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...

The protozoan parasite Toxoplasma gondii blocks the innate aversion of rats for cat urine, instead producing an attraction to the pheromone; this may increase the likelihood of a cat predating a rat. This is thought to reflect adaptive, behavioral manipulation by Toxoplasma in that the parasite, although capable of infecting rats, reproduces sexually only in the gut of the cat. The ''behavioral manipulation'' hypothesis postulates that a parasite will specifically manipulate host behaviors essential for enhancing its own transmission. However, the neural circuits implicated in innate fear, anxiety, and learned fear all overlap considerably, raising the possibility that Toxoplasma may disrupt all of these nonspecifically. We investigated these conflicting predictions. In mice and rats, latent Toxoplasma infection converted the aversion to feline odors into attraction. Such loss of fear is remarkably specific, because infection did not diminish learned fear, anxiety-like behavior, olfaction, or nonaversive learning. These effects are associated with a tendency for parasite cysts to be more abundant in amygdalar structures than those found in other regions of the brain. By closely examining other types of behavioral patterns that were predicted to be altered we show that the behavioral effect of chronic Toxoplasma infection is highly specific. Overall, this study provides a strong argument in support of the behavioral manipulation hypothesis. Proximate mechanisms of such behavioral manipulations remain unknown, although a subtle tropism on part of the parasite remains a potent possibility.behavioral manipulation ͉ fear ͉ parasites ͉ predator T he ''behavioral manipulation'' hypothesis states that a parasite can alter host behavior specifically to increase its own transmission efficiency (1, 2). After an acute infection, the protozoan parasite Toxoplasma gondii latently persists in the brain for the life of an infected host, offering an opportunity to study the behavioral manipulation hypothesis (3). Toxoplasma reproduces sexually in a two-species life cycle (4). The sexual phase of its reproduction occurs in the feline intestine, from which highly stable oocysts are excreted in the feces. Grazing animals, including rodents, can then ingest these oocysts. In these hosts, Toxoplasma forms cysts and persists in the central nervous system. The life cycle is completed when a cat eats an infected animal. Recent reports indicate that the parasite blunts the innate aversion of rats for the urine of cats, converting this aversion to an attraction (5), although it does not interfere with energetically costly behaviors related to mating success and social status (6). These findings agree with the behavioral manipulation hypothesis, which predicts that parasites will alter only behaviors that are beneficial to their transmission while leaving other behaviors intact.Several studies have investigated the innate fear of laboratory rodents toward cat odors (7-11). These studies have delineated a neuroanatomical circuit comprising ...

Summary Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either “intracellular” or purified away from host material. Using liquid chromatography and tandem mass-spectrometry we identified over 5,000 and 10,000 previously unknown phosphorylation sites in P. falciparum and T. gondii respectively, revealing that protein phosphorylation is an extensively used regulation mechanism both within and beyond parasite boundaries. Unexpectedly both parasites have phosphorylated tyrosines and P. falciparum has unusual phosphorylation motifs that are apparently shaped by its A:T-rich genome. This dataset provides important information on the role of phosphorylation in the host-pathogen interaction, and clues to the evolutionary forces operating on protein phosphorylation motifs in both parasites.

The protozoan Toxoplasma gondii is a prevalent parasite in wild and domestic animals worldwide, being transmitted through the food chain by carnivorous feeding and scavenging. Toxoplasma normally divides asexually to yield a haploid form that can infect virtually any vertebrate but it also has a well defined sexual cycle that occurs exclusively in cats. Toxoplasma has become important as an often fatal opportunistic pathogen in patients with AIDS, although the 15-85% of adult human populations that are chronically infected with T. gondii are typically asymptomatic. Infections in immunocompromised hosts have variable outcomes. For example, only 30 to 50% of AIDS patients that are chronically infected with the parasite develop toxoplasmic encephalitis and only about half of acute maternal infections result in congenital disease of the newborn. T. gondii strains differ in their virulence in animals, but the extent to which different strains are related has not been determined. Here we analyse 28 strains from a variety of hosts on five continents and find that the ten virulent strains have an essentially identical genotype, whereas the nonvirulent strains are moderately polymorphic. These data strongly suggest that virulent strains of T. gondii originated from a single lineage which has remained genetically homogeneous despite being globally widespread, and despite the ability of this organism to reproduce sexually.