Author Summary The determinants of virulence are rarely defined for eukaryotic parasites such as T. gondii, a widespread parasite of mammals that also infects humans, sometimes with serious consequences. Recent laboratory studies have established that variation in a single secreted protein, a serine/threonine kinase known as ROPO18, controls whether or not mice survive infection. Here, we establish the extent and nature of variation in ROP18 among a collection of parasite strains from geographically diverse regions. Compared to other genes, ROP18 showed extremely high levels of diversification and changes in expression level, which correlated with severity of infection in mice. Comparison with an out-group demonstrated that changes in the upstream region that regulates expression of ROP18 led to an historical increase in the expression and exposed the protein to diversifying selective pressure. Surprisingly, only three atypically distinct protein variants exist despite marked genetic divergence elsewhere in the genome. These three forms of ROP18 are likely adaptations for different niches in nature, and they confer markedly different virulence to mice. The widespread distribution of a single mouse-virulent allele among geographically and genetically disparate parasites may have consequences for transmission and disease in other hosts, including humans. lineages differ markedly in their virulence to outbred laboratory mice: type I strains are acutely virulent with an estimated 100% lethal dose (LD100) of a single organism, while types II and III are relatively nonvirulent (LD50.105) [14]. Mice are a natural host for T. gondii, and the laboratory mouse provides an excellent model for establishing differences in pathogenesis between parasite strains. Type I strain show faster rates of replications in vitro [15], enhanced migration in vitro and in vivo [16], and reach higher tissue burdens [17] in laboratory mice. While acute mortality exhibited in mice may not be directly extrapolated to other hosts, such experimental models can identify candidate factors that may influence the outcome of infection in other hosts, potentially affecting transmission and disease. Various factors are likely to influence the outcome of human infection, including the genotype of the parasite. However, because human-to-human transmission plays little role in the evolution of T. gondii, any contribution of parasite genotype to pathogenesis is an indirect consequence of adaptation in other hosts. The vast majority of human cases of toxoplasmosis that have been described in North America and Europe belong to the type II genotype [4,18]. Type II strains often cause mild infection in healthy individuals, and yet can very severe congenital infection and acute severe toxoplasmic encephalitis in AIDS patients. While relatively uncommon in the wild, type I strains have been associated with several small cohorts of human congenital toxoplasmosis and opportunistic infection in AIDS patients [19,20], suggesting type I strains may be more likely to cause disease in permissive hosts. In other regions of the world, genetically more diverse strains have been associated with severe infection. For example, atypical genotypes of T. gondii predominate in French Guyana where severe infections have been described in otherwise healthy adults [21,22]. Divergent genotypes of T. gondii have also been associated with severe ocular toxoplasmosis in southern Brazil [23] and in the United States [24]. Notably, strains from both of these South American regions are acutely virulent in the mouse model, similar to the type I lineage. The marked phenotypic differences expressed in the murine model have been utilized to explore the contribution of parasite Population Strucure and Virulence of T. gondii genotype to pathogenesis. Excellent forward and reverse genetic systems have recently supported genome-wide analyses of the factors that control phenotypic differences between the clonal lineages. Independent screens have converged on secretory proteins, discharged from apical organelles called rhoptries, as key modulators of both host gene transcription [25] and of acute virulence in the mouse model [26,27]. During host cell invasion, the parasite discharges the contents of rhoptries into the host cell, thereby delivering a variety of kinases, phosphatases, and other potential effectors directly into the host cell [28,29]. Genetic mapping studies have shown that the primary determinant of acute mouse virulence in type I strains of T. gondii is a polymorphic rhoptry kinase known as ROP18, which relies on serine/threonine (S/T) kinase activity to promote growth [30], and enhance virulence in the mouse model [27]. ROP18 and other polymorphic ROPs have been implicated in the more subtle differences in pathogenicity that occur between types II and III strains in the mouse model [25,26]. Despite these advances, the phenotypic consequences of variation in ROP18 in genetically diverse, natural isolates of T. gondii remain uncertain. Here, we examined the contribution of genetic variation at ROP18 to the natural variation in mouse virulence and the population structure of T. gondii. Results/Discussion ROP18 Is among the Most Divergent Genes in T. gondii The clonal lineages of T. gondii are 98% identical at most loci, reflecting the close similarity of parental lineages that gave rise to them [9]. Within lineage polymorphism is significantly lower, on the order of 1 change in 10,000 base pairs (bp), consistent with the very recent ancestry of the three clonal lineages [5]. In comparing inter-lineage diversity, certain genes exhibit more polymorphism and hence might underlie phenotypic variation between strain types. We compared the diversity of genes encoding polymorphic surface and secretory antigens (i.e. SAGs, ROPs, GRAs) with ROP18 and other putative virulence factors identified by previous genetic mapping studies [25–27]. For comparison, we included several housekeeping genes and introns, previously used for phylogenetic studies [7]. Polymorphism was characterized in a total of 32 separate loci using the recently completed genome sequences from archetypal strains of the type I, II and III (Table S1). Four of the five most variable genes identified belonged to the ROP family, with ROP18 being the most diverse (Figure 1A). Interestingly, three of these belong to the ROP2 family of proteins that contain a serine threonine kinase domain [31]. While most are predicted to be catalytically inactive, ROP18 retains activity [30] and this is required for its virulence enhancing potential [27]. Although variable, most of the ROPs showed little evidence of positive selection (based on the proportion of nonsynonymous (pNS)/ synonymous (pS) substitutions) regardless of whether they are predicted to be pseudokinases (i.e. ROP2, ROP5) or active members of this family (i.e. ROP16, ROP17). In contrast, strong evidence of positive selection was identified for ROP18 and several polymorphic dense granule (GRA) antigens (i.e. GRA3, GRA6, GRA7) [10,12,13] (Figure 1B). While most genes conformed to the expected biallelic pattern of inheritance [9], three atypically divergent alleles were found at ROP18 (Figure 1C). ROP18 is thus distinguished by uncommonly abundant polymorphism, which may be at least partially attributed to strong, positive selection. Only Three Common ROP18 Alleles Account for Its Exceptional Genetic Diversity Previous studies have shown that ROP18 plays a prominent role in mediating virulence differences between laboratory isolates of clonal PLoS Genetics | www.plosgenetics.org 2 March 2009 | Volume 5 | Issue 3 | e1000404