CyPA在杜氏利什曼原虫胞内寄生过程中的生物学功能研究

Leishmania donovani, a protozoan parasite, is a pathogen that causes the major zoonotic disease visceral leishmaniasis. If left untreated, Leishmania can cause fatal visceral disease in humans. At present, visceral Leishmaniasis is highly resistant and has no vaccine. In our previous study 527 amastigotes highly expressed proteins were found and these proteins were identified to be closedly related to the infection of Leishmania. After screened and verificated by serum of 100 visceral leishmaniasis patients, CyPA was found to be the main antigen of this disease and closely related to intracellular parasitism of Leishmania. Based on the previous studies, this study intends to identify the high expression of CyPA in amastigotes by identifying CyPA expression of amastigotes in macrophages. After that, the CyPA gene knockout and overexpression Leishmanias will be constructed to infect the macrophages and animal models. Then, the biological functions of CyPA on growth and reproduction of Leishmania in vitro and infection of host cells/experimental animals will be explored. In this research, we will clarify systematically the important biological functions of CyPA during Leishmania infection against host cell and exploring the value of CyPA in the early diagnosis of leishmaniasis, drug targets and the construction of vaccine. Eventually providing an important theoretical basis for further exploring Leishmaniasis.

内脏利什曼病为严重危害人体健康的人畜共患病，目前该病严重耐药，且无有效的疫苗预防。因此，筛选和鉴定利什曼病早期诊断、药物治疗和疫苗构建靶点具有重要意义。前期，我们筛选到527个杜氏利什曼原虫胞内寄生相关蛋白，经100位内脏利什曼病人血清筛选验证后发现CyPA为杜氏利什曼原虫致病后机体产生的主要抗原，与利什曼原虫胞内寄生密切相关。在前期研究的基础上，本研究拟通过鉴定巨噬细胞内无鞭毛体CyPA表达量，进一步鉴定CyPA在无鞭毛体中的高表达情况；构建杜氏利什曼原虫CyPA基因敲除虫株和过表达虫株，并和正常虫株一起感染巨噬细胞和动物模型，探究CyPA在利什曼原虫体外生长繁殖以及感染宿主细胞和实验动物过程中的生物学功能。通过本课题的研究将较系统地阐明CyPA在利什曼原虫感染宿主细胞过程中的重要生物学功能，为进一步探讨CyPA作为利什曼病诊断、药物治疗和疫苗构建靶标提供重要的理论依据。

杜氏利什曼原虫亲环素A（L. donovani cyclophilin A，LdCyPA），具有肽酰脯氨酸顺反异构酶（PPIase）活性，但其是否为利什曼原虫胞内增殖的重要毒力蛋白此前并无论证。前期研究发现，LdCyPA蛋白在杜氏利什曼原虫进入细胞转化为致病性无鞭毛体后显著高表达，且临床耐药强毒虫株在两个生命阶段LdCyPA蛋白表达量均显著高于敏感虫株；敲除虫体LdCyPA后，胞内杜氏利什曼原虫无鞭毛体在24h内被完全清除，而过表达LdCyPA后，杜氏利什曼原虫在胞内生长和增殖的能力较标准虫株明显增强。这些结果表明LdCyPA是利什曼原虫的一个重要毒力蛋白，值得进一步研究。在此基础上，申请人研究还发现LdCyPA可通过宿主琥珀酰化转移酶p300介导发生赖氨酸琥珀酰化修饰，进而增强LdCyPA异构酶活性；而抑制LdCyPA赖氨酸琥珀酰化可阻碍虫体在细胞内的生长与增殖。后续将继续对这一关键位点进行机制的进一步研究，为研发有效防治药物和疫苗提供新思路。