结核分枝杆菌CRISPR/Cas系统中Cas蛋白毒力机制研究

CRISPR/Cas systems are known as prokaryotic adaptive immune systems against invading nucleic acids. CRISPR/Cas systems show diversity and have been classified into three basic types (Types I-III) and at least 10 subtypes based on their Cas proteins. In recent years, CRISPR/Cas9 has attracted intense interest and is increasingly being applied in genome editing. Alongside this explosion of interest in its defensive role and applications, reports on moonlighting roles functions of CRISPR/Cas proteins (and crRNA) in virulence, commensalism and broader bacterial physiology are raising the possibility that CRISPR/Cas systems may also contribute to the ability of bacterial pathogens to evade and dampen host defenses, and ultimately survive and replicate within the host..M. tuberculosis (MTB), the aetiological agent of tuberculosis (TB), a disease that still has an estimated annual death toll of 1.9 million, is considered to be one of the most successful pathogens and has evolved a range of mechanisms for evading detection by the host immune system. MTB harbors a Class I, Type III-A CRISPR/Cas system, but neither its defensive function against foreign nucleic acids, nor any potential alternative roles in virulence or pathogenesis have been explored. The applicant has completed a series of preliminary experiments on CRISPR/Cas mechanisms in MTB during which he identified a new role of Cas proteins as virulence factors; deletion of CRISPR/Cas proteins significantly attenuated MTB virulence in a murine model..Further research on how CRISPR/Cas proteins act as virulence factors will provide helpful insights for the diagnosis, prevention and treatment of tuberculosis.

CRISPR/Cas系统是一种细菌获得性免疫防御系统，通过CRISPR RNA和Cas蛋白形成复合体，对外源入侵核酸进行切割，完成抵御噬菌体等外源核酸入侵的功能。CRISPR/Cas系统类型复杂，可分为TypeI，TypeII，TypeIII三种类型。近几年，基于TypeII型的CRISPR/Cas系统原理发展了Cas9基因编辑技术，推动了基因编辑领域的快速发展。最近，研究发现CRISPR/Cas系统不局限于参与细菌的免疫防御系统，其中的Cas蛋白在细菌的致病性上也发挥重要作用。.结核病是世界三大传染病之一，病原菌结核分枝杆菌CRISPR系统属于TypeIII型，分子机制未见报道。申请者通过一系列预实验，初步鉴定了结核分枝杆菌CRISPR/Cas系统的部分作用机制，同时，首次发现结核分枝杆菌Cas蛋白是重要的毒力因子。本课题通过研究其毒力发生机制，有助于为结核病诊断、预防、治疗提供新思路。

原核CRISPR/Cas系统蛋白在抵御外源核酸防御作用已被广泛研究。一些I型和II型CRISPR/Cas蛋白在毒力相关的作用被报道，但对于III型系统蛋白尚未发现。我们发现CRISPR系统蛋白与结核分枝杆菌毒力密切相关。而且发现结核分枝杆菌（III-A型系统）CRISPR/Cas蛋白Csm1、Csm3、Csm5、Csm6和Cas6是分泌的，并诱导宿主免疫反应。重组MTBCas6以不依赖于caspase和TLR-2的方式诱导巨噬细胞凋亡。使用MTBCas6刺激的THP-1巨噬细胞进行的转录组学和信号通路研究表明，MTBCas6上调了与NF-κB通路相关的基因表达，从而导致更高水平的IL-6、IL-1β和TNF-α释放，这些细胞因子已知可激活免疫系统细胞以应对结核分枝杆菌感染。我们的研究结果表明，在除了细胞内屏蔽作用外，结核分枝杆菌CRISPR/Cas蛋白还具有非标准的细胞外作用，其功能类似于一把毒剑，并激活宿主免疫反应。