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SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle.

ABSTRACT: Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK complexes with an elongin-like protein to regulate the stability of PKA and PKG in the model apicomplexan Toxoplasma gondii. Defects attributed to SPARK depletion develop after PKG and PKA are down-regulated. Parasites lacking SPARK differentiate into the chronic form of infection, which may arise from reduced activity of a coccidian-specific PKA ortholog. This work delineates the signaling topology of AGC kinases that together control transitions within the asexual cycle of this important family of parasites.

SUBMITTER: Herneisen AL

PROVIDER: S-EPMC11321763 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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SPARK regulates AGC kinases central to the <i>Toxoplasma gondii</i> asexual cycle.

Herneisen Alice L AL Peters Michelle L ML Smith Tyler A TA Shortt Emily E Lourido Sebastian S

eLife 20240813

Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK co ...[more]

PMID: 39136687

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Project description:Toxoplasma gondii is an intracellular parasitic protozoan that poses a significant risk to the fetus carried by a pregnant woman or to immunocompromised individuals. T. gondii tachyzoites duplicate rapidly in host cells during acute infection through endodyogeny. This highly regulated division process is accompanied by complex gene regulation networks. TgAP2XII-9 is a cell cycle-regulated transcription factor, but its specific role in the parasite cell cycle is not fully understood. In this study, we demonstrate that TgAP2XII-9 is identified as a nuclear transcription factor and is dominantly expressed during the S/M phase of the tachyzoite cell cycle. Cleavage Under Targets and Tagmentation (CUT&Tag) results indicate that TgAP2XII-9 targets key genes for the moving junction machinery (RON2, 4, and 8) and daughter cell inner membrane complex (IMC). TgAP2XII-9 deficiency resulted in a significant downregulation of rhoptry proteins and rhoptry neck proteins, leading to a severe defect in the invasion and egress efficiency of tachyzoites. Additionally, the loss of TgAP2XII-9 correlated with a substantial downregulation of multiple IMC and apicoplast proteins, leading to disorders of daughter bud formation and apicoplast inheritance and further contributing to the inability of cell division and intracellular proliferation. Our study reveals that TgAP2XII-9 acts as a critical S/M-phase regulator that orchestrates the endodyogeny and apicoplast division in T. gondii tachyzoites. This study contributes to a broader understanding of the complexity of the parasite's cell cycle and its key regulators.ImportanceThe intracellular apicoplast parasite Toxoplasma gondii poses a great threat to the public health. The acute infection of T. gondii tachyzoites relies on efficient invasion by forming a moving junction structure and also fast replication by highly regulated endodyogeny. This study shows that an ApiAP2 transcription factor, TgAP2XII-9, acts as an activator for the S/M-phase gene expression, including genes related to daughter buds and moving junction formation. Loss of TgAP2XII-9 results in significant growth defects and disorders in endodyogeny and apicoplast inheritance of the parasites. Our results provide valuable insights into the transcriptional regulation of the parasite cell cycle and invading machinery in T. gondii.

Project description:Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii, is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in Toxoplasma gondii has remained largely unknown without understanding its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. By contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which binds GIPLs, exhibits an enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that Δpigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by Δpigj early in the infection in wild-type mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with Δpigj parasites indicate an advantage over wild-type strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcomes in the infected host.IMPORTANCEThe functional significance of sidechain modifications to the glycosylphosphatidylinositol (GPI) anchor in parasites has yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both Toxoplasmsa gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased host susceptibility to infection. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on disease outcomes. This indicates GPI sidechains are fundamental to host-parasite interactions.

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Project description:Apicomplexan parasites mobilize ionic calcium (Ca2+) from intracellular stores to promote microneme secretion and facilitate motile processes including gliding motility, invasion, and egress. Recently, a multipass transmembrane protein, ICM1, was found to be important for calcium mobilization in Plasmodium falciparum and P. berghei. Comparative genomics and phylogenetics have revealed putative ICM orthologs in Toxoplasma gondii and other apicomplexans. T. gondii possesses two ICM-like proteins, which we have named TgICM1-L (TGGT1_305470) and TgICM2-L (TGGT1_309910). TgICM1-L and TgICM2-L localized to undefined puncta within the parasite cytosol. TgICM1-L and TgICM2-L are individually dispensable in tachyzoites, suggesting a potential compensatory relationship between the two proteins may exist. Surprisingly, mutants lacking both TgICM1-L and TgICM2-L are fully viable, exhibiting no obvious defects in growth, microneme secretion, invasion, or egress. Furthermore, loss of TgICM1-L, TgICM2-L, or both does not impair the parasite's ability to mobilize Ca2+. These findings suggest that additional proteins may participate in Ca2+ mobilization or import in Apicomplexa, reducing the dependence on ICM-like proteins in T. gondii. Collectively, these results highlight similar yet distinct mechanisms of Ca2+ mobilization between T. gondii and Plasmodium.IMPORTANCECa2+ signaling plays a crucial role in governing apicomplexan motility; yet, the mechanisms underlying Ca2+ mobilization from intracellular stores in these parasites remain unclear. In Plasmodium, the necessity of ICM1 for Ca2+ mobilization raises the question of whether this mechanism is conserved in other apicomplexans. Investigation into the orthologs of Plasmodium ICM1 in T. gondii revealed a differing requirement for ICM proteins between the two parasites. This study suggests that T. gondii employs ICM-independent mechanisms to regulate Ca2+ homeostasis and mobilization. Proteins involved in Ca2+ signaling in apicomplexans represent promising targets for therapeutic development.

Dataset Information

SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle.

Publications

SPARK regulates AGC kinases central to the <i>Toxoplasma gondii</i> asexual cycle.

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