胰岛素降解酶在隐孢子虫入侵中的作用机制

Cryptosporidium spp. are important diarrhea-causing parasites in animals. The recent development of continuous cell culture and genetic manipulation models of Cryptosporidium spp. has greatly facilitated studies on their invasion mechanism. Insulin-degrading enzymes (IDEs) were reported to play important roles in the invasion of other apicomplexans, but their role in the invasion of Cryptosporidium is unclear. Our previous studies indicated that IDEs are common in Cryptosporidium spp., with many expressing at high levels during early infection, and some being related to host specificity of Cryptosporidium species. In the proposed research, we will focus on the following specific aims: (1) expression patterns of all IDEs at different life stages of Cryptosporidium and subcellular localization of about 10 IDEs in continuous cell culture and animal models, using transcriptomic and immuno-histochemical tools; (2) identification of at least 5 rhoptry- or microneme-associated IDEs that are involved in invasion and adhesion and verification of their functions using neutralization experiments and gene knock-out approaches; (3) studies of the structure, processing and trafficking of 2-3 invasion-related IDEs and identification of their action mechanisms on target proteins using proteomics and mass spectrometry; and (4) evaluations of IDEs as potential drug targets and characterization of the action mechanisms of highly active inhibitors of IDEs using structure analysis of key IDEs, rational design of inhibitors, in vivo and in vitro invasion neutralization experiments, and computational analysis. Data of the research should improve our understanding of the invasion mechanism of Cryptosporidium and facilitate the development of drugs against cryptosporidiosis.

隐孢子虫是重要的动物腹泻病原，最近体外培养模型和遗传操作平台的建立促进了对其入侵机制的研究。胰岛素降解酶(IDE)在顶复门原虫入侵中起重要作用，但它们在隐孢子虫入侵中的作用未知。我们前期工作表明，IDE在隐孢子虫中广泛存在，许多在感染早期表达，且有些与虫种入侵特定宿主相关。本研究拟开展：(1) 基于转录组学和免疫组化，在新型细胞培养和动物模型上，研究各IDE在隐孢子虫的表达规律并完成约10个早期表达IDE的亚细胞定位；(2) 选择至少5个定位在子孢子棒状体和微线体的IDE，通过中和实验和基因敲除，研究其与入侵的相关性；(3) 通过蛋白质组学-质谱分析等，研究2-3个IDE的结构、加工和运输及对靶蛋白的作用机制；(4) 通过对关键酶的结构解析、抑制剂的理性设计、体内外实验和计算分析，阐明高活性抑制剂的作用机制，以判断IDE作为药物靶标的可能性。研究结果有助于了解隐孢子虫的入侵机制和药物研制。

隐孢子虫是重要的腹泻病原，对养殖业和公共卫生造成严重危害。由于对它的入侵机制了解有限，导致目前尚无高效药物。胰岛素降解酶在隐孢子虫中广泛存在，而且在其它顶复门原虫的入侵过程中起重要作用，但它们在隐孢子虫入侵中的作用未知。为此，本项目首先通过组学分析，明确了隐孢子虫胰岛素降解酶的结构特征；进而基于定量RT-PCR和免疫定位，建立了12个胰岛素降解酶的表达规律和亚细胞定位数据库，包括具有完整结构域的3个酶，结构域不完整但具有活性结构域的7个酶，以及缺乏活性结构域的2个酶；之后基于酶的表达规律、亚细胞定位以及抗体中和数据，发现6个胰岛素降解酶（INS-4、INS-6、INS-15、INS-21、INS-19-20和INS-23）可能与隐孢子虫的入侵相关；进一步通过基因敲除和酶活性中心的定点突变，验证了4个酶（INS-1、INS-6、INS-16和INS-19-20）在隐孢子虫生长发育中的作用，并着重解析了INS-1的作用机制，明确了其与有性生殖阶段中雌配子体的发育密切相关；最后通过同源建模和分子对接，筛选到针对INS-4和INS-16的小分子抑制剂121个，其中21个抑制率达50%以上，并从中筛选到4个抑制剂具有较高安全性和有效性，其中1个能够显著抑制INS-4的酶活性。研究结果有助于阐明隐孢子虫的入侵和发育机制，并有望为隐孢子虫的新药研发提供先导化合物。培养博士后和研究生22名，发表SCI论文29篇，会议论文20篇。研究成果多次在国内外做大会报告。