A forward genetic approach to identifying novel calcium regulators in Toxoplasma Gondii

Abstract

Toxoplasma gondii is an obligate intracellular eukaryotic pathogen that causes severe neurologic disease in immunocompromised adults and congenitally infected neonates. Events critical to the propagation of T. gondii, such as invasion and egress, are regulated by calcium-dependent signaling. In order to identify unique components of the parasite’s calcium signaling networks, members of the Arrizabalaga laboratory have used a forward genetics approach to isolate mutants with altered sensitivity to the calcium ionophore A23187. Exposing extracellular parasites to A23187 induces protein secretion, motility and cytoskeletal rearrangements and prolonged treatment causes exhaustion of factors required for invasion, which results in what is referred to as ionophore induced death (iiDeath). Mutants capable of surviving this treatment were isolated from a chemically mutagenized population. Whole genome sequencing of one such mutant, MBD2.1, identified a nonsense mutation in a protein of unknown function (TGGT1_069070, ToxoDBv7.2) Complementation of MBD 2.1 with a wild-type copy of TGGT1_069070 restored sensitivity to iiDeath treatment. Endogenous tagging of this locus revealed that the encoded protein is secreted from a unique parasite secretory organelle known as the dense granule into the parasitophorous vacuole, leading to its designation as TgGRA41. Complete knockout of TgGRA41 recapitulates the resistance to iiDeath observed in MBD2.1 but also exhibits a dramatic decrease in propagation in tissue culture not seen in the original mutant. The knockout shows defects in multiple steps of the lytic including compromised invasion efficiency and premature egress of parasites from host cells. Cytosolic calcium measurements of extracellular parasites show enhanced uptake of calcium in the knockout strain as compared to parental and complemented, suggesting that the loss of TgGra41 results in calcium dysregulation. Together, these results provide a novel insight into the role that the parasitophorous vacuole of T. gondii plays in calcium homeostasis and calcium-dependent signaling processes.