Toxoplasma gondii Parasitophorous Vacuole Membrane-Associated Dense Granule Proteins Regulate Maturation of the Cyst Wall

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. 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.

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

“…Bright s-WGA-stained puncta were visible at the cyst periphery in 6-h-differentiated cysts, suggesting that N-acetylglucosamine-modified molecules preferentially accumulated at the cyst periphery/wall early after differentiation. This phenotype mirrors the recent phenotypes that we reported for DBA-stained cyst wall glycoprotein(s) (CST1) and the cyst wall components GRA1, GRA2, GRA4, GRA5, GRA6, GRA7, GRA9, and GRA12 (12,15). The bright puncta present at the cyst periphery 6 h after differentiation may mark the presence of vesicles secreted by bradyzoites that carry cyst wall cargo for wall building to the cyst periphery.…”

“…Future studies with electron and high-resolution microscopy are needed to identify the mechanisms that could traffic cyst wall cargo to the cyst wall. After differentiation is triggered, the initial tachyzoite-stage PVM develops into the limiting cyst membrane (12). The limiting cyst membrane is ruffled (13,16,19).…”

“…The limiting cyst membrane is ruffled (13,16,19). Cyst wall proteins accumulate at the cyst periphery during cyst wall development and localize in the cyst wall beneath the limiting cyst membrane (12,15). The cyst wall structure has a densely compacted outer layer and a less densely compacted inner layer that faces the cyst matrix and is filled with vesicles and membrane tubules (13).…”

Acquisition of Host Cytosolic Protein byToxoplasma gondiiBradyzoites

“…Here we provide evidence that Toxoplasma bradyzoites can ingest host-cytosolic mCherry. This acquisition of host protein by bradyzoites occurs within 4 h of invasion as well as after 24 h of bradyzoite infection, when the cyst wall would have started forming (7). Our findings are therefore consistent with host proteins potentially serving as a nutrition source for bradyzoite cysts.…”

“…During developmental switching from tachyzoite to bradyzoite, parasite proteins are secreted and localized adjacent to the parasitophorous vacuolar membrane (PVM) to form the thick glycan-rich cyst wall (6). As the cyst matures, the composition and localization of T. gondii proteins in the wall is altered while bradyzoites continue to replicate (7,8). These processes require a continued source of nutrients, yet it is unclear from where bradyzoites acquire such resources and what they are.…”