Abstract
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.IMPORTANCEToxoplasma 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.
Highlights
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
To determine whether a similar cyst wall defect is observed in mutants that lack expression of PVM GRAs, we measured Dolichos biflorus agglutinin (DBA) fluorescence intensity at the cyst peripheries of Δgra3, Δgra5, Δgra7, Δgra8, and Δgra14 cysts [57, 58, 61, 62] (Fig. 1A)
DBA fluorescence intensity was quantitatively measured at the cyst periphery, which reflects CST1 cargo delivered to the cyst periphery, and was compared to that in the cyst interior, which reflects CST1 cargo not yet delivered to the cyst wall/membrane
Summary
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. 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 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. We visualized and tracked the localization of Toxoplasma parasitophorous vacuole membrane (PVM) dense granules (GRA) proteins during cyst development in vitro. While the PVM translocation system is crucial for host cell manipulation and parasite survival [27], recent evidence suggests that after stage differentiation into bradyzoites, GRA proteins do not translocate past the cyst wall and cyst membrane, indicating either that the cyst wall is a barrier to GRA translocation or that the cyst membrane lacks a functional translocon [28]. A similar transport mechanism appears to be active in the cyst stage and is mediated by a GRA17 permeability pore in the cyst membrane [31]
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