Toxoplasma gondii is an opportunistic pathogen infecting humans and a variety of vertebrate animals. Secretory dense-granule proteins (GRAs) play diverse roles in the mediation of host–parasite interactions and facilitate parasitism, but many of them still remain to be identified. Here, we used two proximity-based protein labeling techniques to identify novel GRA proteins. Taking GRA1 as bait, transgenic strains expressing GRA1-BirA* or GRA1-APEX were constructed to biotinylate GRAs. Using these methods, a total of 46 proteins were identified, 20 of which were known GRA proteins. Among these 46, 17 were identified by both strategies, and 14 out of the 17 were known GRAs. The other three were all confirmed to localize to dense granules. Nonetheless a significant portion of the proteins were only identified by either APEX or BirA*, indicating that there are differences between these methods. Of the 26 novel GRAs, 5 were validated as bona fide GRAs by localization studies. The majority of these novel GRAs are only present in coccidian parasites and are likely dispensable for parasite growth in vitro; they may play roles during animal infections. The identification of novel GRAs laid the foundation for further studies investigating the mechanisms underlying parasite–host interactions.

中文翻译：

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两种基于邻近的生物素化方法鉴定弓形虫中的新型密集颗粒蛋白

弓形虫是一种机会性病原体，可感染人类和各种脊椎动物。分泌型致密颗粒蛋白（GRAs）在宿主-寄生虫相互作用的介导中起多种作用，并促进寄生，但其中许多仍有待确定。在这里，我们使用了两种基于邻近度的蛋白质标记技术来识别新型GRA蛋白质。以GRA1为诱饵，构建表达GRA1-BirA *或GRA1-APEX的转基因菌株以生物素化GRA。使用这些方法，总共鉴定出46种蛋白质，其中20种是已知的GRA蛋白。在这46种方法中，两种策略都鉴定出17种，在17种中有14种是已知的GRA。其他三个都被证实定位于致密颗粒。但是，大部分蛋白质只能通过APEX或BirA *进行鉴定，表示这些方法之间存在差异。在26份新的GRA中，有5份被验证为通过本地化研究获得真正的GRA。这些新的GRAs多数仅存在于球虫寄生虫中，并可能在体外寄生虫生长中消失; 它们可能在动物感染期间发挥作用。新型GRAs的鉴定为进一步研究寄生虫-宿主相互作用的机制奠定了基础。