17th International Congress on Toxoplasmosis

Deciphering the core complex and regulatory elements of the rhoptry secretory machinery

Cell Biology

Rhoptries are specialized secretory organelles at the heart of Apicomplexa pathogenesis. They inject parasite proteins directly into the cytoplasm of host cells not only for invasion but also for hijacking host functions crucial to establishing and sustaining infection. Rhoptries are connected to an apical vesicle (AV), which is docked to the parasite plasma membrane (PPM) by a complex machinery called the rhoptry secretory apparatus (RSA). This arrangement suggests the requirement for two membrane-fusion events during rhoptry secretion, yet little is known about the proteins that regulate them. Nd complex members (Nd6, Nd9, NdP1, NdP2, and Fer2) are essential for rhoptry exocytosis. Nd9, NdP1, and NdP2 are required to build the RSA, and Nd6 is linked to the exocytic site at the PPM/AV in Toxoplasma. These observations suggest a role for the Nd complex in the fusion event between the AV and the PPM. Here, we performed immunoprecipitation coupled with mass spectrometry to obtain a more comprehensive inventory of the rhoptry secretion machinery. We identified 9 additional NdPs, of which almost all are required for invasion of T. gondii in vitro, yet they are not essential. When tested for virulence in mice, the mutants appear highly attenuated, even the ones with no or mild in vitro defect. Like Nd6, most of these proteins are present at the exocytic site. A better visualization of their spatial organization is being achieved by iterative U-ExM. Two of the NdPs are predicted to be GTPases. One is an Arf-like GTPase and directly interacts with the predicted GAP-domain of Nd6. The other one, named NdP-GTPase, is a direct partner of Nd9. Phylogenetic analyses revealed that NdP-GTPase is part of an Alveolata-specific distinct clade that does not fall into any of the classical small GTPase sub-families. Further characterization of both Toxoplasma and Tetrahymena NdP-GTPases showed that they are present at the exocytic site, and their mutants are defective in rhoptry and mucocysts exocytosis, respectively. A GDP-locked mutant in Tetrahymena revealed the essentiality of GDP to GTP switch for mucocysts release upon stimulation. Similar experiments in Toxoplasma are currently ongoing. Collectively, these findings support the notion that rhoptry secretion is governed by GTPase activities, and certain Nd/NdP proteins may act as regulators or effectors of GTPase activity.

Oral presentation

Untangling the functional hierarchal architecture of the Toxoplasma gondii apical annuli proteins

The Toxoplasma gondii cytoskeleton contains a network of intermediate filaments, supporting a quilt of alveolar membrane compartments, together known as the inner membrane complex (IMC). Embedded within the IMC are the apical annuli: 5-6 donut shaped pores that facilitate secretion of dense granules. Annuli component LMBD3 spans from IMC to plasma membrane, as do several annuli localized SNARE proteins (Stx1, Stx20, Stx21). Here we describe a novel apical annuli protein, AAP7, which we functionally dissected by conditional knock-down (AAP7-cKD). AAP7 localizes to the annuli of mature parasites only, appearing when new daughters emerge. AAP7 is present in the second largest donut with an average diameter of 300 nm. However, in other parasites AAP7 is present in smaller diameter donuts, which hints at two distinct annuli pore states. Depletion of AAP7 causes a severe fitness defect but ~10% of parasites survive and develop a compensatory phenotype. To follow up on this resistance mechanism, we performed scRNA-seq to reveal any gene candidates that could be associated with overcoming the loss of AAP7. To further probe apical annuli function, we created a lethal double annuli deletion (DAD) strain, combining AAP7-cKD with a direct knock-out of AAP4 (AAP4-KO), the largest and most conserved annuli protein. DAD parasites have a slow death phenotype, and we are currently deciphering how the DAD parasites die. Dense granule secretion is reduced in the absence of AAP7, AAP4, and in the DAD parasites. In the absence of AAP4, annuli component Centrin2 is not recruited to the apical annuli. AAP7 is mis-localized in the AAP4-KO background, demonstrating AAP4 recruitment of AAP7. AAP2 is unaffected by the loss of AAP4, indicating independent recruitment. In the absence of AAP7, Centrin2 and AAP4 localize normally. Overall, our results demonstrate that Centrin2 and AAP7 recruitment to the annuli require AAP4, which is not essential to parasite viability. Both AAP4 and AAP7 are needed for the essential function of the apical annuli. Hence, we have provided insights into the functional hierarchy of apical annuli architecture and established the tool needed to probe the fitness-conferring mechanism of the T. gondii apical annuli.

TOXOPLASMA OXYGEN SENSING: PHDs HARD AT WORK

Toxoplasma gondii is an obligate intracellular parasite that can infect any nucleated cell in any tissue of warm-blooded animals. How does the parasite have such a wide host cell and tissue tropism? One mechanism is by the parasite expressing its own host cell receptor complex that it injects into the host cell. But as the parasite disseminates through its host, it encounters diverse environments that it that it must adapt to so that it can replicate, grow, and cause disease. Oxygen is one environmental factor whose levels change dramatically in tissues and all cells must detect and respond to changes in oxygen availability. Prolyl hydroxylases are dioxygenases that function as key cellular oxygen sensing proteins. In mammals, the transcription factor Hypoxia-Inducible Factor-1a is the best recognized prolyl hydroxylase substrate and our earlier work revealed that parasite growth at low oxygen requires HIF-1 activation. These studies led us to study how Toxoplasma itself senses oxygen? To address this question, we used BLAST analysis and identified 2 Toxoplasma-encoded prolyl hydroxylase homologs that we named PHYa and PHYb. PHYa prolyl hydroxylates SKP1, which is an adaptor in the Skp1/Cullin-1/F-box protein (SCF)-E3 ubiquitin ligase complex. SKP1 prolyl hydroxylation alters its binding preferences for F-Box proteins leading to changes in which proteins are ubiquitinated by the SCF-E3. PHYa is required for growth at low oxygen and is required for virulence as it is critically required to resist IFNγ-dependent killing. PHYb, on the other hand, is required for growth and survival of parasites exposed to stress at elevated oxygen levels and does so by inhibiting phosphorylation of elongation factor 2 (eEF2). eEF2 phosphorylation inhibits it from catalyzing the translocation of the peptidyl-tRNA complex from the A-site to the P-site of the ribosome. We have shown that PHYb promotes protein synthesis to ensure that the parasite proteins are available to ensure that the stress response is properly executed. In this talk, we will discuss new data regarding how PHYa and PHYb function. Taken together, these data indicate that the two prolyl hydroxylases work together as an oxygen sensing rheostat in regulating protein synthesis and degradation to ensure that the parasite proteome is programmed to enable Toxoplasma to grow, survive, and cause disease at whatever oxygen tension it faces.

From the plasma membrane to the plasma membrane, following recycling and flow of Toxoplasma gondii membrane proteins.

The plasma membrane (PM) plays a critical role in numerous functions, from motility to cell communication, and serves as a gateway between intracellular content and the extracellular environment1,2. This is particularly true for intracellular parasites, which switch between drastically different environments during their life cycles3. Endocytosis is an important mechanism involved in the regulation of plasma membrane surface proteins and uptake of environmental molecules. We recently demonstrated endocytosis in Toxoplasma gondii (T. gondii) by labelling endogenous Halo-tagged SAG1 with a non-permeable dye4. This uptake occurring at the micropore is important for intracellular development, as knockdown of the protein composing the micropore impairs endocytosis and disturbs plasma membrane dynamics, leading to membrane accumulation and death of the parasite5,6. However, little is known about the normal behaviour of PM in T. gondii. Here, using SAG1-Halo as a proxy, we followed the dynamics of the PM using a dual (spatial and/or temporal) labelling strategy, allowing the differentiation between internal and surface proteins, but also between the mother and de novo synthesized material during replication. Using this strategy, we showed the presence of a complete cycle of endo-exocytosis in Toxoplasma gondii, regulating PM dynamics during the parasite lytic cycle. Thottacherry, J. J., Sathe, M., Prabhakara, C. &amp; Mayor, S. Spoiled for Choice: Diverse Endocytic Pathways Function at the Cell Surface. Annu Rev Cell Dev Biol 35, 55–84 (2019). Redpath, G. M. I., Betzler, V. M., Rossatti, P. &amp; Rossy, J. Membrane Heterogeneity Controls Cellular Endocytic Trafficking. Frontiers in Cell and Developmental Biology 8, (2020). Attias, M. et al. The life-cycle of Toxoplasma gondii reviewed using animations. Parasit Vectors 13, 588 (2020). Gras, S. et al. An endocytic-secretory cycle participates in Toxoplasma gondii in motility. PLoS Biol 17, e3000060 (2019). Koreny, L. et al. Stable and ancient endocytic structures navigate the complex pellicle of apicomplexan parasites. bioRxiv 2022.06.02.494549 (2022) doi:10.1101/2022.06.02.494549. Wan, W. et al. The Toxoplasma micropore mediates endocytosis for selective nutrient salvage from host cell compartments. Nat Commun 14, 977 (2023).

Professor Dr.

Intrinsically low arachidonic acid synthesis in cat cells, its potential role in ferroptosis resilience and its relevance for Toxoplasma gondii

Recent studies have shown that Toxoplasma gondii requires iron for growth, but that it is also sensitive to iron overload. Felines, the definitive hosts of T. gondii, are strict carnivores. Their heme-iron-rich diet may pose a particular challenge, as it could cause iron-dependent lipid peroxidation, leading to intestinal cell death by ferroptosis. In other cellular systems, sensitivity to ferroptosis was dependent on the level of fatty acid desaturase 2 (FADS2) expression, due to the enzyme&quot;s involvement in arachidonic acid (AA) synthesis from linoleic acid. Although most studies have suggested minimal or absent FADS2 activity in cats and other felids, others have provided contrasting results. Strikingly, FADS2 transcripts are annotated in the published genomes of felids. Given the potential importance of host cell longevity on the sexual development of T. gondii in the cat intestine, we have started to study ferroptosis in a cat in vitro system using stem cell-derived intestinal organoids (IO) and cell lines. We generated continuous, long-term IO cultures from cats, which will enable us to address the following questions: (i) are cat IOs deficient in FADS2 activity; (ii) are they sensitive to ferroptosis, and (iii) how do parasites react to ferroptosis induction in this cellular system? Indeed, we observed FADS2 transcription in all cat cell systems tested. Despite this, metabolic labeling showed that feline cells have very limited, but not lacking, FADS2 activity, resulting in only very little AA synthesis. Intriguingly, we found that cat IOs are surprisingly resistant to usual ferroptosis inducers. The observed low amounts of AA due to FADS2 deficiency could thus play a role in ferroptosis resilience. Ongoing studies are examining the interplay of the parasite and feline intestinal cells in the context of ferroptosis. Overall, this study provides new perspectives on the molecular factors determining T. gondii&quot;s definitive host specificity.

Stuck Together: Investigating Interorganellar Communication in Toxoplasma gondii

In conventional model organisms, it has been shown that organellar interactions are essential for metabolite and lipid exchange, organelle dynamics, cellular homeostasis, and cell fate. While organelles allow for specialized biochemical processes to occur, this necessitates communication between organelles to share signals and metabolites. Originally, it was thought this occurred simply through cytoplasmic diffusion or vesicular trafficking before recent work highlighted the importance of direct contact between organelles. These interactions are supported by areas of close proximity between the two organelles, maintained by protein tethers, and referred to as membrane contact sites. Currently, the majority of our knowledge on membrane contact sites is limited to yeast and mammals, which are part of the Opisthokont clade. Thanks to its genetic amenability, Toxoplasma gondii provides the unique opportunity to investigate membrane contact sites in a divergent model eukaryote. Additionally, T. gondii possesses a phylum-specific organelle, the apicoplast, which has long been observed in close proximity to the mitochondrion. The two organelles have co-evolved in Apicomplexans, resulting in shared biosynthetic and biochemical pathways. We hypothesize that phylum-specific contacts mediate the proximity of these organelles and promote the exchange of materials. To identify membrane contact site candidates in T. gondii, we have taken an unbiased proximity biotinylation-based approach by generating localization handles to anchor a biotin ligase to the surface of the apicoplast, mitochondrion, and also the ER. Preliminary biotinylation experiments followed by mass spectrometry analysis uncovered membrane contact site protein candidates. Our results include several proteins with expected membrane contact site functions, such as lipid-transfer proteins and small GTPases. We also found apicomplexan-specific proteins with no predictable functions or domains that may serve as potential drug targets. Finally, we will also use our data to generate surface proteomes for the ER, mitochondrion, and apicoplast. The results of this work will both expand our knowledge of membrane contact sites across the evolutionary tree, potentially uncover apicomplexan-specific ones, and provide an additional protein localization resource for the community.

Session IX: Cell Biology II

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