阻断GPI-锚定蛋白合成增强白念珠菌免疫原性的机制研究

Candida albicans is the major opportunistic fungal pathogen of humans. The basic research focused on C. albicans immunogenicity is of great significance for the development of immunotherapy. In our previous study, we demonstrated that abolishing cell wall glycosylphosphatidylinositol-anchored proteins of C. albicans through blocking GPI anchor synthesis could expose its surface β-(1,3)-glucan, then host phagocyte Dectin-1 could recognize the "uncloaked" C. albicans cells and mediate effective immune recognition. Our preliminary results also indicated vaccination with gpi7 gene mutant strain, which is abolished cell wall glycosylphosphatidylinositol-anchored proteins, could confer significant protection against experimental disseminated candidiasis by allergizing CD8+ T lymphocytes. The above results suggested inhibiting cell wall glycosylphos-phatidylinositol-anchored proteins synthesis in C. albicans could enhance its immunogenicity and induce host effective immune response to fungal infection. In the present project, we intend to further study mechanism of enhancing Candida albicans immunogenicity by blocking GPI-anchored protein synthesis. First, we will confirm the CD8+ T lymphocytes mediated immune response which was induced by gpi7 gene mutant strain in experimental disseminated candidiasis. Second, we will further study T lymphocyte differentiation induced by gpi7 gene mutant strain in vitro and in vivo. Third, we will also separate the antigen protein from the gpi7 null mutant presenting by antigen presenting cells through co-immunoprecipitation technology, and combine gpi7 gene mutant cell wall proteomics study to identify the antigen which contribute to the enhanced immunogenicity of C. albicans. The present project will provide theoretical and experimental basis for new effective strategy and antifungal agent development.

白念珠菌是侵袭性真菌感染的主要病原菌。针对提高白念珠菌免疫原性进行深入的基础研究对于临床发现免疫治疗新策略具有重要意义。本项目前期发现小鼠巨噬细胞对抑制GPI-锚定蛋白合成的白念珠菌（GPI7基因缺失菌）表现出更强的免疫识别；GPI7基因缺失减毒株免疫小鼠可诱导CD8+ T细胞依赖的免疫反应，并对再次感染白念珠菌具有保护作用，表明抑制白念珠菌GPI-锚定蛋白合成能够增强其免疫原性、诱导宿主产生有效的抗感染免疫反应。本项目拟在前期工作基础上进一步研究抑制GPI-锚定蛋白增强白念珠菌免疫原性的机制，在整体动物水平、体内及体外细胞水平研究GPI7基因缺失菌诱导宿主产生的CD8+ T细胞介导的免疫效应；利用免疫共沉淀技术分离GPI7基因缺失菌被抗原提呈细胞提呈的抗原肽，并关联细胞壁蛋白质组学研究，分析其免疫原性增强的抗原基础，为研究侵袭性白念珠菌感染的免疫治疗新策略提供理论依据。

白念珠菌是侵袭性真菌感染的主要病原菌。侵袭性白念珠菌感染主要发生于免疫功能缺陷人群。针对提高白念珠菌免疫原性并增强宿主抗真菌免疫应答反应进行深入的基础研究，对于临床发现免疫治疗新策略具有重要意义。GPI7基因缺失菌（细胞壁GPI-锚定蛋白缺失）是本课题组前期构建的一株无毒力白念珠菌。本项目研究发现GPI7基因缺失菌在小鼠体内的免疫原性增强，GPI7基因缺失菌免疫小鼠对再次感染白念珠菌和非白念珠菌（光滑念珠菌、热带念珠菌、克柔念珠菌、近平滑念珠菌）具有免疫保护作用；在胸腺缺陷的裸鼠体内GPI7基因缺失菌不能诱导抗真菌免疫应答的形成，表明上述抗真菌免疫保护效应依赖于宿主胸腺功能，属于适应性免疫应答；采用抗IFN-γ中和抗体和抗IL-17中和抗体阻断小鼠体内Th1和Th17细胞免疫应答，不影响GPI7基因缺失菌诱导的抗真菌免疫应答形成，提示上述免疫保护作用不依赖于Th1和Th17细胞；进一步的研究发现GPI7基因缺失菌诱导的抗真菌免疫应答依赖于CD8+T细胞，GPI7基因缺失菌致敏的CD8+T淋巴细胞对野生白念珠菌的吞噬率显著增强、体外感染白念珠菌后促炎细胞因子的释放量显著增加；并且GPI7基因缺失菌致敏的CD8+T淋巴细胞具有动物体内抗白念珠菌活性；我们发现小鼠树突状细胞对GPI7基因缺失菌抗原提呈和免疫识别能力增强，GPI7基因缺失菌能够刺激树突状细胞表面成熟分子CD83、MHC Ⅱ分子和协同刺激分子CD86表达显著升高；蛋白质组学研究表明，GPI7基因缺失菌表面moonlighting蛋白表达增多，并且能够被树突状细胞提呈。综上，本项目研究发现宿主树突状细胞能够提呈GPI7基因缺失菌细胞壁表面moonlighting蛋白，诱导CD8+T淋巴细胞激活，增强宿主抗真菌感染免疫应答，为临床发现免疫治疗念珠菌感染提供了理论基础和实验依据。