Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

Three Toxoplasma gondii dense granule proteins are required for induction of Lewis rat macrophage pyroptosis

Upon invasion of Lewis rat macrophages, Toxoplasma rapidly induces programmed cell death (pyroptosis), which prevents Toxoplasma replication, possibly explaining the resistance of the Lewis rat to Toxoplasma Using a chemical mutagenesis screen, we identified Toxoplasma mutants that no longer induced pyroptosis. Whole-genome sequencing led to the identification of three Toxoplasma parasitophorous vacuole-localized dense granule proteins, GRA35, GRA42, and GRA43, that are individually required for induction of Lewis rat macrophage pyroptosis. Macrophage infection with Δgra35, Δgra42, and Δgra43 parasites led to greatly reduced cell death rates and enhanced parasite replication. Lewis rat macrophages infected with parasites containing a single, double, or triple deletion of these GRAs showed similar levels of cell viability, suggesting that the three GRAs function in the same pathway. Deletion of GRA42 or GRA43 resulted in GRA35 (and other GRAs) being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane. Despite having greatly enhanced replication in Lewis rat macrophages in vitro, Δgra35, Δgra42, and Δgra43 parasites did not establish a chronic infection in Lewis rats. Toxoplasma did not induce F344 rat macrophage pyroptosis, but F344 rats infected with Δgra35, Δgra42, and Δgra43 parasites had reduced cyst numbers. Thus, these GRAs determined parasite in vivo fitness in F344 rats. Overall, our data suggest that these three Toxoplasma dense granule proteins play a critical role in establishing a chronic infection in vivo, independently of their role in mediating macrophage pyroptosis, likely due to their importance in regulating protein localization to the parasitophorous vacuole membrane.IMPORTANCE Inflammasomes are major components of the innate immune system and are responsible for detecting various microbial and environmental danger signals. Upon invasion of Lewis rat macrophages, the parasite rapidly activates the NLRP1 inflammasome, resulting in pyroptosis and elimination of the parasite's replication niche. The work reported here revealed that Toxoplasma GRA35, GRA42, and GRA43 are required for induction of Lewis rat macrophage pyroptosis. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. These three GRAs were also found to be important for parasite in vivo fitness in a Toxoplasma-susceptible rat strain, independently of their role in NLRP1 inflammasome activation, suggesting that they perform other important functions. Thus, this study identified three GRAs that mediate the induction of Lewis rat macrophage pyroptosis and are required for pathogenesis of the parasite

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages

The Role of GRA7, a Component in the ROP18 Protein Complex, in Toxoplasma gondii

The obligate intracellular pathogen, Toxoplasma gondii, is adept at creating a safe niche within the rough environment of the host cytosol. By enclosing itself within a non-fusogenic parasitophorous vacuolar membrane (PVM), it cleverly resists lysosomal fusion. Despite this leverage, avirulent strains of Toxoplasma are cleared effectively from their host cells by the host\u27s intrinsic cellular defenses. Forward genetic analysis has identified a virulence factor, the serine/threonine kinase ROP18, which plays an important role in the survival of virulent strains of Toxoplasma. In an infected cell ROP18 localizes to the PVM along with many members of the rhoptry (ROP) and dense granule (GRA) family of proteins. On the PVM, ROP18 kinase targets the host\u27s dynamin-related immunity related GTPases (IRG) for phosphorylation on critical residues, leading to their inactivation. Avirulent strains that lack ROP18 are successfully targeted by the IRG proteins that are recruited to the PVM, resulting in ruffling and disruption of the membrane and hence death of the parasite within it. Although ROP18 is necessary and sufficient to subvert the IRG pathway, we were interested in whether it associated with other parasite proteins to carry out its virulence function in vivo. Using immunoprecipitation and mass spectrometric analysis we found that ROP18 partners with two parasite proteins, the rhoptry pseudokinase ROP2, and the dense granule protein GRA7. While GRA7 arises from a distinct organelle intracellularly, upon infection, we were able to localize GRA7 to the cytosolic face of the PVM similar to ROP18, using selectively permeable detergents and immunoflorescence microscopy. To resolve the biological significance of this complex, we constructed single as well as double deletion strains, using Δku80, a genetically malleable parent parasite line. Although the single deletion strains did not affect virulence, the double deletion strain Δgra7Δrop18 displayed considerable attenuation. By performing cellular assays, we ascertained that the Δgra7Δrop18 parasites defective as they were significantly more vulnerable to the IRG pathway. Although GRA7 has been suspected to play a role in nutrient acquirement, we were unable to find a defect in the Δgra7parasites growth in vitro, or in virulence in vivo. To establish a mechanism for GRA7 in defense against the IRGs, we performed in vitro biochemical assays testing the effect on GRA7 on the activity of Irga6. We found that sub molar amounts of GRA7 were capable of stimulating rapid oligomerzation of recombinant GTP-activated Irga6 to a much higher extent than without GRA7. In addition, GRA7-driven oligomers of Irga6 also resolved back into monomers much faster than oligomers without GRA7. Collectively, we propose that GRA7 is involved in mediating IRG resistance by stimulating the turnover of Irga6 near the PVM, and in effect, preventing them from accumulating and destroying the PVM, and/or providing its partner kinase, ROP18, with substrate. This finding adds to the growing list of parasite factors that subvert the IRG pathway, and suggests a novel mechanism for its action

Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission

International audienceTransmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers "rounding up" followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission

Plasmodium falciparum ookinetes require mosquito midgut chondroitin sulfate proteoglycans for cell invasion.

Contains fulltext : 51536.pdf (publisher's version ) (Closed access)Malaria transmission entails development of the Plasmodium parasite in its insect vector, the Anopheles mosquito. Parasite invasion of the mosquito midgut is the critical first step and involves adhesion to host epithelial cell ligands. Partial evidence suggests that midgut oligosaccharides are important ligands for parasite adhesion; however, the identity of these glycans remains unknown. We have identified a population of chondroitin glycosaminoglycans along the apical midgut microvilli of Anopheles gambiae and further demonstrated ookinete recognition of these glycans in vitro. By repressing the expression of the peptide-O-xylosyltransferase homolog of An. gambiae by means of RNA interference, we blocked glycosaminoglycan chain biosynthesis, diminished chondroitin sulfate levels in the adult midgut, and substantially inhibited parasite development. We provide evidence for the in vivo role of chondroitin sulfate proteoglycans in Plasmodium falciparum invasion of the midgut and insight into the molecular mechanisms mediating parasite-mosquito interactions