效应蛋白VceA参与布鲁氏菌调控细胞自噬的分子机制研究

Brucellosis is a serious zoonosis that endangers human health and affects the development of animal husbandry. Brucella Type Fourth Secretion System (T4SS) plays a pivotal role in the regulation of intracellular survival and replication, and 15 effectors have been identified, but their functions and molecular mechanisms remain unclear. Autophagy is of great significance in maintaining homeostasis and resistance to pathogen infection. Autophagy favors Brucella replication, and T4SS deletion Brucella will fuse with lysosomes and then degraded, indicating that T4SS and effectors are involved in the utilization autophagy for promoting replication. Brucella and autophagy interaction mechanism and functions of effectors have not been elucidated, through yeast two hybrid system and screening we found the interactions between VceA and autophagy related proteins p62 and NDP52. Therefore, the project intends to adopt the construction of Brucella mutant strain, RNA interference to analysis on the effect of VceA on autophagy and lysosomal formation, and also evaluate whether the interactions between VceA and p62/NDP52 are involved in helping Brucella from degradation. To clarify the molecular details and biological significance of VceA in the regulation of autophagy and lay the foundation of Brucella pathogenesis.

布鲁氏菌病是严重危害人类身体健康及影响畜牧业发展的人畜共患传染病。布鲁氏菌IV型分泌系统（T4SS）在调控布鲁氏菌胞内生存和复制过程中起到重要作用，目前已鉴定到15个作为T4SS底物的效应蛋白，其功能和作用机制还未阐明。布鲁氏菌可利用自噬完成自身复制，并且T4SS缺失的布鲁氏菌与溶酶体融合后被降解，表明T4SS及效应蛋白参与调节布鲁氏菌利用自噬促进自身复制。关于布鲁氏菌与自噬相互作用机制及效应蛋白的生物学功能还未阐明，本项目从效应蛋白出发，通过酵母双杂交及前期的筛选发现VceA蛋白与自噬相关的p62及NDP52存在相互作用。因此本项目拟采用构建布鲁氏菌突变株、RNA干扰等技术，系统分析VceA在布鲁氏菌调节细胞自噬体形成及自噬体与溶酶体的融合中所起的作用，以及与p62和NDP52的相互作用是否参与了这种调节。从分子水平上解释布鲁氏菌调控细胞自噬的分子细节，为阐明布鲁氏菌致病机理奠定基础。

布鲁氏菌作为典型的胞内寄生菌，可以利用自噬在液泡中进行复制。现阶段关于布鲁氏菌究竟是如何利用自噬及其具体机制还不清楚。因此，进一步深入研究布鲁氏菌对自噬的调控机制具有重大意义，这有助于阐明布鲁氏菌在宿主细胞内生存繁殖及致病机制。本课题从布鲁氏菌重要的效应蛋白VceA入手，探讨其对细胞自噬水平的影响及其作用机制，现取得以下结果：1. 将布鲁氏菌野生株与缺失株（2308 ΔVceA）分别侵染细胞，发现VceA缺失后自噬相关基因蛋白表达上升，表明VceA对细胞自噬有抑制作用。2. 在细胞内过量表达VceA后自噬相关基因蛋白表达下降，表明VceA对细胞自噬有抑制作用。3. 证实VceA通过与p62之间的相互作用实现对自噬水平的调节。4. 通过RNA干扰技术证实p62可以促进布鲁氏菌的胞内生存。本课题通过以上结果表明布鲁氏菌通过VceA与p62发生相互作用，实现对细胞自噬水平以及胞内生存的调节。该结果为揭示布鲁氏菌免疫逃逸机制提供理论基础。