Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii.

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The fungus Leptosphaeria maculans causes blackleg of Brassica species. Here, we report the mapping and subsequent cloning of an avirulence gene from L. maculans. This gene, termed AvrLmJ1, confers avirulence towards all three Brassica juncea cultivars tested. Analysis of RNA-seq data showed that AvrLmJ1 is housed in a region of the L. maculans genome which contains only one gene that is highly expressed in planta. The closest genes are 57 and 33 kb away and, like other avirulence genes of L. maculans, AvrLmJ1 is located within an AT-rich, gene-poor region of the genome. The encoded protein is 141 amino acids, has a predicted signal peptide and is cysteine rich. Two virulent isolates contain a premature stop codon in AvrLmJ1. Complementation of an isolate that forms cotyledonary lesions on B. juncea with the wild-type allele of AvrLmJ1 confers avirulence towards all three B. juncea cultivars tested, suggesting that the gene may confer species-specific avirulence activity.

Apicomplexan parasites are pathogens responsible for major human diseases such as toxoplasmosis caused by and malaria caused by 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. possesses a single cytosolic acetyl-CoA synthetase (ACS). Its role in the parasite lipid synthesis is unclear. Here, we generated an inducible ACS KO parasite line and confirmed the cytosolic localization of the protein. We conductedC-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 ACS 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.

Venturia inaequalis and V. pirina are Dothideomycete fungi that cause apple scab and pear scab disease, respectively. Whole genome sequencing of V. inaequalis and V. pirina isolates has revealed predicted proteins with sequence similarity to AvrLm6, a Leptosphaeria maculans effector that triggers a resistance response in Brassica napus and B. juncea carrying the resistance gene, Rlm6. AvrLm6-like genes are present as large families (>15 members) in all sequenced strains of V. inaequalis and V. pirina, while in L. maculans, only AvrLm6 and a single paralog have been identified. The Venturia AvrLm6-like genes are located in gene-poor regions of the genomes, and mostly in close proximity to transposable elements, which may explain the expansion of these gene families. An AvrLm6-like gene from V. inaequalis with the highest sequence identity to AvrLm6 was unable to trigger a resistance response in Rlm6-carrying B. juncea. RNA-seq and qRT-PCR gene expression analyses, of in planta- and in vitro-grown V. inaequalis, has revealed that many of the AvrLm6-like genes are expressed during infection. An AvrLm6 homolog from V. inaequalis that is up-regulated during infection was shown (using an eYFP-fusion protein construct) to be localized to the sub-cuticular stroma during biotrophic infection of apple hypocotyls.

To efficiently enter host cells, apicomplexan parasites such as Toxoplasma gondii rely on an apical complex composed of tubulin‐based structures as well as two sets of secretory organelles named micronemes and rhoptries. The trafficking and docking of these organelles to the apical pole of the parasite is crucial for the discharge of their contents. Here, we describe two proteins typically associated with microtubules, Centrin 2 (CEN2) and Dynein Light Chain 8a (DLC8a), that are required for efficient host cell invasion. CEN2 localizes to four different compartments, and remarkably, conditional depletion of the protein occurs in stepwise manner, sequentially depleting the protein pools from each location. This phenomenon allowed us to discern the essential function of the apical pool of CEN2 for microneme secretion, motility, invasion and egress. DLC8a localizes to the conoid, and its depletion also perturbs microneme exocytosis in addition to the apical docking of the rhoptry organelles, causing a severe defect in host cell invasion. Phenotypic characterization of CEN2 and DLC8a indicates that while both proteins participate in microneme secretion, they likely act at different steps along the cascade of events leading to organelle exocytosis.

SummaryPlasmodium falciparum and Toxoplasma gondii are obligate intracellular parasites that belong to the phylum of Apicomplexa and cause major human diseases. Their access to an intracellular lifestyle is reliant on the coordinated release of proteins from the specialized apical organelles called micronemes and rhoptries. A specific phosphatidic acid effector, the acylated pleckstrin homology domain-containing protein (APH) plays a central role in microneme exocytosis and thus is essential for motility, cell entry, and egress. TgAPH is acylated on the surface of the micronemes and recruited to phosphatidic acid (PA)-enriched membranes. Here, we dissect the atomic details of APH PA-sensing hub and its functional interaction with phospholipid membranes. We unravel the key determinant of PA recognition for the first time and show that APH inserts into and clusters multiple phosphate head-groups at the bilayer binding surface.

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.