溶组织内阿米巴凝集素复合体介导的致病及其免疫调控研究

Amebiasis is a global health concern with large domestic impacts. The World Health Organization (WHO) estimates that approximately 50 million people worldwide suffer from invasive amebic infection each year, resulting in 40–100 thousand deaths annually. Studies conducted in China indicated it may affect over 1% of the population. It is an infection caused by the protozoan parasite Entamoeba histolytica, and could be classified as amebic colitis and/or liver abscess clinically. Early studies demonstrated that tissue surface adhesion of Trophozoites on colonic mucus and epithelial cells is the first and most critical step of infection, and a Gal/GalNAc-specific lectin was implicated in this process. .The E. histolytica Gal/GalNAc lectin was originally identified as a heterodimer of transmembrane heavy (Hgl) and GPI-anchored light (Lgl) glycoproteins linked by disulfide bonds. In 1998, the applicant identified a 150-kD protein that was associated with the Gal/GalNAc lectin by non-covalent bond. The protein, also known as the heterodimer intermediate subunit (Igl) of the Gal/GalNAc lectin complex, was a useful target antigen in diagnosis of amoebiasis and a suitable vaccine for its prevention. It is of interesting to note that the Igl gene contained special CXXC and CXC motifs shared with transmembrane protein kinases (TMK) genes. As the collective data produced in our laboratory indicated that Igl is a critical component for both tissue surface adhesion and pathogenesis, we hypothesized that CXXC motifs may function as a second and critical signal that complement the lectin signal for surface adhesion, invasion and pathogenesis of E. histolytica. It will interact with the host immune cells, and caused host cell and tissue lesions. For another, it will start immune regulation of signal transduction system, occur specific antigen variation and induction of anti-complement function, make the disease become to persistent infection..A series of studies will be included in the current project. Both mRNA and protein expression levels of Igl and TMKs will be monitored at different stages or single trophozoites of infection in the context of the other known virulence factors. The effects of Igl and TMK on immune systems will be assayed by in vitro expressed proteins. Through gene transfection, gene mutation and gene replacement technique, the influence of both genes on in vivo and in vitro virulence of amoeba will be researched. The results will provide required evidences to verify whether surface CXXC Igl and TMK are the core factors in the pathogenicity of Entamoeba histolytica.

溶组织内阿米巴引起每年约10万人死于阿米巴病，是WHO持续关注的公共卫生问题，但其致病机理尚未完全阐明。我们前期研究发现在该原虫表面存在一种富含CXXC基序半乳糖／乙酰氨基半乳糖特异性凝集素中间亚单位，证实其具有疫苗和靶抗原作用，但其致病功能有待深入。为此假设：共有CXXC基序的凝集素中间亚单位与跨膜蛋白激酶形成复合体而成为致病的“双信号模式”，一方面与宿主免疫细胞相互作用引起宿主细胞病变，另一方面启动免疫调控信号传导系统，引起特异性的抗原变异和诱导抗补体作用，使疾病呈持续性感染状态。本项目将动态解析中间亚单位与跨膜蛋白激酶复合体在阿米巴不同培养阶段及单个滋养体的变化，研究其对宿主细胞免疫调节作用和抗补体的作用；进行该基因转染、结构突变和基因置换研究其对滋养体体内外毒力的影响，分析其与下游效应分子的关系和免疫保护作用。揭示中间亚单位与跨膜蛋白激酶复合体在溶组织内阿米巴的致病和免疫调控作用。

全世界每年约有五千万人受到溶组织内阿米巴侵袭而患阿米巴病，并导致每年约10 万人死于阿米巴病；我国传染病防治法将阿米巴病列为乙类传染病。溶组织内阿米巴感染和阿米巴病是国际社会持续关注的公共卫生问题。本项目研究本项目利用组学技术、免疫学技术、细胞生物学技术、分子生物学技术和基因置换技术等开展溶组织内阿米巴凝集素中间亚单位（Igl）和溶组织内阿米巴跨膜蛋白激酶(TMKs)致病及其分子机制的研究。分析了Igl和TMKs的基因组学及蛋白组学以及Igl与TMKs结合的特异性；解析了滋养体单细胞转录组，证明其转录组在体外增殖呈周期性的变化是其毒力下降的重要原因之一；验证了溶组织内阿米巴滋养体保留有不完整的硫代谢通路，半胱氨酸蛋白酶可以调控蛋白酶体通路，半胱氨酸合酶与半胱氨酸代谢有关，在滋养体的存活和致病性中起重要作用； 证明了溶组织内阿米巴重组凝集素复合体调控宿主细胞相关免疫信号通路， Igl和TMK3/40诱导巨噬细胞的极化，从清除杀伤效应逐渐向抵抗过度炎症反应和组织修复转变，这是造成病原体在宿主体内发生慢性持续感染的原因之一；发现了溶组织内阿米巴Igl/TMKs的CXXC基序蛋白诱导宿主细胞代谢重编程,证实差异蛋白主要位于染色体区域和核糖体中，参与碳代谢、mTOR信号通路和自噬等的相关通路，宿主细胞的核心碳代谢相关酶均受到抑制，表现出类似 Warburg 效应；阐明了Igl/TMK复 合体参与溶组织内阿米巴滋养体入侵宿主细胞及其CXXC活性结构突变对其功能的影响，证实Igl 的第1035/1049 号氨基酸所在CXXC 模体结构组成的功能域在溶组织内阿米巴侵袭过程中发挥了重要作用；筛选了能够与Igl的羧基端结合的溶组织内阿米巴过氧化物氧还酶（Prx），证实其可以激活宿主细胞NLRP3炎症小体和自噬；该激活过程不依赖于滋养体和细胞的接触；Prx的C末端可作为抗溶组织内阿米巴感染的潜在药物靶点。项目的完成揭示Igl/TMK复合体在溶组织内阿米巴的致病和免疫调控作用,同时揭示溶组织内阿米巴新的致病机制，得出科学结论具有非常重要的理论意义和实践的指导意义。