The majority of known Toxoplasma gondii isolates from Europe and North America belong to three clonal lines that differ dramatically in their virulence, depending on the host. To identify the responsible genes, we mapped virulence in F1 progeny derived from crosses between type II and type III strains, which we introduced into mice. Five virulence (VIR) loci were thus identified, and for two of these, genetic complementation showed that a predicted protein kinase (ROP18 and ROP16, respectively) is the key molecule. Both are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. These results suggest that secreted kinases unique to the Apicomplexa are crucial in the host-pathogen interaction.Toxoplasma gondii is an obligate intracellular parasite capable of infecting a wide variety of warm-blooded animals. Infections are widespread in humans and can lead to severe disease in utero or in individuals with a suppressed immune system. The majority of European and North American isolates belong to three distinct clonal lines, referred to as types I, II, and III (1,2). Types I and III appear to be the result of just one or two matings between an ancestral type II strain and, respectively, one or other of a pair of closely related strains that are distinct from type II (3-6). The three major Toxoplasma lines differ in a number of phenotypes (7), the best described of which is virulence in mice: type I strains are the most virulent with a lethal dose (LD 100 ) of one parasite (8,9), whereas types II and III have values for median lethal dose (LD 50 ) that range from 10 2 to 10 5 . There may also be differences in the virulence of the three strains in humans (10-12).Previously (3), we demonstrated that a cross between a type II and a type III strain produced F 1 progeny (S23 and CL11) that were more virulent (up to 3 logs) than 14 of their siblings (3). Because only two of the 16 progeny showed this difference, it was likely that multiple loci controlled virulence in these strains, and to identify these loci, we phenotyped 23 additional recombinant F 1 progeny from II × III crosses (13,14). Progeny with high virulence were identified by infecting mice with 100 tachyzoites; progeny with very low virulence were identified by infecting mice with 100,000 parasites. †To whom correspondence should be addressed.

Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, can cause severe disease in humans with an immature or suppressed immune system. The outcome of Toxoplasma infection is highly dependent on the strain type, as are many of its in vitro growth properties 1 . Here we use genetic crosses between type II and III lines to show that strain-specific differences in the modulation of host cell transcription are mediated by a putative protein kinase, ROP16. Upon invasion by the parasite, this polymorphic protein is released from the apical organelles known as rhoptries and injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signalling pathways and consequent downstream effects on a key host cytokine, interleukin (IL)-12. Our findings provide a new mechanism for how an intracellular eukaryotic pathogen can interact with its host and reveal important differences in how different Toxoplasma lineages have evolved to exploit this interaction.Most Toxoplasma gondii isolates that have been identified in Europe and North America belong to three distinct clonal lines 2,3 , referred to as types I, II and III. The three types differ widely in a number of phenotypes in mice such as virulence, persistence, migratory capacity, attraction of different cell types and induction of cytokine expression 1 . Recent results indicate that such differences might also exist in human infection 4-9 . To test the hypothesis that some of these strain-specific differences are a result of how the strains interact with the host cell, we infected human foreskin fibroblasts (HFFs) with each of the three types and used microarray analysis to investigate differences in host gene expression 24 h later. Significance analysis of microarrays 10 (SAM) identified 105 human complementary DNAs, representing at least 88 unique genes that were regulated in a strain-specific manner (false discovery rate 15%) (Fig. 1a).If the strain-specific regulation of a host gene has a genetic basis, it should segregate among F1 progeny derived from a cross between two strains that differ in its regulation. We thereforeCorrespondence and requests for materials should be addressed to J.B. (john.boothroyd@stanford.edu). * These authors contributed equally to this work.Author Contributions J.P.J.S. and S.C. contributed equally to this work. J.P.J.S. performed the microarrays and pathway analyses. S.C. and J.P.J.S. performed the experiments in Fig. 3. S.C. performed the experiments in Fig. 4 and Fig 5. J.P.B., M.E.J. and M.W.W. performed the genetic crosses that produced the progeny D3X1 and JD4. J.P.B. genotyped D3X1 and JD4. J.P.J.S., S.C., J.P.B. and J.C.B. wrote the paper. All authors discussed the results and commented on the manuscript. Author InformationThe microarray data have been deposited in ArrayExpress with the accession number E-MEXP-783. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financia...

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.

Macrophages are centrally involved in the host immune response to infection with Trypanosoma brucei rhodesiense, a protozoan parasite responsible for human sleeping sickness in Africa. During trypanosome infections, the host is exposed to parasite-derived molecules that mediate macrophage activation, specifically GPI anchor substituents associated with the shed variant surface glycoprotein (VSG), plus the host-activating agent IFN-γ, which is derived from activated T cells and is essential for resistance to trypanosomes. In this study, we demonstrate that the level and timing of exposure of macrophages to IFN-γ vs GPI ultimately determine the macrophage response at the level of induced gene expression. Treatment of macrophages with IFN-γ followed by GIP-sVSG (the soluble form of VSG containing the glycosylinositolphosphate substituent that is released by parasites) stimulated the induction of gene expression, including transcription of TNF-α, IL-6, GM-CSF, and IL-12p40. In contrast, treatment of macrophages with GIP-sVSG before IFN-γ stimulation resulted in a marked reduction of IFN-γ-induced responses, including transcription of inducible NO synthase and secretion of NO. Additional experiments revealed that the inhibitory activity of GIP-sVSG was associated with reduction in the level of STAT1 phosphorylation, an event required for IFN-γ-induced macrophage activation. These results suggest that modulation of specific aspects of the IFN-γ response may be one mechanism by which trypanosomes overcome host resistance during African trypanosomiasis.

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