Cyrille Y. Botte

Toxoplasma gondii is an apicomplexan parasite which can infect a diverse range of warm-blooded host species. This promiscuous lifestyle requires metabolic flexibility yet our understanding of the metabolic pathways in this parasite remains incomplete. This gap includes the endoplasmic reticulum localised fatty acid elongation (FAE) pathway, which is responsible for synthesis of long (LCFA) and very long chain fatty acids (VLCFA), thus contributing to critical functions such as lipid storage and membrane biogenesis. Analyses of FAE in the commonly studied type I T. gondii strain revealed its contribution to the production of LCFA and VLCFA in vitro . However, the cystogenic type II T. gondii, which is responsible for most toxoplasmosis cases worldwide, was not studied. These type II parasites can readily differentiate between virulent and immune-evasive stages whereas type I cannot. This ability to readily transition between life stages, which are metabolically fundamentally different, prompts the question of whether the FAE pathway may play different roles in their distinct phenotype. Here we address this question, first confirming the role of the FAE enzymes, TgELO-A & B, in the production of LCFA and VLCFA in a type II T. gondii strain. Next, we demonstrated that their deletion leads to defects in replication and invasion when grown under lipid-restricted conditions. The mutants were further shown to be attenuated in mice as demonstrated by extended survival. Importantly, FAE enzyme deletion caused reduced brain cyst burdens. These findings highlight the importance of FAE for parasite survival under metabolic stress and in vivo . Importance: Toxoplasma gondii infects a wide range of hosts and persists for life in multiple tissues, making metabolic flexibility essential for its success. Fatty acid elongation (FAE), mediated by elongase (ELO) enzymes, enables the parasite to modify both de novo synthesised and host-scavenged fatty acids to support membrane biogenesis, lipid storage, and adaptation to changing nutrient environments. Previous studies in the type I strain established the importance of FAE for parasite metabolism, but its role in the clinically relevant, cystogenic type II lineage—which causes most cases of human toxoplasmosis and forms long-lived, immune-evasive bradyzoite cysts— remained unknown. Here, we address this gap by characterising type II ELO mutants and show that disruption of FAE remodels the parasite lipidome, impairs fitness under fatty acid-limiting conditions, and reduces virulence and chronic persistence in mice. These findings identify FAE as a key metabolic pathway underpinning adaptation and persistence during chronic infection.

A complex transcriptional cascade drives the two-day intraerythrocytic developmental cycle of the most virulent human malaria parasite, Plasmodium falciparum , in the human host. Genes are rapidly activated at specific times, then silenced as quickly. Transcriptional activity correlates with histone acetylation, which is modulated by acetyltransferases that use acetyl-CoA and deacetylases that produce acetate. Acetyl-CoA synthetase (ACAS) uses acetate to produce acetyl-CoA and has been implicated in histone acetylation, providing an interesting link between parasite metabolism and transcription. Here, we show that ACAS becomes enriched in the nucleus at a time during the life cycle when the parasite undergoes rapid growth and increased levels of transcription. We use mass spectrometry to show that ACAS inhibition results in a rapid and global depletion of histone acetylation, which leads to a general decrease in chromatin accessibility at gene promoters. These widespread alterations in chromatin composition disrupted the transcriptional cascade, resulting in cell cycle arrest. Our study provides evidence that ACAS plays an important nuclear role in the histone acetylation cycle and insight into the dynamic nature and essentiality of histone acetylation in the parasite’s complex transcriptional program driving infection of the human host.

Lipid droplets (LD) are dynamic organelles largely distributed in all living cells that are essential notably for membrane biogenesis and cell proliferation. In Trypanosoma brucei , a protozoan parasite responsible for major lethal infectious diseases in humans and cattle, lipid acquisition and mobilization are essential for its survival and adaptation to its different hosts, but the roles of LD in these processes remain largely unknown. To address this gap, we identified and characterized a unique functional orthologue of the human adipose triacylglycerol lipase (ATGL), responsible for the initiation of neutral lipid mobilization, in T. brucei . In this study, we present TbPatatin-like (TbPat), the first ATGL ortholog in protozoan parasites. By combining reverse genetics, microscopy and –omics analysis, we deciphered the role of TbPat in lipolysis and neutral lipid maintenance in both insect and mammalian stages of African trypanosomes and imaged its distribution on the LD surface at an unprecedented resolution by using Cryo-Expansion microscopy. We found that inactivation of TbPat in mammalian form T. brucei led to a global upregulation of the mitochondrial metabolic proteome; furthermore, these cells appeared remarkably unable to differentiate into the procyclic insect stage. Through its expression and localization, TbPat appears to be at an essential checkpoint linking lipid environment and metabolism to more complex cell programs involving proteome, compartment remodeling and lifecycle completion.

Aquaglyceroporins (AQPs) are integral membrane channel proteins that facilitate transport of water and other solutes such as glycerol, thereby playing vital roles in cellular homeostasis. Here, we functionally characterized Plasmodium falciparum aquaglyceroporin (PfAQP) through localization, inducible knockdown, and comprehensive metabolomic analyses revealing its key role in lipid homeostasis and metabolomic flux. PfAQP localizes to the parasite plasma membrane and food vacuole membrane, at critical interfaces for nutrient and metabolite exchange. A glmS-ribozyme mediated inducible knock-down of PfAQP severely impairs parasite development from trophozoite to schizont, leading to marked suppression in parasite growth. Ultra-Expansion Microscopy (UExM) analyses show profound defects in membrane biogenesis, along with disruption in cytokinesis under PfAQP knock-down conditions. Detailed lipidomic analyses suggests that PfAQP disruption cause altered lipid homeostasis, characterized by reduced membrane phospholipid biosynthesis, impaired lipid scavenging, and accumulation of storage lipids. Metabolomic analyses further indicate metabolic dysregulation characterized by reduction in amino acid levels and dysregulated mitochondrial metabolism, including impaired tricarboxylic acid (TCA) cycle. Strikingly, PfAQP expression gets transiently upregulated under nutrient starvation condition, and its ablation drastically compromise parasite recovery, highlighting its role in adaptive responses. Overall, these results establish PfAQP as a critical regulator of glycerol-dependent metabolic pathways, orchestrating membrane biogenesis, energy metabolism, and stress resilience-functions indispensable for parasite growth and survival.

Malaria is caused by Plasmodium parasites and claims 610,000 deaths annually. Parasite invasion of red blood cells (RBCs) involves secretion of multiple proteins from specialised apical organelles (micronemes, rhoptries, and dense granules). These collectively facilitate host cell entry, establish a protective parasitophorous vacuole (PV), and directly and indirectly mediate interactions between the infected RBC (iRBC) and the endothelial wall. The cytoadherence of iRBCs results in sequestration within organs and plays a critical role in pathogenesis and virulence, but the function of many secreted parasite proteins remains poorly characterised. Here, we leverage spatial proteomics from Plasmodium falciparum to identify two novel Plasmodium berghei orthologue proteins containing hydrolase domains (MAP1, PBANKA_1425900, and RhoSH, PBANKA_1001500). Ultrastructure expansion microscopy reveals their localisation to the rhoptries in late schizogony, where co-immunoprecipitation assays show they interact with each other. In vivo studies demonstrate that these proteins help the parasite evade spleen-mediated clearance and contribute to disease progression. Deletion of both genes disrupts lipid metabolism and PV membrane morphology, suggesting a role in establishing or maintaining the protective PV membrane. Loss of map1 reduces sequestration of P. berghei iRBCs to adipose tissue, and conditional knockdown of the P. falciparum map1 orthologue results in reduced CD36-mediated cytoadhesion, suggesting a cross-species role in sequestration and cytoadherence. Our findings identify MAP1 and RhoSH as key mediators of Plasmodium virulence.

Toxoplasma gondii, an apicomplexan parasite and agent of the human disease toxoplasmosis, possesses a non-photosynthetic relic plastid, named the apicoplast. Thought to be evolved from a red algal plastid, the apicoplast houses major metabolic pathways, such as heme, isoprenoid and lipid synthesis, crucial for parasite survival, and thus considered attractive drug targets. However, despite similarities with plant chloroplast lipid synthesis pathways, the apicoplast lacks canonical plant/chloroplast lipid transporters and so metabolite import/export is at present, poorly characterised. Here we identify TgFLP12, a newly identified P5-ATPase transporter localised to the Toxoplasma apicoplast. TgFLP12 is found in the SAR (Stramenopile-Alveolata-Rhizaria) supergroup (to which belong Apicomplexa parasites and chromerids) but absent in higher plants. Disruption of TgFLP12 causes major defects on apicoplast morphology. Lipidomic analyses and stable isotope labelling reveal a unique accumulation of C14:0 in the apicoplast, which is then lacking in most major lipid classes subsequently synthesized in the ER. Successful complementation of a yeast mutant deficient in fatty acid transport with TgFLP12 validates TgFLP12 as a fatty acid transporter. Overall, we identify a potentially important drug target: the apicoplast fatty acid exporter, specific to Apicomplexa which unexpectedly also highlights Toxoplasma’s utility as a model organism for investigating algal biology.

Mitosis in eukaryotes involves reorganisation of the nuclear envelope (NE) and microtubule-organising centres (MTOCs). During male gametogenesis in Plasmodium, the causative agent of malaria, mitosis is exceptionally rapid and highly divergent. Within 8 min, the haploid male gametocyte genome undergoes three replication cycles (1N to 8N), while maintaining an intact NE. Axonemes assemble in the cytoplasm and connect to a bipartite MTOC-containing nuclear pole (NP) and cytoplasmic basal body, producing eight flagellated gametes. The mechanisms coordinating NE remodelling, MTOC dynamics, and flagellum assembly remain poorly understood. We identify the SUN1-ALLAN complex as a novel mediator of NE remodelling and bipartite MTOC coordination during Plasmodium berghei male gametogenesis. SUN1, a conserved NE protein, localises to dynamic loops and focal points at the nucleoplasmic face of the spindle poles. ALLAN, a divergent allantoicase, has a location like that of SUN1, and these proteins form a unique complex, detected by live-cell imaging, ultrastructural expansion microscopy, and interactomics. Deletion of either SUN1 or ALLAN genes disrupts nuclear MTOC organisation, leading to basal body mis-segregation, defective spindle assembly, and impaired spindle microtubule-kinetochore attachment, but axoneme formation remains intact. Ultrastructural analysis revealed nuclear and cytoplasmic MTOC miscoordination, producing aberrant flagellated gametes lacking nuclear material. These defects block development in the mosquito and parasite transmission, highlighting the essential functions of this complex.

Intracellular parasites like Toxoplasma gondii scavenge host nutrients, particularly lipids, to support their growth and survival. Although Toxoplasma is known to adjust its metabolism based on nutrient availability, the mechanisms that mediate lipid sensing and metabolic adaptation remain poorly understood. Here, we performed a genome-wide CRISPR screen under lipid-rich (10% Fetal Bovine Serum (FBS)) and lipid-limited (1% FBS) conditions to identify genes critical for lipid-responsive fitness. We identified the Toxoplasma protein GRA38 as a lipid-dependent regulator of parasite fitness. GRA38 exhibits phosphatidic acid (PA) phosphatase (PAP) activity in vitro, which is significantly reduced by mutation of its conserved DxDxT/V catalytic motif. Disruption of GRA38 led to the accumulation of PA species and widespread alterations in lipid composition, consistent with impaired PAP activity. These lipid imbalances correlated with reduced parasite virulence in mice. Our findings identify GRA38 as a metabolic regulator important for maintaining lipid homeostasis and pathogenesis in Toxoplasma gondii.