The ionophore monensin displays potent activities against several coccidian parasites of veterinary and medical importance including the opportunistic pathogen of humans,Toxoplasma gondii. While monensin is used widely in animals, toxicity impedes its use in humans. Nonetheless, given its potency, understanding its mode of action would reveal vulnerable aspects of the parasite that can be exploited for drug development. We previously established that monensin inducesToxoplasmato undergo cell cycle arrest and an autophagy-like cell death. Interestingly, these effects are dependent on the mitochondrion-localized TgMSH-1 protein, suggesting that monensin disrupts mitochondrial function. We demonstrate that monensin treatment results in decreased mitochondrial membrane potential and altered morphology. These effects are mitigated by the antioxidant compound N-acetyl-cysteine suggesting that monensin causes an oxidative stress, which was indeed the case based on direct detection of reactive oxygen species. Moreover, over-expression of the antioxidant proteins glutaredoxin and peroxiredoxin 2 protectToxoplasmafrom the deleterious effects of monensin. Thus, our studies show that the effects of monensin onToxoplasmaare due to a disruption of mitochondrial function caused by the induction of an oxidative stress and implicate parasite redox biology as a viable target for the development of drugs againstToxoplasmaand related pathogenic parasites.

Summary: The balance between phosphorylation and de‐phosphorylation, which is delicately regulated by protein kinases and phosphatases, is critical for nearly all biological processes. The Apicomplexa are a large phylum which contains various parasitic protists, including human pathogens, such as Plasmodium, Toxoplasma, Cryptosporidium and Babesia species. The diverse life cycles of these parasites are highly complex and, not surprisingly, many of their key steps are exquisitely regulated by phosphorylation. Interestingly, many of the kinases and phosphatases, as well as the substrates involved in these events are unique to the parasites and therefore phosphorylation constitutes a viable target for antiparasitic intervention. Most progress on this realm has come from studies in Toxoplasma and Plasmodium of their respective kinomes and phosphoproteomes. Nonetheless, given their likely importance, phosphatases have recently become the focus of research within the apicomplexan parasites. In this review, we concentrate on serine/threonine phosphatases in apicomplexan parasites, with the focus on comprehensively identifying and naming protein phosphatases in available apicomplexan genomes, and summarizing the progress of their functional analyses in recent years.

The single mitochondrion of Toxoplasma gondii is highly dynamic, being predominantly in a peripherally distributed lasso-shape in intracellular parasites and collapsed in extracellular parasites. The peripheral positioning of the mitochondrion is associated with apparent contacts between the mitochondrion membrane and the parasite pellicle. The outer mitochondrial membrane-associated protein LMF1 is critical for the correct positioning of the mitochondrion. Intracellular parasites lacking LMF1 fail to form the lasso-shaped mitochondrion. To identify other proteins that tether the mitochondrion of the parasite to the pellicle, we performed a yeast two-hybrid screen for LMF1 interactors. We identified 70 putative interactors localized in different cellular compartments, such as the apical end of the parasite, mitochondrial membrane and the inner membrane complex (IMC), including with the pellicle protein IMC10. Using protein–protein interaction assays, we confirmed the interaction of LMF1 with IMC10. Conditional knockdown of IMC10 does not affect parasite viability but severely affects mitochondrial morphology in intracellular parasites and mitochondrial distribution to the daughter cells during division. In effect, IMC10 knockdown phenocopies disruption of LMF1, suggesting that these two proteins define a novel membrane tether between the mitochondrion and the IMC in Toxoplasma. This article has an associated First Person interview with the first author of the paper.

The propagation of Toxoplasma gondii is accomplished by repeated lytic cycles of parasite attachment to a host cell, invasion, replication within a parasitophorous vacuole, and egress from the cell. This lytic cycle is delicately regulated by calcium-dependent reversible phosphorylation of the molecular machinery that drives invasion and egress. While much progress has been made elucidating the protein kinases and substrates central to parasite propagation, little is known about the relevant protein phosphatases. In this study, we focused on the five protein phosphatases that are predicted to be membrane-associated either integrally or peripherally. We have determined that of these only PPM5C, a PP2C family member, localizes to the plasma membrane of Toxoplasma. Disruption of PPM5C results in a slow propagation phenotype in tissue culture. Interestingly, parasites lacking PPM5C divide and undergo egress at a normal rate, but have a deficiency in attaching to host cells. Both membrane localization and phosphatase activity are required for PPM5C’s role in attachment. Phosphoproteomic analysis show relatively few phosphorylation sites being affected by PPM5C deletion in extracellular parasites of which several are found on proteins involved in signaling cascades. This implies that PPM5C is part of a wider regulatory network important for attachment to host cells.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication, and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, although many of the calcium‐dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation, we have determined that a nonsense mutation in a previously uncharacterised protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localises to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network. Our findings support a connection between the tubulovesicular network and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.