肝期约氏疟原虫ΔCSP下调自噬相关蛋白在其抑制IFN-γ杀伤EEF中的作用和机制

Understanding the underlying mechanism of malaria parasite to escape or suppress the host innate immune responses will shed us with new light on the designation of prophylactic strategy against pre-erythrocytic stage. It is well known that the main sporozoites surface protein CSP(circumsporozoites protein), which is translocated into cytoplasma of hepatocyte after it is cleaved into ΔCSP, is essential for the development of liver stage. However, the underlying mechanism is still unclear. Our preliminary data showed that transfection of ΔCSP couldn’t promote the development of EEF in vitro, but could resist to the IFN-γ-induced killing of EEFs. RNA-seqencing and Western blotting results indicated that transfection of ΔCSP could resist to the IFN-γ-induced killing of EEFs through down-regulation of autophagy-related proteins, and pull-down assay showed ΔCSP downregulates the autophagy-related proteins possibly through binding to the HSPA8, but the underlying mechanism is still unknown. In this study, we would like to confirm the abovementioned hypothesis by using CSP-mutant sporozoites and mice with ATG5 conditional knockout in hepatocyte; and test whether ΔCSP down regulates the autophagy-related proteins through binding to the HSPA8. This study could not only shed new light on the escape strategy of exo-erythrocytic stage, but also provide novel clues to design prophylactic strategies against the liver-stage.

探讨肝期疟原虫的免疫逃避机制是设计有效预防疟疾感染手段的前提。研究发现，疟原虫子孢子蛋白CSP被水解生成ΔCSP后，可进入肝细胞胞浆并促进疟原虫EEF的发育，但其机制并不清楚。课题组研究发现，转染ΔCSP重组质粒并不能直接促进EEF的发育，却能够显著抑制IFN-γ对EEF的杀伤作用；转录组测序、Western和ATG回补实验提示ΔCSP可能通过下调自噬相关蛋白抑制IFN-γ的杀伤；而Pull-down实验发现ΔCSP可能通过HSPA8下调自噬相关蛋白，但其中的机制尚不清楚。鉴此，本项目拟在此基础上，首先利用CSP突变子孢子和肝细胞ATG5 条件敲除小鼠，进一步证实ΔCSP可通过下调自噬相关蛋白抑制IFN-γ的杀伤；然后采用分子截短、pull-down，探讨HSPA8在ΔCSP下调自噬相关蛋白中的分子机制。这不但有助于揭示肝期疟原虫免疫逃避新机制，为寻找新的疟疾防治干预靶点提供重要理论依据。

疟疾仍然是世界上危害最为严重的三大传染病之一。肝期疟原虫的感染并不引起患者出现临床表现，但是阻断肝期疟原虫的发育可从源头上控制疟疾的感染和复发，因此是设计疟疾预防手段的理想时期。研究发现，肝期疟原虫会被宿主的免疫系统所识别，并受到宿主免疫系统的强烈攻击。然而，疟原虫最终还是能成功地完成肝期的发育，提示肝期疟原虫存在强大的免疫逃避或抑制机制。目前，有关肝期疟原虫的免疫逃避机制还非常不清楚。研究发现，位于肝细胞纳虫空泡内的子孢子，其表面蛋白CSP可借助pexel 功能域从纳虫空泡逸出到肝细胞胞浆内，促进肝期疟原虫的发育，其中的机制可能与CSP能竞争抑制NFκ-B入核有关。然而，本项目的研究发现，CSP蛋白并不能直接促进肝期疟原虫的发育，而是通过抑制IFN-γ对肝期疟原虫的杀伤，从而间接促进肝期疟原虫的发育。其中的机制并不是通过调节肝细胞内的NO的产生实现，而是与CSP能抑制肝细胞对自噬相关蛋白(ATGs)的特异性泛素化有关；而且发现自噬和ATGs是调节IFN-γ对肝期疟原虫的杀伤的关键成分。本项目最终证实，从纳虫空泡逸出到肝细胞胞浆内的CSP是通过促进肝细胞对自噬相关蛋白(ATGs)的特异性泛素化，从而降解ATGs，抑制IFN-γ对肝期疟原虫的杀伤，促进肝期疟原虫的存活和发育；而CSP是通过其核转位序列(NLS) 入核上调E3泛素连接酶NEDD4的表达，从而促进对ATGs的泛素化的。因此，本项目的研究首次揭示了子孢子表面的CSP能抑制肝细胞清除EEFs的新功能，可为设计针对CSP或NEDD4的疟疾预防手段提供重要的理论依据。