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Apicomplexan parasites have complex metabolic networks that coordinate acquisition of metabolites by de novo synthesis and by scavenging from the host. Toxoplasma gondii has a wide host range and may rely on the flexibility of this metabolic network. Currently, the literature categorizes genes as essential or dispensable according to their dispensability for parasite survival under nutrient-replete in vitro conditions. However, recent studies revealed correlations between medium composition and gene essentiality. Therefore, nutrient availability in the host environment likely determines the requirement of metabolic pathways, which may redefine priorities for drug target identification in a clinical setting. Here we review the recent work characterizing some of the major Toxoplasma metabolic pathways and their functional adaptation to host nutrient content.

Fundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of egress. Biochemical analysis of 4 phosphodiesterases (PDEs) identified in our phosphoproteomes establishes PDE2 as a cAMP-specific PDE which regulates Ca2+ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca2+ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways.

Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7., Author summary In this study, we demonstrate that protein kinase TgCDPK7 regulates cellular processes like vesicular trafficking and the synthesis of phospholipids, which are critical for the development of the parasite Toxoplasma gondii. It regulates the localization of a small GTPase TgRab11a by phosphorylating it at a specific site, which is critical for trafficking of important parasite proteins and is important for parasite division. TgCDPK7 may regulate key enzymes involved biogenesis of phosphatidylethanolamine, which may contribute to the synthesis of this important phospholipid. These and other studies shed light on a novel signaling pathway in apicomplexan parasite Toxoplasma gondii.

Host cell invasion and subsequent egress byToxoplasmaparasites is regulated by a network of cGMP, cAMP, and calcium signalling proteins. Such eukaryotic signalling networks typically involve lipid second messengers including phosphatidylinositol phosphates (PIPs), diacylglycerol (DAG) and phosphatidic acid (PA). However, the lipid signalling network inToxoplasmais poorly defined. Here we present lipidomic analysis of a mutant of central flippase/guanylate cyclase TgGC inToxoplasma, which we show has disrupted turnover of signalling lipids impacting phospholipid metabolism and membrane stability. The turnover of signalling lipids is extremely rapid in extracellular parasites and we track changes in PA and DAG to within 5 seconds, which are variably defective upon disruption of TgGC and other signalling proteins. We then identify the position of each protein in the signal chain relative to the central cGMP signalling protein TgGC and map the lipid signal network coordinating conoid extrusion and microneme secretion for egress and invasion.

Summary: Apicomplexan parasites are unicellular eukaryotic pathogens that must obtain and combine lipids from both host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the role and regulation of each lipid source upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase, TgATS2, shows that the apicoplast provides (lyso)phosphatidic acid, required for the recruitment of a critical dynamin (TgDrpC) during parasite cytokinesis. Disruption of TgATS2 also leads parasites to shift metabolic lipid acquisition from de novo synthesis toward host scavenging. We show that both lipid scavenging and de novo synthesis pathways in wild-type parasites exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (1) upregulation of de novo fatty acid synthesis capacities in the apicoplast and (2) parasite-driven host remodeling to generate multi-membrane-bound structures from host organelles that are imported toward the parasite. : Apicoplast de novo lipid synthesis and lipid host scavenging are both critical for apicomplexan intracellular development. Amiar et al. show that the parasite adapts to the fluctuations of host nutritional content to regulate the metabolic activity of both apicoplast and scavenging pathways and maintain parasite development and division. Keywords: Apicomplexa, toxoplasmosis, malaria, apicoplast, lipid synthesis, phosphatidic acid, host-parasite interaction, lipidomics, host nutritional environment, cytokinesis

Plasmodium parasites, the causative agents of malaria, possess a distinctive membranous structure of flattened alveolar vesicles supported by a proteinaceous network, and referred to as the inner membrane complex (IMC). The IMC has a role in actomyosin-mediated motility and host cell invasion. Here, we examine the location, protein interactome and function of PhIL1, an IMC-associated protein on the motile and invasive stages of both human and rodent parasites. We show that PhIL1 is located in the IMC in all three invasive (merozoite, ookinete-, and sporozoite) stages of development, as well as in the male gametocyte and locates both at the apical and basal ends of ookinete and sporozoite stages. Proteins interacting with PhIL1 were identified, showing that PhIL1 was bound to only some proteins present in the glideosome motor complex (GAP50, GAPM1–3) of both P. falciparum and P. berghei. Analysis of PhIL1 function using gene targeting approaches indicated that the protein is required for both asexual and sexual stages of development. In conclusion, we show that PhIL1 is required for development of all zoite stages of Plasmodium and it is part of a novel protein complex with an overall composition overlapping with but different to that of the glideosome.

The myosin superfamily comprises of actin‐dependent eukaryotic molecular motors important in a variety of cellular functions. Although well studied in many systems, knowledge of their functions in Plasmodium, the causative agent of malaria, is restricted. Previously, six myosins were identified in this genus, including three Class XIV myosins found only in Apicomplexa and some Ciliates. The well characterized MyoA is a Class XIV myosin essential for gliding motility and invasion. Here, we characterize all other Plasmodium myosins throughout the parasite life cycle and show that they have very diverse patterns of expression and cellular location. MyoB and MyoE, the other two Class XIV myosins, are expressed in all invasive stages, with apical and basal locations, respectively. Gene deletion revealed that MyoE is involved in sporozoite traversal, MyoF and MyoK are likely essential in the asexual blood stages, and MyoJ and MyoB are not essential. Both MyoB and its essential light chain (MCL‐B) are localised at the apical end of ookinetes but expressed at completely different time points. This work provides a better understanding of the role of actomyosin motors in Apicomplexan parasites, particularly in the motile and invasive stages of Plasmodium during sexual and asexual development within the mosquito., Very little was known about the family of six myosins in malaria‐causing parasites, except for a role of one in motility. We show here that myosins have a diverse pattern of expression and subcellular location through the parasite life cycle in both the mammalian host and mosquito vector. Three are essential for parasite development and growth in the mammalian bloodstream and one has a role in parasite migration to the mosquito salivary gland.

International audience; Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca2+] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca2+ ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K+] to activate cytosolic [Ca2+] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca2+ signaling and motility in Toxoplasma.

Apicomplexan parasites are unicellular eukaryotes responsible for major human diseases including malaria and toxoplasmosis. Apicomplexan parasites must obtain and combine lipids both from host cell scavenging andde novosynthesis to maintain parasite propagation and survival within their human host. Major questions on the actual role for each lipid source or how these are regulated upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase TgATS2, shows that the apicoplast provides local (lyso)phosphatidic acid balance, which is required for the recruitment of a novel dynamin (TgDrpC) critical during parasite cytokinesis. Disruption of TgATS2 led parasites to shift metabolic lipid acquisition fromde novosynthesis towards host scavenging. Importantly, both lipid scavenging andde novosynthesis pathways exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (i) up-regulation ofde novofatty acid synthesis capacities in the apicoplast, and (ii) parasite-driven host cell remodelling to generate multi-membrane-bound structures from host organelles that are imported towards the parasite.

Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca2+ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca2+, and we show that this Ca2+‐mediated response is contingent on the function of calcium‐dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca2+ signalling plays in the invasion biology of apicomplexan parasites.

The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating stages and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not understood. Here, we show that the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that PKAc1 is sequestered to the parasite periphery by dual acylation of PKA regulatory subunit 1 (PKAr1). Upon genetic depletion of PKAc1 we show that newly invaded parasites exit host cells shortly thereafter, in a perforin-like protein 1 (PLP-1)-dependent fashion. Furthermore, we demonstrate that loss of PKAc1 prevents rapid down-regulation of cytosolic [Ca2+] levels shortly after invasion. We also provide evidence that loss of PKAc1 sensitises parasites to cyclic GMP (cGMP)-induced Ca2+ signalling, thus demonstrating a functional link between cAMP and these other signalling modalities. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity., Author summary Central to pathogenesis and infectivity of Toxoplasma and related parasites is their ability to move through tissue, invade host cells, and establish a replicative niche. Ca2+-dependent signalling pathways are important for activating motility, host cell invasion, and egress, yet how this signalling is turned off after invasion is unclear. Here, we show that a cAMP-dependent protein kinase A (PKA) is essential for rapid suppression of Ca2+ signalling upon completion of host cell invasion. Parasites lacking this kinase rapidly invoke an egress program to re-exit host cells, thus preventing the establishment of a stable infection. This finding therefore highlights the first factor required for Toxoplasma (and any related apicomplexan parasite) to switch from invasive to the replicative forms.

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca(2+)] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca(2+) ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K(+)] to activate cytosolic [Ca(2+)] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca(2+) signaling and motility in Toxoplasma.

Apicomplexan parasites includingToxoplasma gondiiandPlasmodiumspp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite’s invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca2+act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca2+, and we show that this Ca2+-mediated response is contingent on the function of calcium-dependent protein kinasesTgCDPK1 andTgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca2+signalling plays in the invasion biology of apicomplexan parasites.

Apicomplexan parasites are pathogens responsible for major human diseases such as toxoplasmosis caused by Toxoplasma gondii and malaria caused by Plasmodium spp. Throughout their intracellular division cycle, the parasites require vast and specific amounts of lipids to divide and survive. This demand for lipids relies on a fine balance between de novo synthesized lipids and scavenged lipids from the host. Acetyl-CoA is a major and central precursor for many metabolic pathways, especially for lipid biosynthesis. T. gondii possesses a single cytosolic acetyl-CoA synthetase (TgACS). Its role in the parasite lipid synthesis is unclear. Here, we generated an inducible TgACS KO parasite line and confirmed the cytosolic localization of the protein. We conducted (13)C-stable isotope labeling combined with mass spectrometry-based lipidomic analyses to unravel its putative role in the parasite lipid synthesis pathway. We show that its disruption has a minor effect on the global FA composition due to the metabolic changes induced to compensate for its loss. However, we could demonstrate that TgACS is involved in providing acetyl-CoA for the essential fatty elongation pathway to generate FAs used for membrane biogenesis. This work provides novel metabolic insight to decipher the complex lipid synthesis in T. gondii.

The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating forms and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is not understood. Here we show that the cAMP-dependent Protein Kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that that PKAc1 is sequestered to the parasite periphery by dual acylation of its regulatory subunit PKAr1. Newly invaded PKAc1-deficient parasites exit host cells shortly thereafter in a perforin-like protein 1 (PLP-1)-dependent fashion. We demonstrate that loss of PKAc1 results in an inability to rapidly downregulate cytosolic Ca2+ levels shortly after invasion. Furthermore, we demonstrate that PKAc1 also specifically negatively regulates resting cytosolic Ca2+ in conditions that mimic intracellularity. We also show that cAMP and cGMP have opposing role in microneme secretion, further supporting evidence that cAMP signalling has a suppressive role during motility. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.

The SAS6-like (SAS6L) protein, a truncated paralogue of the ubiquitous basal body/centriole protein SAS6, has been characterised recently as a flagellum protein in trypanosomatids, but associated with the conoid in apicomplexan Toxoplasma. The conoid has been suggested to derive from flagella parts, but is thought to have been lost from some apicomplexans including the malaria-causing genus Plasmodium. Presence of SAS6L in Plasmodium, therefore, suggested a possible role in flagella assembly in male gametes, the only flagellated stage. Here, we have studied the expression and role of SAS6L throughout the Plasmodium life cycle using the rodent malaria model P. berghei. Contrary to a hypothesised role in flagella, SAS6L was absent during gamete flagellum formation. Instead, SAS6L was restricted to the apical complex in ookinetes and sporozoites, the extracellular invasive stages that develop within the mosquito vector. In these stages SAS6L forms an apical ring, as we show is also the case in Toxoplasma tachyzoites. The SAS6L ring was not apparent in blood-stage invasive merozoites, indicating that the apical complex is differentiated between the different invasive forms. Overall this study indicates that a conoid-associated apical complex protein and ring structure is persistent in Plasmodium in a stage-specific manner.

The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga., King Abdullah University of Science and Technology (KAUST; Council of Scientific and Industrial Research; National Institute of Allergy and Infectious Diseases (NIAID); Australian Research Council (ARC); Monash University; National Health and Medical Research Council (NHMRC); Czech Science Foundation (Grantova´ agentura Ceske´ republiky)

The apical complex is the definitive cell structure of phylum Apicomplexa, and is the focus of the events of host cell penetration and the establishment of intracellular parasitism. Despite the importance of this structure, its molecular composition is relatively poorly known and few studies have experimentally tested its functions. We have characterized a novel Toxoplasma gondii protein, RNG2, that is located at the apical polar ring—the common structural element of apical complexes. During cell division, RNG2 is first recruited to centrosomes immediately after their duplication, confirming that assembly of the new apical complex commences as one of the earliest events of cell replication. RNG2 subsequently forms a ring, with the carboxy- and amino-termini anchored to the apical polar ring and mobile conoid, respectively, linking these two structures. Super-resolution microscopy resolves these two termini, and reveals that RNG2 orientation flips during invasion when the conoid is extruded. Inducible knockdown of RNG2 strongly inhibits host cell invasion. Consistent with this, secretion of micronemes is prevented in the absence of RNG2. This block, however, can be fully or partially overcome by exogenous stimulation of calcium or cGMP signaling pathways, respectively, implicating the apical complex directly in these signaling events. RNG2 demonstrates for the first time a role for the apical complex in controlling secretion of invasion factors in this important group of parasites., Author Summary Apicomplexan parasites comprise major human pathogens, including the malaria-causing parasites Plasmodium spp., and Toxoplasma gondii that causes birth defects and neurological disorders. Key to the success of this group was the evolution of the apical complex, a structure at the focus of the events of host cell invasion. This structure was recently shown to derive from elements of the flagellar apparatus, and rudiments of an apical complex are used for feeding in related protists. Evolution of host cell invasion in Apicomplexa has entailed development of a coordinated secretion of invasion factors from the cell apex. Little is known, however, of the behaviour or function of the components of the apical complex during invasion. We have characterized a new protein, RNG2, that forms a ring at the heart of the apical complex in T. gondii. This is a dynamic ring that links the mobile conoid with the apical polar ring, and is assembled as one of the first structures in replicating parasites. When RNG2 is artificially depleted, cells become insensitive to the molecular cues for secretion, and invasion of host cells is blocked. This reveals that the apical complex participates directly in regulating secretion, and controlling the events of invasion.