A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity inToxoplasma gondii

Abstract

SUMMARY Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, while many of the calcium-dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation we have determined that a nonsense mutation in a previously uncharacterized protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localizes to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network (TVN). Our findings support a connection between the TVN and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.

Identification of Fis1 Interactors in Toxoplasma gondii Reveals a Novel Protein Required for Peripheral Distribution of the Mitochondrion

“…Plaque and doubling assays were performed with 12-well plates using standard methods (50). Briefly, for the plaque assays, 500 freshly egressed parasites were added to confluent HFF monolayers.…”

A Window to Toxoplasma gondii Egress

“…This protein localizes within a recognized candidate for Ca 2+ storage, the TVN. The TVN is also known to be crucial for T. gondii cellular cycle regulation inside the PV, and the absence of GRA41 results in altered morphology of this structure [ 33 ].…”

Toxoplasma gondii LCAT Primarily Contributes to Tachyzoite Egress

“…As for GRA41, a recent study showed that a mutation in this dense granule protein results in aberrations of parasite calcium levels and egress. Parasites lacking GRA41 exhibit dysregulation of calcium ion uptake, leading to altered calcium ion homeostasis and premature egress ( 39 ). Although LCAT and GRA41 appear to affect egress in opposite ways, they both reside in the PV, suggesting an emerging role for PV resident proteins in Toxoplasma egress.…”