本研究成果对于阐明CpsA作为免疫调节蛋白在分枝杆菌致病中的作用和揭示新的细菌和宿主相互作用机制具有重要意义。

本课题组前期的工作证明分枝杆菌CpsA蛋白与其致病性密切相关，但其具体分子机制尚不明确。本课题中我们将以海分枝杆菌为模式菌，利用巨噬细胞体外感染模型及斑马鱼和小鼠感染动物模型，深入研究了CpsA的功能及致病机制。我们构建了cpsA基因完全敲除株（ΔcpsA Mm）进一步证实CpsA的缺失使海分枝杆菌在胞内增殖减弱。通过构建并利用海分枝杆菌ESX分泌系统的4个亚型的敲除和敲低突变株，CpsA蛋白被证明是通过ESX-5分泌系统特异性分泌。值得注意的是我们通过RNA-seq发现CpsA参与激活巨噬细胞的I型干扰素信号通路，其分子机制是CpsA通过激活胞质核酸感应信号通路cGAS-TBK1-IRF3并伴随着诱导吞噬体膜破裂，进而激活I型干扰素的表达。同时，我们也发现CpsA与宿主的相互作用蛋白为半乳糖凝集素家族蛋白Galectin-8（GAL-8），它们如何介导分枝杆菌与宿主的互作值得进一步深入研究。综合上述结果，本研究阐明了CpsA作为毒力蛋白的分泌机制，发现了其激活宿主I型干扰素参与致病过程的功能及机制，同时也发现了其宿主互作蛋白GAL-8，为进一步深入研究CpsA的致病功能及机制指明了新的方向，该成果也为研发新型疫苗及抗结核小分子研发提供了新靶标。