Abstract text
Dimitrios Alexandros Katelas, Rosalba Cruz Miron, Nishith Gupta
Department of Molecular Parasitology, Faculty of Life Sciences, Humboldt University, Berlin, Germany
Introduction
Toxoplasma gondii is a prominent apicomplexan parasite infecting many warm-blood organisms. Its lytic cycle in human host cells requires a range of phospholipids, some of which have emerged as key players in signaling and calcium regulation.
Objectives
Following our previous research, this work aimed to evaluate the synthesis and roles of anionic phospholipids during the lytic cycle of T. gondii.
Materials & Methods
We engineered a repertoire of parasite mutants (knockout, conditional knockdown, and overexpression strains) and phenotyped them by standard molecular biology methods, including immunofluorescence, western blot, and lipidomic assays.
Results
Herein, we show that tachyzoites of T. gondii harbor two similar enzymes in the endoplasmic reticulum, catalyzing the synthesis of phosphatidylserine (PtdSer) and phosphatidylthreonine (PtdThr). Transgenic expression of a gene-encoded biosensor (LactC2-GFP) revealed the distribution of two phospholipids in the parasite ER and inner membrane complex. While tachyzoites could survive deletion of PtdThr synthase (PTS), de novo synthesis of PtdSer is essential for the lytic cycle. Notably, the PTS mutant displayed a defective calcium homeostasis, resulting in impaired gliding motility and concomitant invasion and egress events. Our lipid binding assays disclosed interaction of PtdThr and PtdSer with the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, and its ectopic overexpression in the PTS mutant restored its lytic cycle. Conversely, auxin-mediated knockdown of SERCA and PtdSer synthase abrogated the parasite development with evident defects in replication and locomotion. Our independent work on P4-ATPases (Chen et al, JBC 2021; CSBJ, in review) has discovered a PtdSer/PtdThr flippase located at the apical end of tachyzoites that contributes to the motility-dependent invasion and egress.
Conclusion
PtdSer in mammalian cells is known to regulate the calcium release via IP3 channel, which remains to be identified in T. gondii (ongoing work). These data, taken together, strongly suggest evolutionary speciation and functional repurposing of two analogous glycerophospholipids to cater the lytic cycle of T. gondii.