Lipid kinases are essential for apicoplast homeostasis inToxoplasma gondii

Abstract

Phosphoinositides regulate numerous cellular processes, by recruiting cytosolic effector proteins and acting as membrane signaling entities. The cellular metabolism and localization of phosphoinositides are tightly regulated by distinct lipid kinases and phosphatases. Here, we identify and characterize a unique phosphatidylinositol 3-Kinase (PI3K) in Toxoplasma gondii, a protozoan parasite belonging to the phylum Apicomplexa. Conditional depletion of this enzyme and subsequently of its product, PI(3)P, drastically alters the morphology and inheritance of the apicoplast, an endosymbiontic organelle of algal origin that is a unique feature of many Apicomplexa. We searched the T. gondii genome for PI(3)P binding proteins and identified in total six PX and FYVE-domain containing proteins including a PIKfyve lipid kinase, which phosphorylates PI(3)P into PI(3,5)P2. While depletion of putative PI(3)P binding proteins shows that they are not essential for parasite growth and apicoplast biology, conditional disruption of PIKfyve induces enlarged apicoplasts, as observed upon the loss of PI(3)P. A similar defect of apicoplast homeostasis was also observed by knocking-down the PIKfyve regulatory protein ArPIKfyve, suggesting that in T. gondii, PI(3)P-related function for the apicoplast might mainly be to serve as a precursor for the synthesis of PI(3,5)P2. Accordingly, PI3K is conserved in all apicomplexan parasites whereas PIKfyve and ArPIKfyve are absent in Cryptosporidium species which lack an apicoplast, supporting a direct role of PI(3,5)P2 in apicoplast homeostasis. This study enriches the already diverse functions attributed to PI(3,5)P2 in eukaryotic cells and highlights these parasite lipid kinases as potential drug targets.

The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence

“…RT‐PCR confirmed that the TgArk2 catalytic kinase domain transcript (k) was absent in the Tgark2‐ki strain, while the level of TgFYVE1 mRNA used as a loading control was unchanged (Supporting Information Fig. 3C) (Daher et al ., ). The phenotypic consequence of TgArk2 gene disruption was first examined by plaque assay.…”

“…Plasmids LIC‐CAT‐Ark2Ctg‐3HA and LIC‐CAT‐Ark3Ctg‐3HA were designed to add a sequence coding for a 3HA at the endogenous locus of TGME49_318770 ( TgArk2 ) and TGME49_203010 ( TgArk3 ) open reading frames respectively. A 1101 bp and a 1382 bp fragment corresponding to the 3′ of TgArk2 and TgArk3 respectively were amplified from genomic DNA using primer sets 1/2 and 3/4 respectively and cloned into LIC‐CAT‐3HA vector (Daher et al ., ). Forty micrograms of these plasmids were digested by MluI and ApaI respectively, transfected in the RH‐ku80ko strain and were subjected to chloramphenicol selection.…”

“…Plasmid DHFR-tetO7Sag4-NtArk3 was designed to knockdown the level of expression of Ark3 protein using the tet-off system. A 1498 bp fragment corresponding to the 5′ of the coding region of Ark3 was amplified by PCR from T. gondii genomic DNA using primers 24/25 and cloned BamHI/NotI in the DHFR-TetO7SAG4 plasmid (Sheiner et al, 2011;Morlon-Guyot et al, 2014;Daher et al, 2015;Morlon-Guyot et al, 2015) downstream of the DHFR selection marker, tetO7 operator and pSag4 promoter. The construct was linearized by EcoRV and transfected TATi1-ku80ko parasites with 40 μg of this vector were selected with pyrimethamine and cloned by limiting dilution.…”

Characterisation of two Toxoplasma PROPPINs homologous to Atg18/WIPI suggests they have evolved distinct specialised functions

“…To further verify his, we expressed dT-fused TgPROP1 and TgPROP2 in lipid kinases-deficient parasite cell lines. They were first expressed transiently in a class III phosphatidylinositol 3-kinase (P13k)- deficient cell line, which is unable to generate both PtdIns3P and PtdIns(3,5)P 2 [34], put in nutrient depleted conditions, and puncta were quantified as described previously (Fig 4B). Depletion of TgPI3k activity severely impaired the formation of TgPROP1- and TgPROP2-positive puncta in these conditions (Fig 4B).…”

A Plastid Protein That Evolved from Ubiquitin and Is Required for Apicoplast Protein Import in Toxoplasma gondii

“…Intriguingly, autophagy-related protein 8 and phosphatidylinositol 3-phosphate heavily accumulate on the surface of the apicoplast. However, genetic interference with these mechanisms in T. gondii and Plasmodium falciparum did not produce an unequivocal link to protein import but rather pointed to a broader role of autophagy in apicoplast morphogenesis and inheritance (46–48). While the nature of transport across the outermost membrane remains to be revealed, we know more about the PPM that imported proteins encounter next.…”