Population Strucure and Virulence of T. gondii Figure 5. Expression levels of ROP18 correlate with acute virulence. A) Schematic of ROP18 coding and upstream regions based on sequence analysis of the three clonal lineages. A ,2 kb upstream segment (UPS) shown in black (not to scale) is unique to the type III genotype. Type I and II are similar, although Type II contains three tandem repeats of a 44 bp region that is only found once in type I. B) PCR analysis from genomic DNA of clonal isolates (group) demonstrating only type III strains (group 3) contain the UPS region (primers C/D). C) Differential amplification of the upstream regions from a variety of strains reveals that most strains lack the UPS (positive with primers A/B) and resemble types I and II, while the UPS is limited to types 3, 9 and CASTELLS. D) Acute mortality in outbred mice (% cumulative mortality) is correlated with allele-type and level of expression of ROP18 as determined by qPCR. Strains include those in Table S2 where mortality data is available. Correlation of expression level in type II* stains is plotted as a linear regression, r = 0.68. doi:10.1371/journal.pgen.1000404.g005 sequencing revealed that Types I and II were highly similar, aside from the tandem duplication of three copies of a 44 bp block in type II, while this region only occurs once in type I (Figure 5A). In contrast, the type III lineage contains a ,2 kb segment of DNA upstream of the ROP18 gene that was not present in types I or II (Figure 5A). There is no similarity of this upstream segment (UPS) to other regions of the genome nor is there any homologous sequence in NCBI (data not shown). Using specific PCR primers that distinguished the upstream region of types I and II (primers A/B) from type III (primers C/D), we demonstrated that they amplified mutually exclusive fragments from genomic DNAs derived from a set of representative clonal isolates (Figure 5B,C). This analysis confirmed that the UPS is unique to the type III lineage (Figure 5B). Further analysis of a wider collection of strains from other haplogroups identified the UPS only in members of type III (group 3), group 9, and a single member of group 4 (i.e. CASTELLS). All other strains lacked the UPS (Figure 5C). Sequencing the coding regions of ROP18 III* alleles confirmed that these were always flanked by the UPS, which was invariably lacking in ROP18 I* and ROP18II* alleles (data not shown). We also tested all of the above strains for acute virulence in the mouse model described previously [27], and evaluated expression based on qPCR. Strains containing the UPS expressed low levels of ROP18 and were nonvirulent in outbred mice, whereas the opposite was true of parasites lacking the UPS (Figure 5D). In total, 17/18 type I* strains were acutely virulent while 6 of 8 type III* were avirulent (Table S2). Rare exceptions to this pattern (i.e. BOF for type I*; P89 and CASTELLS for type III*) were not due to differences in either the coding or the upstream regions of ROP18 (sequencing data not shown), and presumably arise from other genetic differences. For example, BOF contains a type I* allele, lacks the UPS, and shows high expression of ROP18, yet is avirulent in the mouse model, presumably due to a defect in some other pathway needed for efficient infection. Importantly, this strain was isolated from an AIDS patient, suggesting it may only be pathogenic in immunocompromised hosts (Table S2). Con- versely, P89 and CASTELLS contain a type III allele and the corresponding UPS, consistent with their low expression of ROP18, yet they both show high levels of virulence. Presumably this is the result of another favorable combination of genes that enhance pathogenicity, as these strains are not conventional members of the type III clonal lineage, but rather divergent isolates. Importantly, the variation in expression level among type I or type III strains differed by more than an order of magnitude, without altering their respective phenotypes (Figure 5D). Whereas virulence in ROP18I* and ROP18III* expressing strains tended to be all or none, virulence among ROP18II* expressing strains increased with the expression level (Figure 5D). Among strains expressing type II* alleles, expression level and mortality were highly correlated (r = 0.68) as shown by linear regression analysis (line in Figure 5D). Consistent with this, previous reports indicate that over-expression of the type II allele in the type III background leads to a dramatic increase in virulence [26]. The low level of expression of ROP18III* may be finely tuned to the genotype as a whole, since it has thus far not been possible to over-express the type III allele or to disrupt ROP18 by homologous recombination (data not shown). Overall, the combination of allele and expression level of ROP18 correlates with acute virulence in 30 of 33 strains included here. Hence, differences in acute virulence among these isolates can be largely explained by the allele of ROP18 and the extent of its expression. DNA Rearrangements Were Associated with Upregulation of ROP18 Expression The striking increase in ROP18 expression in the absence of the UPS, previously established for laboratory strains [32], also characterizes our broader sample of natural isolates. Two scenarios could explain how such difference came to be: 1) the UPS was inserted in the ancestor that gave rise to group 3 (type III) and group 9, or 2) the UPS was deleted from (or rearranged in) the common ancestor to the ROP18I* and ROP18II* alleles. To test these two models, we used an out-group comparison to infer the ancestral condition of this genomic region, employing the homologous sequence from the animal pathogen Neospora caninum [33]. Previous estimates based on small subunit RNA and the internal transcribed spacer (ITS) region of the ribosomal RNA indicate T. gondii shared a common ancestor with N. caninum ,10 million years ago [5]. A syntenic block encompassing ROP18, and the two genes flanking it, was identified in N. caninum (available whole genome sequence assembly generated by The Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/sequencing/ Neospora/caninum/), allowing comparison to T. gondii (Figure 6). Like ROP18III*, the ROP18 gene in N. caninum included a UPS region not present in either ROP18I* or ROP18II* (Figure 6). Clustal analyses revealed that N. caninum was ,54% identical to ROP18III* in this upstream region, and ,68% identical in the coding region. Thus, the UPS region, evidently present in the common ancestor of all T. gondii, was lost by deletion or rearrangement in the ancestor to the more recently derived ROP18I* and ROP18II* alleles. ROP18 Shows a Biphasic Ancestry Associated with Expansion of Virulence The high degree of divergence among allelic lineages of ROP18 suggests it has either undergone faster change or evolved over a longer period of time, relative to most regions of the genome. To differentiate among these alternative explanations, we compared the extent intra-lineage variation in ROP18 to that present in several surface antigen genes in 17 representative members of the clonal lineages. ROP18 did not contain more polymorphism than did other genes or introns, suggesting no elevation in its rate of molecular evolution (Table S4). Indeed, as previously derived from putatively neutral loci such as introns [5], the scarcity of unique polymorphisms in ROP18 within each of the three clonal types is consistent with the model that they originated within the last ,10,000 years (Table S4). Because ROP18 has not been changing at an especially rapid rate, our data instead suggest that the principal alleles have been maintained for an especially long period of time. PLoS Genetics | www.plosgenetics.org 8 March 2009 | Volume 5 | Issue 3 | e1000404