17th International Congress on Toxoplasmosis

Professor

A Novel Pyruvate Transporter in the Apicoplast of Apicomplexan Parasites

Metabolism, Biochemistry & Drug Development

Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its inter-organelle shuttling is crucial for cell fitness. Many Apicomplexan parasites harbor a unique organelle called the apicoplast that houses metabolic pathways like fatty acid and isoprenoid precursor biosyntheses, requiring pyruvate as a substrate. However, how pyruvate is supplied in the apicoplast remains enigmatic. Here, deploying the zoonotic parasite Toxoplasma gondii as a model apicomplexan, we discovered two proteins residing in the apicoplast membranes that together constitute a functional apicoplast pyruvate carrier (APC) to mediate the import of cytosolic pyruvate. Depletion of APC results in reduced activities of metabolic pathways in the apicoplast and impaired integrity of this organelle, leading to parasite growth arrest. APC is a novel pyruvate transporter in diverse apicomplexan parasites, suggesting a common strategy for pyruvate acquisition by the apicoplast in these clinically-relevant intracellular pathogens.

Oral presentation

Professor Dr.

Exploring Apicoplast Functions through Toxoplasma gondii lacking the apicoplast

Apicomplexan parasites, notorious for causing severe diseases like malaria (Plasmodium spp.) and toxoplasmosis (Toxoplasma gondii), harbor a distinctive organelle known as the apicoplast. The apicoplast is derived from secondary endosymbiosis and, while no longer photosynthetic, plays a pivotal role in housing metabolic functions critical for the survival of T. gondii. In this study, we aimed to dissect apicoplast functions and identify potential drug targets by bypassing the four metabolic pathways generating essential output for the parasite, namely de novo fatty acid (FA) synthesis via a prokaryotic fatty acid synthase II (FASII), synthesis of the lipid precursor lysophosphatidic acid (LPA), parts of the heme synthesis and synthesis of isopentenyl pyrophosphate (IPP) via a 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway. To achieve this, we supplemented the culture medium with palmitate, myristate, LPA, 5-aminolevulinic acid, and mevalolactone, after genetically engineering parasites to express an MVA cassette1,2, enabling parasites to synthesize IPP from mevalonolactone independent of their endogenous pathway. Under these apicoplast-bypassing conditions, pharmacological disruption of apicoplast-resident protease FtsH1 with actinonin, led to a rapid loss of the organelle but did not kill parasites. The continuous culture of T. gondii devoid of its plastid organelle serves as a unique platform to uncover novel apicoplast functions. Comparative proteomic analysis between wildtype and apicoplast-less parasites revealed a set of downregulated proteins residing in the apicoplast. Intriguingly, some of these proteins were identified as fitness-conferring critical components involved in the biology of the organelle. In conclusion, this study sheds light on the adaptability of T. gondii to survive in the absence of the apicoplast and uncovers novel functions associated with the organelle. 1Swift R, et al. A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites PLoS Pathog. 2020 Feb 14;16(2): e1008316. 2Niu Z, et al. Two apicoplast dwelling glycolytic enzymes provide key substrates for metabolic pathways in the apicoplast and are critical for Toxoplasma growth. PLoS Pathog. 2022 Nov 30;18(11):e1011009.

A multitude of small mitochondrial RNAs are used in the mitoribosome of T. gondii

Cell Biology

The mitochondrial genome of T. gondii is made up of 21 sequence blocks that are found in various combinations. It comprises merely three protein-coding genes, alongside a set of thirty to forty genes encoding small RNAs (sRNAs), many of which exhibit homologies to rRNA from E. coli. The expression status and integration of these short RNAs into ribosomes remains unclear and direct evidence for active ribosomes within apicomplexan mitochondria is still lacking. We conducted small RNA sequencing on the apicomplexan Toxoplasma gondii to investigate the occurrence and function of mitochondrial sRNAs. We find that many small RNAs originated from the junction sites between sequence blocks in the mitochondrial genome. Surprisingly, such block border sRNAs were incorporated into polysomes together with canonical rRNA fragments and mRNAs. In conclusion, apicomplexan ribosomes are active within polysomes and are indeed assembled through the integration of sRNAs, including previously undetected sRNAs with merged mRNA-rRNA sequences. Our findings lead to the hypothesis that T. gondii's block-based genome organization enables the dual utilization of mitochondrial sequences as both messenger RNAs and ribosomal RNAs, potentially establishing a link between the regulation of rRNA and mRNA expression.

TgORP2, an Oxysterol binding protein Related Protein putatively involved in Endoplasmic reticulum and Mitochondrion Membrane Contact Site formation and lipid exchange in Toxoplasma gondii.

Apicomplexa are obligate intracellular parasites responsible for major human diseases such as toxoplasmosis, caused by Toxoplasma gondii (Tg.). Its intracellular survival is closely linked to fatty acids (FAs) acquisition and usage to produce essential complex lipids for membrane biogenesis, signal transduction, membrane stability and energy storage (1). To fulfil its needs in FAs, the parasite is able to produce some FAs de novo through the apicoplast FASII pathway as well as massively scavenging FAs from its host (1). To maintain this critical need for FA to maintain parasite propagation and to avoid their toxic accumulation, the trafficking of lipids is regulated by key proteins that act during their synthesis/import, their storage, and their transport between organelles (2). Endoplasmic reticulum (ER), Mitochondria (Mito) and Lipid droplets (LD) are key organelles for lipid trafficking and homeostasis. Recent work show that there is intense lipid exchanges happening between these organelles through membrane contact sites (MCSs) and specific lipid transfer proteins. In eukaryotic cells, ER MCSs involvement in lipid homeostasis have been shown to be pivotal for survival (3,4), these include ER-LD and ER-Mito. We have identified such pivotal LTPs, and we characterized the members of the Oxysterol Binding Protein Related Protein (ORP) in Toxoplasma. Among them proteins, we found that TgORP2 was located at the ER-Mito MCS and is responsible for lipid exchange between the two organelles. Here, I will be presenting my PhD project data deciphering on the key function of TgORP2 to maintain lipid trafficking and homeostasis, allowing parasite propagation during tachyzoite life stage. Amiar, S. et al. Cell Rep 30, 3778-3792.e9 (2020). 2. Dass, S. et al. Nat Commun 12, 2813 (2021). 3. Wu, H. et al. Science (1979) 361, (2018). 4. Rakotonirina-Ricquebourg, R. et al. Prog Lipid Res 85, 101141 (2022).

To inhibit or not to inhibit: characterization of the Toxoplasma gondii homolog of ATPase inhibitory factor 1 (IF1)

In many eukaryotes, mitochondria play a key role in the generation of cellular energy as the so-called powerhouses of the cell. Within the powerhouse, the ATP synthase is the true power generator; it harvests the proton gradient created by the complexes of the electron transport chain to produce ATP through oxidative phosphorylation. In mammals and yeast, the activity of the ATP synthase is tightly controlled via many mechanisms, including by the binding of interactor proteins. One such interactor is ATPase inhibitory factor 1 (IF1), which can inhibit the ATP synthase and mediate the induction of cytoprotective gene expression pathways through a process known as mitohormesis. As we know little about how Toxoplasma gondii ATP synthase activity is regulated, we wanted to investigate whether the T. gondii homolog of IF1 (TgIF1) played similar roles to the IF1 found in other organisms. Using CRISPR/Cas9, we tagged TgIF1 and generated TgIF1 knockout and overexpression parasite strains. With these strains, we found that TgIF1 knockout and overexpression had no observable impact on metabolism, mitochondrial membrane potential, or ATP synthase dimerization. We did, however, find that TgIF1 overexpression appeared to increase higher order oligomerization of the ATP synthase and that knockout significantly reduced cristae density. Although TgIF1 levels had no impact on parasite growth under normal culture conditions, we found that TgIF1 knockout and overexpression both negatively impacted growth under hypoxia. Further, we illustrated that TgIF1 overexpression improved growth when parasites were exposed to oxidative stress, suggesting a mitohormetic role of TgIF1. This phenotypic observation was corroborated by bulk RNAseq data which showed that the main group of genes impacted by TgIF1 overexpression is involved in gene expression regulation and RT-qPCR data indicating that TgIF1 levels correlate with expression levels of several antioxidants. In summary, it appears that while TgIF1 does not have significant effects on parasite metabolism and growth under normal conditions, it plays a key role in mitochondrial cristae biogenesis and is important for mediating the response to various stressors.

Fragmented and on the move - the enigmatic mitochondrial genome sequences of Toxoplasma and Sarcocystis

Genomics

Recently we have reported on two remarkable aspects of the Toxoplasma gondii mitochondrial genome. First it exists as a redundant and highly fragmented sequence of unknown topology1. Second, ~10,000 relatively small pieces of the mitochondrial genome (NUMTs) have been transferred to the nuclear genome2. These transferred fragments account for an astonishing 1.4% of the nuclear genome sequence and appear to have contributed to evolutionary innovation. We find very similar mtDNA genome sequence novelties in Hammondia and Neospora, including very large numbers of NUMTs1. Thus, we decided to examine the more distantly related Sarcocystis neurona SN3. We assembled a new S. neurona SN3 genome sequence using ONT, Illumina and Hi-C. It is 127 Mb and we were able to observe at least 10 putative chromosomes, the largest of which is 24 Mb. The assembly has been annotated using orthology, as well as RNA-seq and ISO-seq from different lifecycle stages. We used this new assembly to study the distribution of NUMTs and characterize the mtDNA. We find a markedly reduced number of mtDNA insertions, 1077 or about 0.06% of the nuclear genome content. Interestingly, we find a redundant, fragmented mtDNA that cannot be assembled, as was the case in T. gondii. However, the fragments in S. neurona are quite different from those observed in other related tissue coccidians. This is significant, because as reported by Tetzlaff et al.,3 the T. gondii mtDNA breakpoints are associated with transcription initiation for the generation of small ribosomal RNAs in Toxoplasma. 1Namasivayam et al., doi: 10.1101/gr.266403.120 2Namasivayam et al., doi: 10.1101/2023.05.22.539837 3Tetzlaff et al., doi: 10.1101/2023.09.02.556019

Session XI: Mitochondrion & Apicoplast

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