阻断GPI锚去酰基化抑制白念珠菌侵染宿主分子机制研究

Invasive fungal infections are associated with significant morbidity and mortality. Candida albicans is the major opportunistic fungal pathogen of humans. The fungal cell wall, as the outermost cellular structure, is a complex of cross-linked polysaccharides and glycoproteins which are critical for maintenance of the integrity and shape of the cell, but also a key determinant of virulence because its surface bears glycoproteins that mediate pathogenic fungi adhesion and invasion of host tissues. The first and most abundant class of cell wall proteins (CWPs) covalently attached to cell wall structural fibrillar polysaccharide is glycosylphosphatidyl inositol (GPI) anchored proteins (GPI-CWP), linking to β-1, 6-glucan through a GPI remnant. GPI anchor is responsible for all of the GPI-anchored proteins transporting and attaching to the cell wall of the C. albicans. Given that deleting one of the GPI-anchored proteins, such as Ecm33p, can cause reduced virulence, it is reasonable to expect that deletions in the GPI biosynthetic pathway to block all of the GPI-anchored proteins attaching to the cell wall will be fatal for C.albicans and may provide a novel mechanism for antifungal therapy. However, few previous studies pubished on the proteins which involved in GPI biosynthesis. In our previous study, we performed series study on the GPI biosynthetic pathway. We functionally dissected the BST1 gene, which was responsible for GPI inositol deacylation in C. albicans. We showed that blocking GPI inositol deacylation in C. albicans by deletion of BST1 gene could markedly change the cell wall structure, including GPI-anchored protein abolishing and pathogen-associated molecular patterns (PAMP) exposure of cell wall. These cell wall structure changes induced C. albicans avirulence and enhanced the immune recognition by host innate immune cells. To our knowledge, this work is the first study with BST1 gene in C. albicans, and clearly shows a critical role for GPI inositol deacylation in virulence and immune recognition by host cell. In the present project, we mainly intend to investigate the cellular and molecular mechanisms on inhibition of C.albican infecting host by blocking GPI inositol deacylation, using BST1 gene deletion strains as a tool. One hand, we will screen the critical proteins on cell wall of C.albicans, which are responsible for avirulence induced by blocking GPI inositol deacylation, by iTRAQ-based quantitative proteomics technology. On the other hand, we will investigate the molecular mechanisms of the enhanced host defense against C.albicans bst1Δ/Δ, of which GPI inositol deacylation is blocked. Finally, we will explore the structure-activity relationship of Bst1p involving in virulence and host immune recognition using site-directed mutagenesis technique. The present project will provide theoretical and experimental basis for new effective strategy and antifungal agent development.

侵袭性真菌感染发病率逐年上升，病死率居高不下。白念珠菌是最主要的条件致病真菌。大部分白念珠菌细胞壁蛋白包括许多毒力因子都属于GPI-锚定蛋白，且均通过GPI锚转运并锚定于细胞壁。虽然GPI锚生物合成对于白念珠菌侵染宿主具有重要作用，但目前研究尚较少。本项目前期对白念珠菌GPI锚合成通路开展了系列研究，首次发现GPI锚前体转移到新生蛋白质上后肌醇去酰基化反应对其侵染宿主能力有重要影响，该步反应由Bst1p催化。本项目拟在前期工作基础上，以BST1基因缺失菌为工具菌，深入研究阻断GPI锚去酰基化抑制白念珠菌侵染宿主的细胞与分子机制，一方面利用iTRAQ等技术研究阻断GPI锚去酰基化后白念珠菌对宿主侵袭力降低的机制；另一方面研究阻断白念珠菌GPI锚去酰基化后宿主对其免疫识别增强的机制；并采用定点突变技术研究Bst1p参与侵染宿主的活性位点，为发现防控侵袭性白念珠菌感染的新靶标提供理论和实验依据。

侵袭性真菌感染发病率逐年上升，其中最常见的念珠菌感染位居院内血源性感染的第四位。白念珠菌细胞壁中多数毒力因子都属于GPI-锚定蛋白，它们均通过GPI锚转运并锚定于细胞壁。我们提出如下科学假设：阻断白念珠菌GPI锚合成的关键步骤能够抑制众多毒力因子在细胞壁上的定位，在很大程度上抑制其侵染宿主。本项目前期发现白念珠菌GPI锚前体转移到新生蛋白质上后的肌醇去酰基化反应是GPI-锚定蛋白合成的关键步骤，该步反应由Bst1蛋白催化。本项目在前期工作基础之上深入研究阻断GPI锚去酰基化抑制白念珠菌侵染宿主的细胞与分子机制。本项目研究表明，通过BST1基因缺失抑制肌醇去酰基化反应后，白念珠菌细胞壁中GPI-锚定蛋白含量显著下降；BST1基因缺失菌对宿主细胞（血管内皮细胞、口腔和肠粘膜上皮细胞、肺上皮细胞）粘附、侵袭（诱导胞吞和主动穿刺）和损伤能力显著下降；BST1基因缺失菌在小鼠血源性念珠菌血症和胃肠道来源感染念珠菌血症模型中丧失毒力。另外一方面，我们发现通过基因缺失突变白念珠菌BST1基因后，细胞壁结构发生明显变化，最外层甘露聚糖成分显著减少，中间层β-(1,3)-D-葡聚糖成分暴露在细胞壁表面，能够诱导天然免疫细胞更强的免疫识别反应（如巨噬细胞NF-κB和MAPKs信号通路的激活及细胞因子释放、中性粒细胞杀伤增强等）。同时，我们研究发现Bst1蛋白202位丝氨酸是其发挥生物学功能的关键位点。综合以上研究数据，我们认为Bst1蛋白催化的肌醇去酰基化反应是白念珠菌GPI-锚定蛋白合成的关键步骤；阻断肌醇去酰基化反应不仅可以抑制毒力因子在白念珠菌细胞壁的定位，而且可以增强宿主的免疫识别反应，从而抑制白念珠菌感染机体。基于该项目的基础研究，本课题组开展了抗真菌感染GPI-锚定蛋白抑制剂的研究，获得了氨基砒啶类候选药物GPI-89，目前正在进一步研究。