The Toxoplasma pseudokinase ROP5 is an allosteric inhibitor of the immunity-related GTPases.
ABSTRACT: 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.
Project description:Polymorphic rhoptry-secreted kinases (ROPs) are essential virulence factors of Toxoplasma gondii. In particular, the pseudokinase ROP5 is the major determinant of acute virulence in mice, but the underlying mechanisms are unclear. We developed a tandem affinity protein tagging and purification approach in T. gondii and used it to show that ROP5 complexes with the active kinases ROP18 and ROP17. Biochemical analyses indicate that ROP18 and ROP17 have evolved to target adjacent and essential threonine residues in switch region I of immunity-related guanosine triphosphatases (GTPases) (IRGs), a family of host defense molecules that function to control intracellular pathogens. The combined activities of ROP17 and ROP18 contribute to avoidance of IRG recruitment to the intracellular T. gondii-containing vacuole, thus protecting the parasite from clearance in interferon-activated macrophages. These studies reveal an intricate, multilayered parasite survival strategy involving pseudokinases that regulate multiple active kinase complexes to synergistically thwart innate immunity.
Project description:The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-? (IFN?)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFN?-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans.
Project description:Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-? mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the ?rop5 and ?rop18 mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3(-/-) mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-? dependent, indicting it plays a general role in governing virulence factors that block immunity.
Project description:The intracellular parasite Toxoplasma gondii infects a wide variety of vertebrate species globally. Infection in most hosts causes a lifelong chronic infection and generates immunological memory responses that protect the host against new infections. In regions where the organism is endemic, multiple exposures to T. gondii likely occur with great frequency, yet little is known about the interaction between a chronically infected host and the parasite strains from these areas. A widely used model to explore secondary infection entails challenge of chronically infected or vaccinated mice with the highly virulent type I RH strain. Here, we show that although vaccinated or chronically infected C57BL/6 mice are protected against the type I RH strain, they are not protected against challenge with most strains prevalent in South America or another type I strain, GT1. Genetic and genomic analyses implicated the parasite-secreted rhoptry effectors ROP5 and ROP18, which antagonize the host's gamma interferon-induced immunity-regulated GTPases (IRGs), as primary requirements for virulence during secondary infection. ROP5 and ROP18 promoted parasite superinfection in the brains of challenged survivors. We hypothesize that superinfection may be an important mechanism to generate T. gondii strain diversity, simply because two parasite strains would be present in a single meal consumed by the feline definitive host. Superinfection may drive the genetic diversity of Toxoplasma strains in South America, where most isolates are IRG resistant, compared to North America, where most strains are IRG susceptible and are derived from a few clonal lineages. In summary, ROP5 and ROP18 promote Toxoplasma virulence during reinfection.Toxoplasma gondii is a widespread parasite of warm-blooded animals and currently infects one-third of the human population. A long-standing assumption in the field is that prior exposure to this parasite protects the host from subsequent reexposure, due to the generation of protective immunological memory. However, this assumption is based on clinical data and mouse models that analyze infections with strains common to Europe infections with strains common to Europe and North America. In contrast, we found that the majority of strains sampled from around the world, in particular those from South America, were able to kill or reinfect the brains of hosts previously exposed to T. gondii. The T. gondii virulence factors ROP5 and ROP18, which inhibit key host effectors that mediate parasite killing, were required for these phenotypes. We speculate that these results underpin clinical observations that pregnant women previously exposed to Toxoplasma can develop congenital infection upon reexposure to South American strains.
Project description:Some strains of the protozoan parasite Toxoplasma gondii (such as RH) are virulent in laboratory mice because they are not restricted by the Immunity-Related GTPase (IRG) resistance system in these mouse strains. In some wild-derived Eurasian mice (such as CIM) on the other hand, polymorphic IRG proteins inhibit the replication of such virulent T. gondii strains. Here we show that this resistance is due to direct binding of the IRG protein Irgb2-b1CIM to the T. gondii virulence effector ROP5 isoform B. The Irgb2-b1 interface of this interaction is highly polymorphic and under positive selection. South American T. gondii strains are virulent even in wild-derived Eurasian mice. We were able to demonstrate that this difference in virulence is due to polymorphic ROP5 isoforms that are not targeted by Irgb2-b1CIM, indicating co-adaptation of host cell resistance GTPases and T. gondii virulence effectors.
Project description:The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.
Project description:In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity-related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6-specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18-specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.
Project description:Ingestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the ?rop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5, ROP17, ROP18, ROP35, or ROP38/29/19 (ROP38, ROP29, and ROP19) severely reduced cyst burdens. ?rop5 and ?rop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The ?rop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the ?rop35 or ?rop38/29/19 strain. Resistance to host IRGs was not affected in the ?rop17, ?rop35, or ?rop38/29/19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondiiReactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections.
Project description: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.
Project description:The ROP5 family is a closely related set of polymorphic pseudokinases that are critical to the ability of Toxoplasma to cause disease. Polymorphisms in ROP5 also make it a major determinant of strain-specific differences in virulence. ROP5 possesses all of the major kinase motifs required for catalysis except for a substitution at the catalytic Asp. We show that this substitution in the catalytic loop of ROP5 is part of a motif conserved in other pseudokinases of both Toxoplasma and human origin, and that this motif is required for the full activity in vivo of ROP5. This suggests evolutionary selection at this site for a biochemical function, rather than simple drift away from catalysis. We present the crystal structures of a virulent isoform of ROP5 both in its ATP-bound and -unbound states and have demonstrated that despite maintaining the canonical ATP-binding motifs, ROP5 binds ATP in a distorted conformation mediated by unusual magnesium coordination sites that would not be predicted from the primary sequence. In addition, we have mapped the polymorphisms spread throughout the primary sequence of ROP5 to two major surfaces, including the activation segment of ROP5. This suggests that the pseudoactive site of this class of pseudokinases may have evolved to use the canonical ATP-binding motifs for non-catalytic signaling through allostery.