After differentiation is triggered, the tachyzoite-stage Toxoplasma gondii parasitophorous vacuole membrane (PVM) has been hypothesized to transition into the cyst membrane that surrounds the cyst wall and encloses bradyzoites. Here, we tracked the localization of two PVM dense granule (GRA) proteins (GRA5 and GRA7) after in vitro differentiation of the tachyzoite stage parasitophorous vacuole into the mature cyst. GRA5 and GRA7 were visible at the cyst periphery at 6 h and at all later times after differentiation, suggesting that the PVM remained intact as it transitioned into the cyst membrane. By day 3 postdifferentiation, GRA5 and GRA7 were visible in a continuous pattern at the cyst periphery. In mature 7- and 10-day-old cysts permeabilized with a saponin pulse, GRA5 and GRA7 were localized to the cyst membrane and the cyst wall regions. Cysts at different stages of cyst development exhibited differential susceptibility to saponin permeabilization, and, correspondingly, saponin selectively removed GRA5 from the cyst membrane and cyst wall region in 10-day-old cysts. GRA5 and GRA7 were localized at the cyst membrane and cyst wall region at all times after differentiation of the parasitophorous vacuole, which supports a previous model proposing that the PVM develops into the cyst membrane. In addition, evaluation of Δgra3, Δgra5, Δgra7, Δgra8, and Δgra14 mutants revealed that PVM-localized GRAs were crucial to support the normal rate of accumulation of cyst wall proteins at the cyst periphery.IMPORTANCE Toxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. Once host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Furthermore, how the cyst membrane and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma parasitophorous vacuole membrane (PVM) dense granules (GRA) proteins during cyst development in vitro. PVM-localized GRA5 and GRA7 were found at the cyst membrane and cyst wall region throughout cyst development, suggesting that the PVM remains intact and develops into the cyst membrane. In addition, our results show that genetic deletion of PVM GRAs reduced the rate of accumulation of cyst wall cargo at the cyst periphery and suggest that PVM-localized GRAs mediate the development and maturation of the cyst wall and cyst membrane.

中文翻译：

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弓形虫弓形虫寄生虫膜相关的致密颗粒蛋白调节囊肿壁的成熟。

分化被触发后，已假设速殖子阶段的弓形虫弓形虫寄生液泡膜（PVM）过渡到囊膜中，囊膜围绕囊壁并包围着缓殖子。在这里，我们跟踪了速殖子阶段寄生虫空泡成成熟囊肿的体外分化后，两个PVM致密颗粒（GRA）蛋白（GRA5和GRA7）的定位。在分化后的6小时及以后的所有时间，在囊肿外围都可以看到GRA5和GRA7，这表明PVM过渡到囊膜后仍保持完整。到分化后第3天，在囊肿外围连续可见GRA5和GRA7。在成熟的7天和10天大的囊肿中，其皂素脉冲可透化，GRA5和GRA7定位于囊膜和囊壁区域。处于囊肿发育不同阶段的囊肿对皂苷通透性表现出不同的敏感性，因此，皂苷选择性地从10天大的囊肿中从囊膜和囊壁区域去除GRA5。寄生虫空泡分化后，GRA5和GRA7始终位于囊膜和囊壁区域，这支持了先前的模型，即PVM发育成囊膜。此外，对Δgra3，Δgra5，Δgra7，Δgra8和Δgra14突变体的评估显示，PVM定位的GRA对支持囊肿外围囊壁蛋白的正常积累速率至关重要。重要的弓形虫通过形成形成人类慢性感染保留在大脑中的厚壁囊肿。一旦宿主免疫力减弱，囊肿就会重新激活，导致严重的，并经常致命，弓形虫性脑炎。目前尚无消除囊肿或防止其重新活化的疗法。此外，对囊膜和囊壁结构如何发展的了解很少。在这里，我们可视化和跟踪在体外囊肿发展过程中弓形虫的寄生虫液泡膜（PVM）致密颗粒（GRA）蛋白的定位。在整个囊肿形成过程中，在囊膜和囊壁区域发现了PVM定位的GRA5和GRA7，这表明PVM保持完整并发展为囊膜。此外，我们的结果表明，PVM GRA的遗传缺失降低了囊壁周边囊壁货物的积累速率，并表明PVM定位的GRA介导了囊壁和囊膜的发育和成熟。目前尚无消除囊肿或防止其重新活化的疗法。此外，对囊膜和囊壁结构如何发展的了解很少。在这里，我们可视化和跟踪在体外囊肿发展过程中弓形虫的寄生虫液泡膜（PVM）致密颗粒（GRA）蛋白的定位。在整个囊肿形成过程中，在囊膜和囊壁区域发现了PVM定位的GRA5和GRA7，这表明PVM保持完整并发展为囊膜。此外，我们的结果表明，PVM GRA的遗传缺失降低了囊壁周边囊壁货物的积累速率，并表明PVM定位的GRA介导了囊壁和囊膜的发育和成熟。目前尚无消除囊肿或防止其重新活化的疗法。此外，对囊膜和囊壁结构如何发展的了解很少。在这里，我们可视化和跟踪在体外囊肿发展过程中弓形虫的寄生虫液泡膜（PVM）致密颗粒（GRA）蛋白的定位。在整个囊肿形成过程中，在囊膜和囊壁区域发现了PVM定位的GRA5和GRA7，这表明PVM保持完整并发展为囊膜。此外，我们的结果表明，PVM GRA的遗传缺失降低了囊壁周边囊壁货物的积累速率，并表明PVM定位的GRA介导了囊壁和囊膜的发育和成熟。此外，对囊膜和囊壁结构如何发展的了解很少。在这里，我们可视化和跟踪在体外囊肿发展过程中弓形虫的寄生虫液泡膜（PVM）致密颗粒（GRA）蛋白的定位。在整个囊肿形成过程中，在囊膜和囊壁区域发现了PVM定位的GRA5和GRA7，这表明PVM保持完整并发展为囊膜。此外，我们的结果表明，PVM GRA的遗传缺失降低了囊壁周边囊壁货物的积累速率，并表明PVM定位的GRA介导了囊壁和囊膜的发育和成熟。此外，对囊膜和囊壁结构如何发展的了解很少。在这里，我们可视化和跟踪在体外囊肿发展过程中弓形虫的寄生虫液泡膜（PVM）致密颗粒（GRA）蛋白的定位。在整个囊肿形成过程中，在囊膜和囊壁区域发现了PVM定位的GRA5和GRA7，这表明PVM保持完整并发展为囊膜。此外，我们的结果表明，PVM GRA的遗传缺失降低了囊壁周边囊壁货物的积累速率，并表明PVM定位的GRA介导了囊壁和囊膜的发育和成熟。我们可视化并跟踪了体外囊肿发育过程中弓形虫的寄生虫液泡膜（PVM）致密颗粒（GRA）蛋白的定位。在整个囊肿形成过程中，在囊膜和囊壁区域发现了PVM定位的GRA5和GRA7，这表明PVM保持完整并发展为囊膜。此外，我们的结果表明，PVM GRA的遗传缺失降低了囊壁周边囊壁货物的积累速率，并表明PVM定位的GRA介导了囊壁和囊膜的发育和成熟。我们可视化并跟踪了体外囊肿发育过程中弓形虫的寄生虫液泡膜（PVM）致密颗粒（GRA）蛋白的定位。在整个囊肿形成过程中，在囊膜和囊壁区域发现了PVM定位的GRA5和GRA7，这表明PVM保持完整并发展为囊膜。此外，我们的结果表明，PVM GRAs的遗传缺失降低了囊壁周围囊壁货物的积累速率，并表明PVM定位的GRAs介导了囊壁和囊膜的发育和成熟。