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

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.

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma.

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

CDPK2A and CDPK1 form a signaling module upstream of Toxoplasma motility.

IMPORTANCE: This work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium-dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating that it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive kinase alleles and conditionally depleted alleles uncovered epistasis between CDPK2A and CDPK1, implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting kinases for anti-parasitic therapies.

Functional profiling of the Toxoplasma genome during acute mouse infection.

Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasite Toxoplasma gondii during mouse infection. Barcoded gRNAs were used to track mutant parasite lineages, enabling detection of bottlenecks and mapping of population structures. We uncovered over 300 genes that modulate parasite fitness in mice with previously unknown roles in infection. These candidates span multiple axes of host-parasite interaction, including determinants of tropism, host organelle remodeling, and metabolic rewiring. We mechanistically characterized three novel candidates, including GTP cyclohydrolase I, against which a small-molecule inhibitor could be repurposed as an antiparasitic compound. This compound exhibited antiparasitic activity against T. gondii and Plasmodium falciparum, the most lethal agent of malaria. Taken together, we present the first complete survey of an apicomplexan genome during infection of an animal host, and point to novel interfaces of host-parasite interaction that may offer new avenues for treatment.

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma.

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways.

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan Taxoplasma gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.

Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress.

Protein kinases regulate fundamental aspects of eukaryotic cell biology, making them attractive chemotherapeutic targets in parasites like Plasmodium spp. and Toxoplasma gondii. To systematically examine the parasite kinome, we developed a high-throughput tagging (HiT) strategy to endogenously label protein kinases with an auxin-inducible degron and fluorophore. Hundreds of tagging vectors were assembled from synthetic sequences in a single reaction and used to generate pools of mutants to determine localization and function. Examining 1,160 arrayed clones, we assigned 40 protein localizations and associated 15 kinases with distinct defects. The fitness of tagged alleles was also measured by pooled screening, distinguishing delayed from acute phenotypes. A previously unstudied kinase, associated with a delayed phenotype, was shown to be a regulator of invasion and egress. We named the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), SPARK lacks a lipid-binding domain, suggesting a rewiring of the pathway in parasites. HiT screening extends genome-wide approaches into complex cellular phenotypes, providing a scalable and versatile platform to dissect parasite biology.

Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii.

Toxoplasma gondii is a single-celled eukaryotic parasite that chronically infects a quarter of the global population. In recent years, phenotypic screens have identified compounds that block parasite replication. Unraveling the pathways and molecular mechanisms perturbed by such compounds requires target deconvolution. In parasites, such deconvolution has been achieved via chemogenomic approaches-for example, directed evolution followed by whole-genome sequencing or genome-wide knockout screens. As a proteomic alternative that directly probes the physical interaction between compound and protein, thermal proteome profiling (TPP), also known as the cellular thermal shift assay (CETSA), recently emerged as a method to identify small molecule-target interactions in living cells and cell extracts in a variety of organisms, including unicellular eukaryotic pathogens. Ligand binding induces a thermal stability shift-stabilizing or destabilizing proteins that change conformationally in response to the ligand-that can be measured by mass spectrometry (MS). Cells are incubated with different concentrations of ligand and heated, causing thermal denaturation of proteins. The soluble protein is extracted and quantified with multiplexed, quantitative MS, resulting in thousands of thermal denaturation profiles. Proteins engaging the ligand can be identified by their compound-dependent thermal shift. The protocol provided here can be used to identify ligand-target interactions and assess the impact of environmental or genetic perturbations on the thermal stability of the proteome in T. gondii and other eukaryotic pathogens. Graphic abstract: Thermal proteome profiling for target identification in the apicomplexan parasite T. gondii.

Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility.

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.

Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling.

Apicomplexan parasites include the causative agents of malaria and toxoplasmosis. Cell-based screens in Toxoplasma previously identified a chemical modulator of calcium signaling (ENH1) that blocked parasite egress from host cells and exhibited potent antiparasitic activity. To identify the targets of ENH1, we adapted thermal proteome profiling to Toxoplasma, which revealed calcium-dependent protein kinase 1 (CDPK1) as a target. We confirmed the inhibition of CDPK1 by ENH1 in vitro and in parasites by comparing alleles sensitive or resistant to ENH1. CDPK1 inhibition explained the block in egress; however, the effects of ENH1 on calcium homeostasis and parasite viability were CDPK1-independent, implicating additional targets. Thermal proteome profiling of lysates from parasites expressing the resistant allele of CDPK1 identified additional candidates associated with the mitochondria and the parasite pellicle-compartments that potentially function in calcium release and homeostasis. Our findings illustrate the promise of thermal profiling to identify druggable targets that modulate calcium signaling in apicomplexan parasites.

The distal cytoplasmic tail of the influenza A M2 protein dynamically extends from the membrane.

The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61-70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle.

A Budding-Defective M2 Mutant Exhibits Reduced Membrane Interaction, Insensitivity to Cholesterol, and Perturbed Interdomain Coupling.

Influenza A M2 is a membrane-associated protein with a C-terminal amphipathic helix that plays a cholesterol-dependent role in viral budding. An M2 mutant with alanine substitutions in the C-terminal amphipathic helix is deficient in viral scission. With the goal of providing atomic-level understanding of how the wild-type protein functions, we used a multipronged site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) approach to characterize the conformational properties of the alanine mutant. We spin-labeled sites in the transmembrane (TM) domain and the C-terminal amphipathic helix (AH) of wild-type (WT) and mutant M2, and collected information on line shapes, relaxation rates, membrane topology, and distances within the homotetramer in membranes with and without cholesterol. Our results identify marked differences in the conformation and dynamics between the WT and the alanine mutant. Compared to WT, the dominant population of the mutant AH is more dynamic, shallower in the membrane, and has altered quaternary arrangement of the C-terminal domain. While the AH becomes more dynamic, the dominant population of the TM domain of the mutant is immobilized. The presence of cholesterol changes the conformation and dynamics of the WT protein, while the alanine mutant is insensitive to cholesterol. These findings provide new insight into how M2 may facilitate budding. We propose the AH-membrane interaction modulates the arrangement of the TM helices, effectively stabilizing a conformational state that enables M2 to facilitate viral budding. Antagonizing the properties of the AH that enable interdomain coupling within M2 may therefore present a novel strategy for anti-influenza drug design.