Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii

et al. (2010) Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii. PLoS Biol 8(12): e1000576. doi:10.1371/journal.pbio.1000576 Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii Tobias Steinfeldt 0 Stephanie Ko nen-Waisman 0 Lan Tong 0 Nikolaus Pawlowski 0 Tobias 0 Lamkemeyer 0 L. David Sibley 0 Julia P. Hunn 0 Jonathan C. Howard 0 Gary E. Ward, University of Vermont, United States of America 0 1 Institute for Genetics, University of Cologne , Cologne, Germany, 2 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany , 3 Department of Molecular Microbiology, Washington University School of Medicine , St. Louis, Missouri , United States of America Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are wellestablished model species with excellent analytical tools available. Current address; Miltenyi Biotec GmbH; Friedrich-Ebert-Strasse 68; 51429 Bergisch Gladbach; Germany - Funding: TL was supported by CECAD. This research was supported by grants in the collaborative research programs SFB635, SFB670, SFB680, and SPP1394 from the Deutsche Forschungsgemeinschaft (to JCH), and grants AI036629 and AI084243 from the NIH (to LDS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: CID, collision-induced dissociation; GBP, guanylate binding protein; IRG, immunity-related GTPase; MEF, mouse embryonic fibroblast; MS, mass spectrometry; PVM, parasitophorous vacuole membrane; wt, wild type Toxoplasma gondii is an intracellular protozoan parasite with a complex life cycle and is distantly related to the malarial genus Plasmodium. The sexual phase occurs only in the true cats (Felidae), while all warm-blooded animals including humans can be intermediate hosts. The infection is transmitted from cats to intermediate hosts via ingestion of oocysts from the faeces of infected cats, and from intermediate hosts back to cats by carnivory. Typically, T. gondii establishes a lifelong chronic infection in intermediate hosts by encysting, after an initial phase of rapid intracellular proliferation and cellcell spread, in brain and muscle. The life cycle is completed when the infected host is eaten by a cat [1]. However, some T. gondii strains are highly virulent for mice, killing the host as early as ten days after initial infection. Of the three clonal lineages of T. gondii commonly found in Eurasia and North America [2,3], the type I strains are highly virulent for mice [4]. In a genetic cross between a type I virulent and a type III avirulent strain, the serine-threonine kinase secreted from rhoptry organelles, ROP18 [5], emerged as a major virulence factor [6]. In another genetic cross, ROP16 kinase and the ROP5 pseudokinases were implicated in virulence differences between type II and type III strains [7]. Comparative studies of ROP18 from multiple T. gondii strains, including the major Eurasian types, established that this virulence protein shows extensive polymorphic sequence variation derived from recent episodes of positive selection [8]. In mice the major resistance factors preventing acute death from avirulent T. gondii infection, and thereby allowing T. gondii transmission, are large GTPases of the immunity-related GTPase (IRG) family [9,10]. These interferon-c (IFNc)-inducible proteins accumulate on the parasitophorous vacuole membrane (PVM) Many pathogens manipulate the immune system of their hosts to facilitate infection and ensure transmission to subsequent hosts. The intracellular protozoan Toxoplasma gondii, a relative of the malaria parasite, is able to infect and persist in a remarkable variety of warm-blooded hosts. Indeed roughly a third of the human race carry live Toxoplasma cysts in their brains with no overt effects. Toxoplasma infection is kept at bay in many mammals (but not in humans) by a resistance system based on a family of proteins known as the immunity-related GTPase (IRG) family. IRG proteins accumulate in infected cells on the vacuoles containing the parasite and ultimately destroy them. In this paper, we show that, in the mouse, Toxoplasma can oppose the IRG system by secreting an enzyme called ROP18 into infected cells, which phosphorylates key amino acids on the IRG proteins, rendering them inactive. Not all strains of Toxoplasma can produce an active form of ROP18, but those strains that do are more virulent. We propose that individual hosts control Toxoplasma with differing efficiency, and the variation we see in ROP18 kinase activity produced by different Toxoplasma strains is an evolutionary response to this. Thus, in different mammalian hosts, each strain seeks a balance between an excess of virulence (resulting in premature death of the host) and resistance that is too efficient (resulting in clearance of the par (...truncated)