结核分枝杆菌PE_PGRS家族抗原PE_PGRS41调控宿主免疫应答的分子机制研究

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a global burden of morbidity and mortality. M. tuberculosis survives and persists in the host by modulating host immune responses. M. tuberculosis genome has a unique multigenic PE_PGRS family with highly conserved N-terminal Pro-Glu (PE) domain and a variable polymorphic GC-rich repetitive sequence (PGRS) domain. PE_PGRS proteins, with functions largely unknown, the distribution of PE_PGRS and its characteristic cell surface associated proteins implicated a role in virulence and pathogen-host interaction. PE_PGRS41, also known as acid and phagosome regulated protein C (aprC), which can modulate pH-driven adaptation to the macrophage phagosome. We showed that PE_PGRS41 is a cell wall associated protein, implicating in mycobacteria-host interaction. Functional studies revealed that PE_PGRS41 significantly suppressed pro-inflammatory cytokines TNF-α, IL-1β and IL-6 expression, while increased anti-inflammatory IL-10 production. Expression of PE_PGRS41 in nonpathogenic Mycobacterium smegmantis significantly increased intracellular survival of M. smegmantis within macrophage. M. tuberculosis blocks phagosome-lysosome fusion via inhibiting intracellular Ca2+, resulting in persistance within macrophage. Ca2+ is a ubiquitous and versatile intracellular second messenger involved in many signaling processes. Disruption of intracellular homeostasis of Ca2+ signaling will alter the ER stress-mediated apoptosis in response to various pathological conditions. PE_PGRS41 with 16 typical Ca2+ binding motifs can suppress cell apoptosis of macrophages, yet its role in Ca2+ signaling regulation and ER stress-mediated apoptosis remains unknown. The central theme of this proposal is thus to elucidate the molecular basis of PE_PGRS41 in regulating host immune response. We will also use the cell and animal model to evaluate if silencing of PE_PGRS41 will mitigate intracellular persistence. Our proposed studies therefore hold value in providing a better understanding on the role of PE_PGRS41 in the pathogenesis of M. tuberculosis.

PE_PGRS抗原是结核分枝杆菌基因组一个独特的多基因家族，与分枝杆菌毒力相关。PE_PGRS41蛋白，又称酸性和吞噬体调控蛋白aprC，调控吞噬体酸化，具体的作用机制未知。我们的前期研究发现PE_PGRS41蛋白定位于分枝杆菌细胞壁，能够抑制巨噬细胞凋亡并调控宿主免疫应答反应，促进非致病性耻垢分枝杆菌在胞内存活。Ca2+作为重要的第二信使参与多个细胞信号的调控，包括吞噬体成熟及内质网应激介导的细胞凋亡。PE_PGRS41蛋白氨基酸序列中含有16个Ca2+结合基序，暗示PE_PGRS41可能调节巨噬细胞内Ca2+水平，阻碍吞噬体活化以及扰乱内质网应激介导的细胞凋亡，最终促进分枝杆菌在巨噬细胞内持留。本项目进一步研究PE_PGRS41蛋白在调控宿主免疫应答反应的分子机制，并在细胞和动物两个层次验证PE_PGRS41蛋白在分枝杆菌毒力中的重要性。

结核杆菌全基因组的解析揭示了一个重要的 PE_PGRS 抗原家族，该家族抗原在病原菌-宿主相互作用中发挥重要的功能且与分枝杆菌毒力和致病性相关。因此，解析该家族抗原的功能及其潜在的分子机制，将有助于了解结核杆菌如何逃逸宿主免疫反应及开发新的疫苗用于结核病的治疗。该抗原家族蛋白是结核病疫苗的潜在且重要靶点。本研究基于结核杆菌PE_PGRS41的前期研究基础，进一步研究PE_PGRS62基因在病原菌-宿主相互作用中的功能。首先本项目研究通过在模式生物耻垢分枝杆菌中成功过表达结核分枝杆菌毒力因子PE_PGRS41和PE_PGRS62，体外抗生素杀菌实验表明表达PE_PGRS41对抗生素比表达空质粒的重组耻垢分枝杆菌更敏感，而表达PE_PGRS62却能够促进耻垢分枝杆菌对多种压力耐受。这两种截然不同的表型促使我们对通过感染宿主巨噬细胞发现该蛋白在转录水平和蛋白水平均抑制了巨噬细胞细胞因子分泌。本项目首先通过构建好实验所需的所有菌株，证明了结核杆菌PE_PGRS62蛋白是潜在的毒力因子，我们通过结合体外和体内实验，充分证实PE_PGRS62蛋白能够调控巨噬细胞免疫反应。进一步证实了PE_PGRS62能够抑制宿主细胞因子的表达与分泌。本项目其次证明了PE_PGRS62蛋白通过影响宿主巨噬细胞内质网应激介导的细胞凋亡，帮助结核杆菌逃逸宿主巨噬细胞的免疫清除，最终在巨噬细胞内生存与持留。本项目最后运用细胞感染模型证实PE_PGRS62促进耻垢分枝杆菌在细胞内存活，阐明PE_PGRS62蛋白帮助分枝杆菌逃逸宿主免疫反应。本项目证实了PE_PGRS62作为一个全新的结核分枝杆菌独立蛋白，在调控宿主天然免疫以及结核病致病过程中发挥重要的功能，其作用机制的解析为结核病疫苗开发提供重要的研究基础。本项目研究取得较好的结果，达到对应项目任务书约定的具体研究内容。