Optogenetic monitoring identifies phosphatidylthreonine-regulated calcium homeostasis in Toxoplasma gondii

Abstract

Toxoplasma gondii is an obligate intracellular parasite, which inflicts acute as well as chronic infections in a wide range of warm-blooded vertebrates. Our recent work has demonstrated the natural occurrence and autonomous synthesis of an exclusive lipid phosphatidylthreonine in T. gondii. Targeted gene disruption of phosphatidylthreonine synthase impairs the parasite virulence due to unforeseen attenuation of the consecutive events of motility, egress and invasion. However, the underlying basis of such an intriguing phenotype in the parasite mutant remains unknown. Using an optogenetic sensor (gene-encoded calcium indicator, GCaMP6s), we show that loss of phosphatidylthreonine depletes calcium stores in intracellular tachyzoites, which leads to dysregulation of calcium release into the cytosol during the egress phase of the mutant. Consistently, the parasite motility and egress phenotypes in the mutant can be entirely restored by ionophore-induced mobilization of calcium. Collectively, our results suggest a novel regulatory function of phosphatidylthreonine in calcium signaling of a prevalent parasitic protist. Moreover, our application of an optogenetic sensor to monitor subcellular calcium in a model intracellular pathogen exemplifies its wider utility to other entwined systems.

Phosphatidylinositol synthesis, its selective salvage, and inter-regulation of anionic phospholipids in Toxoplasma gondii

“…Lytic cycle assays. Standard phenotyping methods were used to determine the impact of genetic manipulation on the lytic cycle of tachyzoites in vitro, as described earlier 10,53 . Plaque assays were performed by infecting confluent HFF cells in 6-well plates (100-200 parasites/well).…”

“…In context of this report, we showed that the acute stage of T. gondii can synthesize several major classes of phospholipids, namely phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylthreonine (PtdThr), and phosphatidylserine (PtdSer). Among these, PtdThr is an exclusive anionic lipid occurring only in coccidian parasites, which regulates calcium homeostasis in T. gondii 10,11 . PtdThr, PtdCho, and PtdSer are made in the endoplasmic reticulum (ER), whereas PtdEtn is produced at multiple locations including the ER, mitochondrion and parasitophorous vacuole (PV) [3][4][5][6][7][8][9] .…”

Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii

“…However, it was the use of Genetically Encoded Ca 2+ Indicators (GECIs) that provided the final and conclusive evidence that there was a cytosolic Ca 2+ increase right before egress [123]. The use of these indicators has vastly impacted the studies of Ca 2+ in intracellular parasites [95, 117, 123, 127]. Secretion from the micronemes of the perforin-like protein TgPLP1, assists in the permeabilization of the PVM and host cell membrane [128].…”

Illuminating pathogen–host intimacy through optogenetics

“…A pioneering study involving expression of a light-activated adenylate cyclase in the protozoan parasite Toxoplasma gondii has already demonstrated the proof of principle for applying optogenetics in infection research [4]. Equally, expression of gene-encoded biosensors has enabled an otherwise challenging monitoring of subcellular calcium in T. gondii and Plasmodium falciparum enclosed within host cells [8,9]. These works have indeed paved a way to tap the vast potential of optogenetic actuators and biosensors.…”