Department of Infectious Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Center for Gender-Specific Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Int J Parasitol. 2020 Nov;50(13):1099-1115. doi: 10.1016/j.ijpara.2020.06.012. Epub 2020 Aug 31.
Toxoplasma gondii has a worldwide distribution and infects virtually all warm-blooded animals, including humans. Ingestion of the environmentally resistant oocyst stage, excreted only in the feces of cats, is central to transmission of this apicomplexan parasite. There is vast literature on the host and T. gondii tachyzoite (proliferative stage of the parasite) but little is known of the host-parasite interaction and conversion of the free-living stage (sporozoite inside the oocyst) to the parasitic stage. Here, we present events that follow invasion of host cells with T. gondii sporozoites by using immunofluorescence (IF) and transmission electron microscopy (TEM). Several human type cell cultures were infected with T. gondii sporozoites of the two genotypes (Type II, ME49 and Type III, VEG) most prevalent worldwide. For the first known time, using anti-rhoptry neck protein 4 (RON4) antibodies, the moving junction was visualized in sporozoites during the invasion process and shortly after its completion. Surprisingly, IF and TEM evaluation revealed that intracellular sporozoites release, at their posterior end, long membranous tails, herein named sporozoite-specific trails (SSTs). Differential permeabilization and IF experiments showed that the SSTs are associated with several dense granule proteins (GRAs) and that their membranous component is of parasite origin. Furthermore, TEM observations demonstrated that SST-associated sporozoites are delimited by a typical parasitophorous vacuole, which is retained during parasite exit from the host cell and during cell-to-cell passage. Our data strongly suggest that host cell traversal by T. gondii sporozoites relies on a novel force-producing mechanism, based on the massive extrusion at the parasite posterior pole of GRA-associated membranous material derived from the same pool of membranes forming the intravacuolar network.
刚地弓形虫分布广泛，几乎感染所有温血动物，包括人类。食入环境中具有抵抗力的卵囊阶段，只有猫的粪便中才会排出，这是这种顶复门寄生虫传播的关键。关于宿主和刚地弓形虫速殖子（寄生虫的增殖阶段）有大量文献，但对宿主-寄生虫相互作用以及自由生活阶段（卵囊内的孢子）向寄生阶段的转化知之甚少。在这里，我们通过免疫荧光（IF）和透射电子显微镜（TEM）研究了刚地弓形虫孢子入侵宿主细胞后的一系列事件。用两种基因型（世界范围内最流行的 II 型 ME49 和 III 型 VEG）的刚地弓形虫孢子感染了几种人类细胞培养物。首次使用抗 rhoptry neck protein 4（RON4）抗体，在入侵过程中和完成后不久，在孢子虫中观察到移动连接。令人惊讶的是，IF 和 TEM 评估显示，细胞内的孢子虫在后端释放长膜状尾巴，我们将其命名为孢子虫特异性轨迹（SST）。差异透化和 IF 实验表明，SST 与几种致密颗粒蛋白（GRAs）相关，并且其膜成分来自寄生虫。此外，TEM 观察表明，SST 相关的孢子虫被典型的寄生质膜包围，在寄生虫从宿主细胞逸出和细胞间传递过程中都保留着该质膜。我们的数据强烈表明，刚地弓形虫孢子虫穿过宿主细胞依赖于一种新的产生力的机制，该机制基于寄生虫后端大量挤出与致密颗粒相关的膜状物质，这些物质来自形成腔内网络的同一膜池。
