分泌型核酸酶在结核分枝杆菌抵抗嗜中性粒细胞胞外网状陷阱中的作用研究

The formation of neutrophils extracellular traps (NETs), a form of programmed cell death, is an important mechanism in the innate immune response. NETs are composed of a chromatin-DNA backbone with attached antimicrobial peptides and enzymes that can capture and kill microbes. However, pathogens produce extracellular DNase to escape this kind of capture and killing traps by degradation of the DNA backbone. Thus the secretory DNase was considered as one of the crucial virulence factors in these pathogens. Recently, it was demonstrated that Mycobacterium tuberculosis induced subcellular changes on infected neutrophils, leading to NETs formation, but was not killed by NETs. Was the resistance due to potential extracellular DNase produced by Mycobacterium tuberculosis? During the previous study of this project, we found that gene Rv0394 of Mycobacterium tuberculosis H37Rv encoded a secretory protein, which was detected to have DNase activity. In this project, analyzing of the interaction between the protein encoded by gene Rv0394 and NETs, we will reveal the role of the secrectory DNase played in the resistance of Mycobacterium tuberculosis to the capture of NETs. Gene knockout mutant of Mycobacterium tuberculosis and Mycobacterium smegmatis strain constitutively express Rv0394 gene will be constructed to estimate the degradation effect to NETS caused by gene Rv0394 in mycobacteria. In addition, in vitro granuloma research model will be constructed and optimized. Comparative analysis of the mycobacterial mutants based on in vitro granuloma model will characterize the role of gene Rv0394 involved in granuloma development. Thus, novel virulence factor of Mycobacterium tuberculosis will be identified and the findings will broaden our knowledge of molecular mechanism of immune evasion applied by Mycobacterium tuberculosis.

嗜中性粒细胞能形成一种由DNA骨架构成的胞外网状陷阱（NETs），具有捕获和杀灭病原的作用。致病菌的分泌型DNase能降解NETs的DNA骨架，逃脱其捕获，因而被定义为新的细菌毒力因子。有研究证明结核分枝杆菌（Mtb）能诱导NETs的形成，但不会被其杀灭。本项目的前期工作发现H37Rv的基因rv0394编码产物有DNase活性，且能分泌至胞外，据此推测Mtb对NETs的耐受与该基因相关。本项目拟通过建立Rv0394蛋白与NETs的互作模型，验证其能降解NETs的DNA骨架；进一步构建Mtb的基因缺失株和耻垢分枝杆菌（Msg）的重组菌，与NETs互作，从不同角度证明该基因产物对NETs的作用效果。同时，建立并优化体外肉芽肿模型，利用Mtb的基因缺失株和Msg的重组菌，浅析该基因对结核肉芽肿病变发展的影响。以此鉴定出Mtb新的毒力因子，丰富人们对Mtb突破机体天然免疫屏障的分子机制的认识。

嗜中性粒细胞能形成一种由DNA骨架构成的胞外网状陷阱（NETs），具有捕获和杀灭病原的作用。致病菌的分泌型DNase能降解NETs的DNA骨架，逃脱其捕获，因而被定义为新的细菌毒力因子。有研究证明结核分枝杆菌（Mtb）能诱导NETs的形成，但不会被其杀灭。本项目的前期工作发现H37Rv的基因rv0394c编码产物有DNase活性，且能分泌至胞外，据此推测结核分枝杆菌对NETs的耐受与该基因相关。本项目的前期工作曾以为Rv0394c蛋白有核酸酶功能，因而立项预期研究Rv0394c的核酸酶活性作用于NETs的生物学意义。但后来的研究中，发现Rv0394c并非核酸酶，而是透明质酸酶。分泌型透明质酸酶同样对病原的致病性有重要意义，因此在立项后的两年里，我们以该酶为研究对象，阐述其对Mtb致病的贡献。同时，我们也继续在Mtb的基因组中分析寻找能编码分泌型核酸酶的基因。最后发现基因rv0888的编码产物有核酸酶活性，能非特异性地降解环状、线性的双链DNA和单链RNA，且该酶能被Mtb分泌至胞外，其功能很可能与降解NETs的DNA骨架有关。此后我们详细分析了Rv0888核酸酶的功能特征，并筛选到四种能抑制其酶活性的中药单体化合物。此外，利用重组耻垢分枝杆菌，通过小鼠的模型证实了Rv0888对分枝杆菌致病性确有重要贡献。