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.

A DRP-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 inner membrane-localized 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 complexes to promote normal mitochondrial morphology and respiratory function. Bioinformatic analyses reveal that Mmc1 is a distant relative of the Dynamin-Related Protein (DRP) family of GTPases, which are well established to shape and remodel membranes. We find that, like DRPs, 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 it does not dynamically remodel membranes. These data are consistent with a model in which 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.

Toxoplasma ERK7 protects the apical complex from premature degradation.

Accurate cellular replication balances the biogenesis and turnover of complex structures. In the apicomplexan parasite Toxoplasma gondii, daughter cells form within an intact mother cell, creating additional challenges to ensuring fidelity of division. The apical complex is critical to parasite infectivity and consists of apical secretory organelles and specialized cytoskeletal structures. We previously identified the kinase ERK7 as required for maturation of the apical complex in Toxoplasma. Here, we define the Toxoplasma ERK7 interactome, including a putative E3 ligase, CSAR1. Genetic disruption of CSAR1 fully suppresses loss of the apical complex upon ERK7 knockdown. Furthermore, we show that CSAR1 is normally responsible for turnover of maternal cytoskeleton during cytokinesis, and that its aberrant function is driven by mislocalization from the parasite residual body to the apical complex. These data identify a protein homeostasis pathway critical for Toxoplasma replication and fitness and suggest an unappreciated role for the parasite residual body in compartmentalizing processes that threaten the fidelity of parasite development.

Cryo-tomography reveals rigid-body motion and organization of apicomplexan invasion machinery.

The apical complex is a specialized collection of cytoskeletal and secretory machinery in apicomplexan parasites, which include the pathogens that cause malaria and toxoplasmosis. Its structure and mechanism of motion are poorly understood. We used cryo-FIB-milling and cryo-electron tomography to visualize the 3D-structure of the apical complex in its protruded and retracted states. Averages of conoid-fibers revealed their polarity and unusual nine-protofilament arrangement with associated proteins connecting and likely stabilizing the fibers. Neither the structure of the conoid-fibers nor the architecture of the spiral-shaped conoid complex change during protrusion or retraction. Thus, the conoid moves as a rigid body, and is not spring-like and compressible, as previously suggested. Instead, the apical-polar-rings (APR), previously considered rigid, dilate during conoid protrusion. We identified actin-like filaments connecting the conoid and APR during protrusion, suggesting a role during conoid movements. Furthermore, our data capture the parasites in the act of secretion during conoid protrusion.

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.

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.

SchistoCyte Atlas: A Single-Cell Transcriptome Resource for Adult Schistosomes.

Schistosomes cause untold disease and disability in the developing world. Here, we introduce SchistoCyte Atlas, a web-based platform for exploring gene expression at single-cell resolution in adult Schistosoma mansoni. Similar resources accessible to non-specialists across the globe will expedite our ability to understand the biology of these devastating parasites.

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.

Multivalent Interactions Drive the Toxoplasma AC9:AC10:ERK7 Complex To Concentrate ERK7 in the Apical Cap.

The Toxoplasma inner membrane complex (IMC) is a specialized organelle that is crucial for the parasite to establish an intracellular lifestyle and ultimately cause disease. The IMC is composed of both membrane and cytoskeletal components, further delineated into the apical cap, body, and basal subcompartments. The apical cap cytoskeleton was recently demonstrated to govern the stability of the apical complex, which controls parasite motility, invasion, and egress. While this role was determined by individually assessing the apical cap proteins AC9, AC10, and the mitogen-activated protein kinase ERK7, how the three proteins collaborate to stabilize the apical complex is unknown. In this study, we use a combination of deletion analyses and yeast two-hybrid experiments to establish that these proteins form an essential complex in the apical cap. We show that AC10 is a foundational component of the AC9:AC10:ERK7 complex and demonstrate that the interactions among them are critical to maintaining the apical complex. Importantly, we identify multiple independent regions of pairwise interaction between each of the three proteins, suggesting that the AC9:AC10:ERK7 complex is organized by multivalent interactions. Together, these data support a model in which multiple interacting domains enable the oligomerization of the AC9:AC10:ERK7 complex and its assembly into the cytoskeletal IMC, which serves as a structural scaffold that concentrates ERK7 kinase activity in the apical cap. IMPORTANCE The phylum Apicomplexa consists of obligate, intracellular parasites, including the causative agents of toxoplasmosis, malaria, and cryptosporidiosis. Hallmarks of these parasites are the IMC and the apical complex, both of which are unique structures that are conserved throughout the phylum and required for parasite survival. The apical cap portion of the IMC has previously been shown to stabilize the apical complex. Here, we expand on those studies to determine the precise protein-protein interactions of the apical cap complex that confer this essential function. We describe the multivalent nature of these interactions and show that the resulting protein oligomers likely tether ERK7 in the apical cap. This study represents the first description of the architecture of the apical cap at a molecular level, expanding our understanding of the unique cell biology that drives Toxoplasma infections.

Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division.

Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis.IMPORTANCEToxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.

A single-cell RNA-seq atlas of Schistosoma mansoni identifies a key regulator of blood feeding.

Schistosomiasis is a neglected tropical disease that infects 240 million people. With no vaccines and only one drug available, new therapeutic targets are needed. The causative agents, schistosomes, are intravascular flatworm parasites that feed on blood and lay eggs, resulting in pathology. The function of the parasite's various tissues in successful parasitism are poorly understood, hindering identification of therapeutic targets. Using single-cell RNA sequencing (RNA-seq), we characterize 43,642 cells from the adult schistosome and identify 68 distinct cell populations, including specialized stem cells that maintain the parasite's blood-digesting gut. These stem cells express the gene hnf4, which is required for gut maintenance, blood feeding, and pathology in vivo. Together, these data provide molecular insights into the organ systems of this important pathogen and identify potential therapeutic targets.

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.

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.

Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure in Toxoplasma gondii.

Primary cilia are important organizing centers that control diverse cellular processes. Apicomplexan parasites like Toxoplasma gondii have a specialized cilium-like structure called the conoid that organizes the secretory and invasion machinery critical for the parasites' lifestyle. The proteins that initiate the biogenesis of this structure are largely unknown. We identified the Toxoplasma orthologue of the conserved kinase ERK7 as essential to conoid assembly. Parasites in which ERK7 has been depleted lose their conoids late during maturation and are immotile and thus unable to invade new host cells. This is the most severe phenotype to conoid biogenesis yet reported, and is made more striking by the fact that ERK7 is not a conoid protein, as it localizes just basal to the structure. ERK7 has been recently implicated in ciliogenesis in metazoan cells, and our data suggest that this kinase has an ancient and central role in regulating ciliogenesis throughout Eukaryota.

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.

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.

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.

Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1.

Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

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.