Functional analysis of calcium sensing proteins in Toxoplasma invasion

Abstract

Toxoplasma gondii – an obligate intracellular parasite – has a complex multistep mechanism for host cell invasion with a pivotal role played by calcium signaling. However, the coordination amongst the players in all the key steps of this signaling pathway, essential for parasitic life cycle of Toxoplasma, is still not entirely known. Given the evolutionary relationship between ciliates and apicomplexans, this work evaluates the functions of three orthologous proteins in cell signaling of alveolates leading to calcium-dependent exocytosis of vesicles such as trichocysts and micronemes. The proteins investigated are calmodulin (CaM) which is a calcium sensor, calcineurin (CN), a protein phosphatase, and parafusin-related protein 1 (PRP1), the Toxoplasma ortholog of ciliate protein parafusin. In the ciliate, CaM activates CN upon rise in intracellular calcium. Activated CN then leads to the dephosphorylation of the secretory vesicle scaffolding protein called parafusin. Parafusin dephosphorylation dissociates it from the calcium-dependent secretory vesicles, which then discharge their contents. As expected upon conditional depletion, we found CaM to be essential for Toxoplasma tachyzoites and CN to be significant for the lytic cycle. Surprisingly, the microneme secretion remained normal in the CN depleted parasites, although there was significant reduction in the attachment of the parasite to the host cell. We also found that PRP1 is dispensable for Toxoplasma lytic cycle, despite its absence affecting the microneme secretion induced by the calcium ionophore, A23187. However the secretion defect is not uniform and remains comparable to the wild type when it is induced using other pharmacological agents like ethanol, zaprinast and propranolol. Therefore despite phylogenetic conservation of the three proteins in alveolates, this work demonstrates their involvement in distinct functional aspects in Toxoplasma compared to other ciliates. Alongside the limited understanding of the molecular mechanism, our knowledge about the cellular sensors in Toxoplasma is also scarce. Our laboratory has previously identified a double C2 (DOC2) domain containing protein called TgDOC2 and demonstrated its role in overall microneme secretion. However, TgDOC2 does not regulate the dosed microneme release associated with varied steps of the egress-invasion trajectory. In general, DOC2 domain containing proteins work in coordination with each other to execute their cellular function, mostly in calcium-dependent manner. We therefore, wanted to expand our knowledge of this domain containing proteins in the parasite. In this work, we investigated a conserved apicomplexan protein called FER2 containing multiple DOC2 domains. Conditional depletion of FER2 appeared detrimental due to significant loss of invasion and attachment of the parasites. FER2 depleted parasites have normal microneme secretion, which is currently the only known calcium-dependent secretory organelle in Toxoplasma, but abrogated the release of rhoptries, the second secretory organelle to be released during invasion. Altogether this work extends the importance of calcium signaling in Toxoplasma gondii and brings into light the novel aspects of some parasitic proteins which are significant in reconstructing our understanding of the signaling pathway in this parasite.