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.
Highlights
Toxoplasma gondii is considered as one of the most successful parasites on Earth, infecting humans as well as a wide range of animals [1]
Using an optogenetic sensor, 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
We generated the parental and PTSknockout strains stably expressing the GCaMP6s fusion protein, which consists of a M13 kinase peptide, a circularly permuted enhanced GFP (CpEGFP) barrel and a calcium-sensitive calmodulin (Fig. 1A)
Summary
Toxoplasma gondii is considered as one of the most successful parasites on Earth, infecting humans as well as a wide range of animals [1]. The loss of PtdThr in a mutant of lipid synthesis (Δtgpts strain) does not affect membrane biogenesis and intracellular replication but compromises the gliding motility, which in turn blights the downstream events of parasite egress and invasion. Tachyzoites egressing at the end of lytic cycle exhibit induced cytosolic calcium levels, which exert conformational changes in EGFP, leading to increased fluorescence.
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