Toxoplasma acyl-CoA synthetase TgACS3 is crucial to channel host fatty acids in lipid droplets and for parasite propagation.

Apicomplexa comprise important pathogenic parasitic protists that heavily depend on lipid acquisition to survive within their human host cells. Lipid synthesis relies on the incorporation of an essential combination of fatty acids (FAs) either generated by a metabolically adaptable de novo synthesis in the parasite or by scavenging from the host cell. The incorporation of FAs into membrane lipids depends on their obligate metabolic activation by specific enzyme groups, acyl-CoA synthetases (ACSs). Each ACS has its own specificity, so it can fulfill specific metabolic functions. Whilst such functionalities have been well studied in other eukaryotic models, their roles and importance in Apicomplexa are currently very limited, especially for Toxoplasma gondii. Here, we report the identification of seven putative ACSs encoded by the genome of T. gondii (TgACS), which localize to different sub-cellular compartments of the parasite, suggesting exclusive functions. We show that the perinuclear/cytoplasmic TgACS3 regulates the replication and growth of Toxoplasma tachyzoites. Conditional disruption of TgACS3 shows that the enzyme is required for parasite propagation and survival, especially under high host nutrient content. Lipidomic analysis of parasites lacking TgACS3 reveals its role in the activation of host-derived FAs that are used for i) parasite membrane phospholipid and ii) storage triacylglycerol (TAG) syntheses, allowing proper membrane biogenesis of parasite progenies. Altogether, our results reveal the role of TgACS3 as the bulk FA activator for membrane biogenesis allowing intracellular division and survival in T. gondii tachyzoites, further pointing to the importance of ACS and FA metabolism for the parasite.

The flexibility of Apicomplexa parasites in lipid metabolism.

Apicomplexa are obligate intracellular parasites responsible for major human infectious diseases such as toxoplasmosis and malaria, which pose social and economic burdens around the world. To survive and propagate, these parasites need to acquire a significant number of essential biomolecules from their hosts. Among these biomolecules, lipids are a key metabolite required for parasite membrane biogenesis, signaling events, and energy storage. Parasites can either scavenge lipids from their host or synthesize them de novo in a relict plastid, the apicoplast. During their complex life cycle (sexual/asexual/dormant), Apicomplexa infect a large variety of cells and their metabolic flexibility allows them to adapt to different host environments such as low/high fat content or low/high sugar levels. In this review, we discuss the role of lipids in Apicomplexa parasites and summarize recent findings on the metabolic mechanisms in host nutrient adaptation.

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in calcium-induced Toxoplasma gondii egress.

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.

Protein kinase TgCDPK7 regulates vesicular trafficking and phospholipid synthesis in Toxoplasma gondii.

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.

Protein kinase A negatively regulates Ca2+ signalling in Toxoplasma gondii.

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.

An apically located hybrid guanylate cyclase-ATPase is critical for the initiation of Ca2+ signaling and motility in Toxoplasma gondii.

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.

Calcium negatively regulates secretion from dense granules in Toxoplasma gondii.

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.

Systematic analysis of Plasmodium myosins reveals differential expression, localisation, and function in invasive and proliferative parasite stages.

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.

Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages.

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 13C-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 apical complex provides a regulated gateway for secretion of invasion factors in Toxoplasma.

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.

The acyl-CoA synthetase TgACS1 allows neutral lipid metabolism and extracellular motility in Toxoplasma gondii through relocation via its peroxisomal targeting sequence (PTS) under low nutrient conditions.

Apicomplexa parasites cause major diseases such as toxoplasmosis and malaria that have major health and economic burdens. These unicellular pathogens are obligate intracellular parasites that heavily depend on lipid metabolism for the survival within their hosts. Their lipid synthesis relies on an essential combination of fatty acids (FAs) obtained from both de novo synthesis and scavenging from the host. The constant flux of scavenged FA needs to be channeled toward parasite lipid storage, and these FA storages are timely mobilized during parasite division. In eukaryotes, the utilization of FA relies on their obligate metabolic activation mediated by acyl-co-enzyme A (CoA) synthases (ACSs), which catalyze the thioesterification of FA to a CoA. Besides the essential functions of FA for parasite survival, the presence and roles of ACS are yet to be determined in Apicomplexa. Here, we identified TgACS1 as a Toxoplasma gondii cytosolic ACS that is involved in FA mobilization in the parasite specifically during low host nutrient conditions, especially in extracellular stages where it adopts a different localization. Heterologous complementation of yeast ACS mutants confirmed TgACS1 as being an Acyl-CoA synthetase of the bubble gum family that is most likely involved in ß-oxidation processes. We further demonstrate that TgACS1 is critical for gliding motility of extracellular parasite facing low nutrient conditions, by relocating to peroxisomal-like area.IMPORTANCEToxoplasma gondii, causing human toxoplasmosis, is an Apicomplexa parasite and model within this phylum that hosts major infectious agents, such as Plasmodium spp., responsible for malaria. The diseases caused by apicomplexans are responsible for major social and economic burdens affecting hundreds of millions of people, like toxoplasmosis chronically present in about one-third of the world's population. Lack of efficient vaccines, rapid emergence of resistance to existing treatments, and toxic side effects of current treatments all argue for the urgent need to develop new therapeutic tools to combat these diseases. Understanding the key metabolic pathways sustaining host-intracellular parasite interactions is pivotal to develop new efficient ways to kill these parasites. Current consensus supports parasite lipid synthesis and trafficking as pertinent target for novel treatments. Many processes of this essential lipid metabolism in the parasite are not fully understood. The capacity for the parasites to sense and metabolically adapt to the host physiological conditions has only recently been unraveled. Our results clearly indicate the role of acyl-co-enzyme A (CoA) synthetases for the essential metabolic activation of fatty acid (FA) used to maintain parasite propagation and survival. The significance of our research is (i) the identification of seven of these enzymes that localize at different cellular areas in T. gondii parasites; (ii) using lipidomic approaches, we show that TgACS1 mobilizes FA under low host nutrient content; (iii) yeast complementation showed that acyl-CoA synthase 1 (ACS1) is an ACS that is likely involved in peroxisomal ß-oxidation; (iv) the importance of the peroxisomal targeting sequence for correct localization of TgACS1 to a peroxisomal-like compartment in extracellular parasites; and lastly, (v) that TgACS1 has a crucial role in energy production and extracellular parasite motility.

Toxoplasma metabolic flexibility in different growth conditions.

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.

Toxoplasma LIPIN is essential in channeling host lipid fluxes through membrane biogenesis and lipid storage.

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.

Division and Adaptation to Host Environment of Apicomplexan Parasites Depend on Apicoplast Lipid Metabolic Plasticity and Host Organelle Remodeling.

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.

Photosensitized INA-Labelled protein 1 (PhIL1) is novel component of the inner membrane complex and is required for Plasmodium parasite development.

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.

SAS6-like protein in Plasmodium indicates that conoid-associated apical complex proteins persist in invasive stages within the mosquito vector.

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.

Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites.

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.