Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease, ASP5, at the Host-Parasite Interface

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite’s ability to modulate host signalling pathways and immune responses.

刚地弓形虫（Toxoplasma gondii）编码多组致密颗粒蛋白（dense granule proteins，GRAs），此类蛋白或组装于纳虫空泡膜（parasitophorous vacuole membrane，PVM），或跨该膜分布，且携带有与此前在一系列输出性疟原虫蛋白中鉴定得到的HT/PEXEL基序相似的保守基序。在疟原虫属（Plasmodium spp.）物种中，内质网驻留蛋白酶Plasmepsin V会先对HT/PEXEL基序进行切割，随后相关致病相关蛋白与宿主细胞重塑相关蛋白才能被转运至纳虫空泡膜，并跨膜输出至宿主细胞内。本研究对刚地弓形虫天冬氨酸蛋白酶5（ASP5）开展了功能表征——该蛋白是一类系统发育上与Plasmepsin V同源的高尔基体驻留蛋白酶。研究证实，敲除ASP5会导致体外培养的弓形虫适合度显著下降，并在宿主体内改变寄生虫的毒力表型。此外，本研究发现ASP5是GRA16、GRA19与GRA20的PEXEL样基序发生特异性切割的必需条件。在ASP5缺失的弓形虫中，空泡内纳米管网络消失，多组致密颗粒蛋白无法正常定位于纳虫空泡膜；而此前已被证实会被靶向运输至宿主细胞核的GRA16与GRA24，则被滞留于寄生虫空泡腔中。此外，树突状细胞的过度迁移与缓殖子囊壁形成过程均受到显著抑制，这会严重损害寄生虫的宿主播散能力与持续感染能力。综上，ASP5的缺失会极大削弱弓形虫调控宿主信号通路与免疫应答的能力。