Third-generation sequencing revises the molecular karyotype for Toxoplasma gondii and identifies emerging copy number variants in sexual recombinants.

Toxoplasma gondii is a useful model for intracellular parasitism given its ease of culture in the laboratory and genomic resources. However, as for many other eukaryotes, the T. gondii genome contains hundreds of sequence gaps owing to repetitive and/or unclonable sequences that disrupt the assembly process. Here, we use the Oxford Nanopore Minion platform to generate near-complete de novo genome assemblies for multiple strains of T. gondii and its near relative, N. caninum We significantly improved T. gondii genome contiguity (average N50 of ∼6.6 Mb) and added ∼2 Mb of newly assembled sequence. For all of the T. gondii strains that we sequenced (RH, ME49, CTG, II×III progeny clones CL13, S27, S21, S26, and D3X1), the largest contig ranged in size between 11.9 and 12.1 Mb in size, which is larger than any previously reported T. gondii chromosome, and found to be due to a consistent fusion of Chromosomes VIIb and VIII. These data were validated by mapping existing T. gondii ME49 Hi-C data to our assembly, providing parallel lines of evidence that the T. gondii karyotype consists of 13, rather than 14, chromosomes. By using this technology, we also resolved hundreds of tandem repeats of varying lengths, including in well-known host-targeting effector loci like rhoptry protein 5 (ROP5) and ROP38 Finally, when we compared T. gondii with N. caninum, we found that although the 13-chromosome karyotype was conserved, extensive, previously unappreciated chromosome-scale rearrangements had occurred in T. gondii and N. caninum since their most recent common ancestry.

Not your Mother's MAPKs: Apicomplexan MAPK function in daughter cell budding.

Reversible phosphorylation by protein kinases is one of the core mechanisms by which biological signals are propagated and processed. Mitogen-activated protein kinases, or MAPKs, are conserved throughout eukaryotes where they regulate cell cycle, development, and stress response. Here, we review advances in our understanding of the function and biochemistry of MAPK signaling in apicomplexan parasites. As expected for well-conserved signaling modules, MAPKs have been found to have multiple essential roles regulating both Toxoplasma tachyzoite replication and sexual differentiation in Plasmodium. However, apicomplexan MAPK signaling is notable for the lack of the canonical kinase cascade that normally regulates the networks, and therefore must be regulated by a distinct mechanism. We highlight what few regulatory relationships have been established to date, and discuss the challenges to the field in elucidating the complete MAPK signaling networks in these parasites.

Anesthetic management of children with medically refractory pulmonary hypertension undergoing surgical Potts shunt.

INTRODUCTION: Pulmonary hypertension in children is associated with high rates of adverse events under anesthesia. In children who have failed medical therapy, a posttricuspid shunt such as a Potts shunt can offload the right ventricle and possibly delay or replace the need for lung transplantation. Intraoperative management of this procedure, during which an anastomosis between the pulmonary artery and the descending aorta is created, is complex and requires a deep understanding of the pathophysiology of acute and chronic right ventricular failure. This retrospective case review describes the intraoperative management of children undergoing surgical creation of a Potts shunt at a single center. METHODS: A retrospective case review of all patients under the age of 18 who underwent Potts shunt between April 2013 and June 2022. Medical records were examined, and clinical data of demographics, intraoperative vital signs, anesthetic management, and postoperative outcomes were extracted. RESULTS: Twenty-nine children with medically refractory pulmonary hypertension underwent surgical Potts shunts with a median age of 12 years (range 4 months to 17.4 years). Nineteen Potts shunts (65%) were placed via thoracotomy and 10 (35%) were placed via median sternotomy with use of cardiopulmonary bypass. Ketamine was the most frequently utilized induction agent (17 out of 29, 59%), and the majority of patients were initiated on vasopressin prior to intubation (20 out of 29, 69%). Additional inotropic support with epinephrine (45%), milrinone (28%), norepinephrine (17%), and dobutamine (14%) was used prior to shunt placement. Following opening of the Potts shunt, hemodynamic support was continued with vasopressin (66%), epinephrine (62%), milrinone (59%), dobutamine (14%), and norepinephrine (10%). Major intraoperative complications included severe hypoxemia (21 out of 29, 72%) and hypotension requiring boluses of epinephrine (10 out of 29, 34.5%) but no patient suffered intraoperative cardiac arrest. There were four in-hospital mortalities. DISCUSSION: A Potts shunt offers another palliative option for children with medically refractory pulmonary hypertension. General anesthesia in these children carries high risk for pulmonary hypertensive crises. Anesthesiologists must understand underlying physiological mechanisms responsble for acute hemodynaic decompensation during acute pulmonary hypertneisve crises. Severe physiological perturbations imposed by thoracic surgery and use of cardiopulmonay bypass can be mitigated by aggresive heodynamic support of ventricle function and maintainence of systemic vascular resistance. Early use of vasopressin, before or immidiately after anesthesia induction, in combination with other inotropes is a useful agent during the perioperative care of thes. Early use of vasopressin during anesthesia induction, and aggressive inotropic support of right ventricular function can help mitigate effects of induction and intubation, single-lung ventilation, and cardiopulmonary bypass. CONCLUSIONS: Our single center expereince shows that the Potts shunt surgery, despite high short-term mortaility, may offer another option for palliation in children with medically refractory pulmonary hypertension.

Divergent kinase WNG1 is regulated by phosphorylation of an atypical activation sub-domain.

Apicomplexan parasites like Toxoplasma gondii grow and replicate within a specialized organelle called the parasitophorous vacuole. The vacuole is decorated with parasite proteins that integrate into the membrane after trafficking through the parasite secretory system as soluble, chaperoned complexes. A regulator of this process is an atypical protein kinase called WNG1. Phosphorylation by WNG1 appears to serve as a switch for membrane integration. However, like its substrates, WNG1 is secreted from the parasite dense granules, and its activity must, therefore, be tightly regulated until the correct membrane is encountered. Here, we demonstrate that, while another member of the WNG family can adopt multiple multimeric states, WNG1 is monomeric and therefore not regulated by multimerization. Instead, we identify two phosphosites on WNG1 that are required for its kinase activity. Using a combination of in vitro biochemistry and structural modeling, we identify basic residues that are also required for WNG1 activity and appear to recognize the activating phosphosites. Among these coordinating residues are the 'HRD' Arg, which recognizes activation loop phosphorylation in canonical kinases. WNG1, however, is not phosphorylated on its activation loop, but rather on atypical phosphosites on its C-lobe. We propose a simple model in which WNG1 is activated by increasing ATP concentration above a critical threshold once the kinase traffics to the parasitophorous vacuole.

Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required for Toxoplasma apical complex biogenesis.

Apicomplexan parasites use a specialized cilium structure called the apical complex to organize their secretory organelles and invasion machinery. The apical complex is integrally associated with both the parasite plasma membrane and an intermediate filament cytoskeleton called the inner-membrane complex (IMC). While the apical complex is essential to the parasitic lifestyle, little is known about the regulation of apical complex biogenesis. Here, we identify AC9 (apical cap protein 9), a largely intrinsically disordered component of the Toxoplasma gondii IMC, as essential for apical complex development, and therefore for host cell invasion and egress. Parasites lacking AC9 fail to successfully assemble the tubulin-rich core of their apical complex, called the conoid. We use proximity biotinylation to identify the AC9 interaction network, which includes the kinase extracellular signal-regulated kinase 7 (ERK7). Like AC9, ERK7 is required for apical complex biogenesis. We demonstrate that AC9 directly binds ERK7 through a conserved C-terminal motif and that this interaction is essential for ERK7 localization and function at the apical cap. The crystal structure of the ERK7-AC9 complex reveals that AC9 is not only a scaffold but also inhibits ERK7 through an unusual set of contacts that displaces nucleotide from the kinase active site. ERK7 is an ancient and autoactivating member of the mitogen-activated kinase (MAPK) family and its regulation is poorly understood in all organisms. We propose that AC9 dually regulates ERK7 by scaffolding and concentrating it at its site of action while maintaining it in an "off" state until the specific binding of a true substrate.

The assembly of lipid droplets and their roles in challenged cells.

Cytoplasmic lipid droplets are important organelles in nearly every eukaryotic and some prokaryotic cells. Storing and providing energy is their main function, but they do not work in isolation. They respond to stimuli initiated either on the cell surface or in the cytoplasm as conditions change. Cellular stresses such as starvation and invasion are internal insults that evoke changes in droplet metabolism and dynamics. This review will first outline lipid droplet assembly and then discuss how droplets respond to stress and in particular nutrient starvation. Finally, the role of droplets in viral and microbial invasion will be presented, where an unresolved issue is whether changes in droplet abundance promote the invader, defend the host, to try to do both. The challenges of stress and infection are often accompanied by changes in physical contacts between droplets and other organelles. How these changes may result in improving cellular physiology, an ongoing focus in the field, is discussed.

Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5.

Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite's protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.

Divergent kinase regulates membrane ultrastructure of the Toxoplasma parasitophorous vacuole.

Apicomplexan parasites replicate within a protective organelle, called the parasitophorous vacuole (PV). The Toxoplasma gondii PV is filled with a network of tubulated membranes, which are thought to facilitate trafficking of effectors and nutrients. Despite being critical to parasite virulence, there is scant mechanistic understanding of the network's functions. Here, we identify the parasite-secreted kinase WNG1 (With-No-Gly-loop) as a critical regulator of tubular membrane biogenesis. WNG1 family members adopt an atypical protein kinase fold lacking the glycine rich ATP-binding loop that is required for catalysis in canonical kinases. Unexpectedly, we find that WNG1 is an active protein kinase that localizes to the PV lumen and phosphorylates PV-resident proteins, several of which are essential for the formation of a functional intravacuolar network. Moreover, we show that WNG1-dependent phosphorylation of these proteins is required for their membrane association, and thus their ability to tubulate membranes. Consequently, WNG1 knockout parasites have an aberrant PV membrane ultrastructure. Collectively, our results describe a unique family of Toxoplasma kinases and implicate phosphorylation of secreted proteins as a mechanism of regulating PV development during parasite infection.

Thinking outside of the cell: Secreted protein kinases in bacteria, parasites, and mammals.

Previous decades have seen an explosion in our understanding of protein kinase function in human health and disease. Hundreds of unique kinase structures have been solved, allowing us to create generalized rules for catalysis, assign roles of communities within the catalytic core, and develop specific drugs for targeting various pathways. Although our understanding of intracellular kinases has developed at a fast rate, our exploration into extracellular kinases has just begun. In this review, we will cover the secreted protein kinase families found in humans, bacteria, and parasites. © 2019 IUBMB Life, 71(6):749-759, 2019.

Feasibility trial of an unsupervised, facility-based exercise programme for depressed outpatients.

This pilot study examined feasibility of an unsupervised, facility-based exercise programme for promoting exercise adherence among depressed adult outpatients. The potential effect of adding physical activity counselling on depressive symptoms and physical activity was also explored. Participants were randomly assigned to a 12-week programme comprising an orientation and access to fitness centre resources (control, n = 18) or that programme plus 6 physical activity counselling sessions (intervention, n = 18). Outcome measures were feasibility (fitness centre attendance over 12 weeks); Beck Depression Inventory (BDI-II) and International Physical Activity Questionnaire (IPAQ) completed at baseline and week 12; and qualitative programme feedback. Fitness centre attendance averaged only 12 days (14% of all possible days) with no differences between study groups. No group differences were found on IPAQ or BDI-II scores at week 12. Increases from baseline in IPAQ moderate/vigorous activity minutes were associated with decreases in BDI-II scores at week 12 (p < 0.001). The most helpful programme aspect reported was connecting participants to fitness centre resources. In this pilot study of depressed outpatients, an unsupervised fitness centre based program was not feasible for promoting exercise adherence and adding physical activity counselling was not useful for increasing physical activity levels or reducing depression.

The coccidian parasites Toxoplasma and Neospora dysregulate mammalian lipid droplet biogenesis.

Upon infection, the intracellular parasite Toxoplasma gondii co-opts critical functions of its host cell to avoid immune clearance and gain access to nutritional resources. One route by which Toxoplasma co-opts its host cell is through hijacking host organelles, many of which have roles in immunomodulation. Here we demonstrate that Toxoplasma infection results in increased biogenesis of host lipid droplets through rewiring of multiple components of host neutral lipid metabolism. These metabolic changes cause increased responsiveness of host cells to free fatty acid, leading to a radical increase in the esterification of free fatty acids into triacylglycerol. We identified c-Jun kinase and mammalian target of rapamycin (mTOR) as components of two distinct host signaling pathways that modulate the parasite-induced lipid droplet accumulation. We also found that, unlike many host processes dysregulated during Toxoplasma infection, the induction of lipid droplet generation is conserved not only during infection with genetically diverse Toxoplasma strains but also with Neospora caninum, which is closely related to Toxoplasma but has a restricted host range and uses different effector proteins to alter host signaling. Finally, by showing that a Toxoplasma strain deficient in exporting a specific class of effectors is unable to induce lipid droplet accumulation, we demonstrate that the parasite plays an active role in this process. These results indicate that, despite their different host ranges, Toxoplasma and Neospora use a conserved mechanism to co-opt these host organelles, which suggests that lipid droplets play a critical role at the coccidian host-pathogen interface.

Clinical and imaging findings, treatments, and outcomes in 27 dogs with imaging diagnosed trigeminal nerve sheath tumors: A multi-center study.

The clinical behavior of canine trigeminal nerve sheath tumors and benefits of previously reported treatments are incompletely defined. Aims of this retrospective, multicenter, observational study were to describe clinical signs, tumor localization characteristics, treatments, and clinical outcomes in a group of dogs with this neoplasm. Databases at four hospitals were reviewed for dogs with a trigeminal nerve sheath tumor diagnosis, magnetic resonance imaging (MRI) studies, and presentation between 2004 and 2014. A single observer recorded medical record findings and two observers recorded MRI characteristics by consensus. A total of 27 dogs met inclusion criteria (15 treated with stereotactic radiation therapy and 12 unirradiated). Two unirradiated dogs were excluded from outcome analyses. The most common presenting signs were masticatory muscle atrophy (26 dogs), neurologic signs referable to intracranial disease (13), and ocular disease (12). Based on MRI findings, all dogs had disease extending centrally at the level of the brainstem. The most commonly affected trigeminal nerve branches were the mandibular (26 dogs), maxillary (22), and ophthalmic (10). Of 15 dogs treated with stereotactic radiation therapy, one had improved muscle atrophy, and six had poor ocular health after treatment. Neurologic signs improved in 4/5 dogs with intracranial signs. Overall median survival time for the 10 unirradiated dogs with available follow-up was 12 days and 441 days for the 15 stereotactic radiation therapy dogs. Mean survival times between these groups were not significantly different (mean 95% CI for unirradiated dogs was 44-424 days and mean 95% CI for stereotactic radiation therapy dogs was 260-518 days).

The Toxoplasma pseudokinase ROP5 is an allosteric inhibitor of the immunity-related GTPases.

The Red Queen hypothesis proposes that there is an evolutionary arms race between host and pathogen. One possible example of such a phenomenon could be the recently discovered interaction between host defense proteins known as immunity-related GTPases (IRGs) and a family of rhoptry pseudokinases (ROP5) expressed by the protozoan parasite, Toxoplasma gondii. Mouse IRGs are encoded by an extensive and rapidly evolving family of over 20 genes. Similarly, the ROP5 family is highly polymorphic and consists of 4-10 genes, depending on the strain of Toxoplasma. IRGs are known to be avidly bound and functionally inactivated by ROP5 proteins, but the molecular basis of this interaction/inactivation has not previously been known. Here we show that ROP5 uses a highly polymorphic surface to bind adjacent to the nucleotide-binding domain of an IRG and that this produces a profound allosteric change in the IRG structure. This has two dramatic effects: 1) it prevents oligomerization of the IRG, and 2) it alters the orientation of two threonine residues that are targeted by the Toxoplasma Ser/Thr kinases, ROP17 and ROP18. ROP5s are highly specific in the IRGs that they will bind, and the fact that it is the most highly polymorphic surface of ROP5 that binds the IRG strongly supports the notion that these two protein families are co-evolving in a way predicted by the Red Queen hypothesis.

A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins.

The ability of mice to resist infection with the protozoan parasite, Toxoplasma gondii, depends in large part on the function of members of a complex family of atypical large GTPases, the interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of T. gondii are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for T. gondii by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, ROP5, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

Expression of the essential Kinase PfCDPK1 from Plasmodium falciparum in Toxoplasma gondii facilitates the discovery of novel antimalarial drugs.

We have previously shown that genetic disruption of Toxoplasma gondii calcium-dependent protein kinase 3 (TgCDPK3) affects calcium ionophore-induced egress. We examined whether Plasmodium falciparum CDPK1 (PfCDPK1), the closest homolog of TgCDPK3 in the malaria parasite P. falciparum, could complement a TgCDPK3 mutant strain. PfCDPK1 is essential and plays critical roles in merozoite development, motility, and secretion. We show that expression of PfCDPK1 in the TgCDPK3 mutant strain rescues the egress defect. This phenotypic complementation requires the localization of PfCDPK1 to the plasma membrane and kinase activity. Interestingly, PfCDPK1-expressing Toxoplasma becomes more sensitive to egress inhibition by purfalcamine, a potent inhibitor of PfCDPK1 with low activity against TgCDPK3. Based on this result, we tested eight small molecules previously determined to inhibit the kinase activity of recombinant PfCDPK1 for their abilities to inhibit ionophore-induced egress in the PfCDPK1-expressing strain. While two of these chemicals did not inhibit egress, we found that six drugs affected this process selectively in PfCDPK1-expressing Toxoplasma. Using mutant versions of PfCDPK1 and TgCDPK3, we show that the selectivities of dasatinib and PLX-4720 are regulated by the gatekeeper residue in the ATP binding site. Importantly, we have confirmed that the three most potent inhibitors of egress in the PfCDPK1-expressing strain effectively kill P. falciparum. Thus, we have established and validated a recombinant strain of Toxoplasma that can be used as a surrogate for the discovery and analysis of PfCDPK1-specific inhibitors that can be developed as antimalarials.

Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence.

Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4-10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma's successful adaptation to a wide host range and has resulted in dramatic differences in virulence.

Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion.

Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis-activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.

A dynamin superfamily-like pseudoenzyme coordinates with MICOS to promote cristae architecture.

Mitochondrial cristae architecture is crucial for optimal respiratory function of the organelle. Cristae shape is maintained in part by the mitochondrial contact site and cristae organizing system (MICOS) complex. While MICOS is required for normal cristae morphology, the precise mechanistic role of each of the seven human MICOS subunits, and how the complex coordinates with other cristae-shaping factors, has not been fully determined. Here, we examine the MICOS complex in Schizosaccharomyces pombe, a minimal model whose genome only encodes for four core subunits. Using an unbiased proteomics approach, we identify a poorly characterized inner mitochondrial membrane protein that interacts with MICOS and is required to maintain cristae morphology, which we name Mmc1. We demonstrate that Mmc1 works in concert with MICOS to promote normal mitochondrial morphology and respiratory function. Mmc1 is a distant relative of the dynamin superfamily of proteins (DSPs), GTPases, which are well established to shape and remodel membranes. Similar to DSPs, Mmc1 self-associates and forms high-molecular-weight assemblies. Interestingly, however, Mmc1 is a pseudoenzyme that lacks key residues required for GTP binding and hydrolysis, suggesting that it does not dynamically remodel membranes. These data are consistent with the model that Mmc1 stabilizes cristae architecture by acting as a scaffold to support cristae ultrastructure on the matrix side of the inner membrane. Our study reveals a new class of proteins that evolved early in fungal phylogeny and is required for the maintenance of cristae architecture. This highlights the possibility that functionally analogous proteins work with MICOS to establish cristae morphology in metazoans.