Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases

Abstract

The population structure of Toxoplasma gondii includes three highly prevalent clonal lineages referred to as types I, II, and III, which differ greatly in virulence in the mouse model. Previous studies have implicated a family of serine/threonine protein kinases found in rhoptries (ROPs) as important in mediating virulence differences between strain types. Here, we explored the genetic basis of differences in virulence between the highly virulent type I lineage and moderately virulent type II based on successful genetic cross between these lineages. Genome-wide association revealed that a single quantitative trait locus controls the dramatic difference in lethality between these strain types. Neither ROP16 nor ROP18, previously implicated in virulence of T. gondii, was found to contribute to differences between types I and II. Instead, the major virulence locus contained a tandem cluster of polymorphic alleles of ROP5, which showed similar protein expression between strains. ROP5 contains a conserved serine/threonine protein kinase domain that includes only part of the catalytic triad, and hence, all members are considered to be pseudokinases. Genetic disruption of the entire ROP5 locus in the type I lineage led to complete attenuation of acute virulence, and complementation with ROP5 restored lethality to WT levels. These findings reveal that a locus of polymorphic pseudokinases plays an important role in pathogenesis of toxoplasmosis in the mouse model.

Fig. 1.

Generation of a type I × II genetic cross and assessment of virulence. (A) Virulent type I GT-1 SNF^R and avirulent type II ME49 FUDR^R parental strains were crossed in the definitive host and recombinant progeny obtained using dual drug selection (S^R/F^R). Informative recombinants were identified using 10 RLFP markers located on 10 separate chromosomes. (B) The parental strains and 45 informative progeny were tested for virulence by inoculation of female CD-1 mice with 100 parasites each and monitoring for 30 d.

Fig. 2.

Genome-wide scan of the virulence phenotype reveals a single major QTL. (A) Progeny SFP marker genotypes were used to perform a genome-wide QTL association scan for virulence plotted on the y axis as log (1/P). A single major QTL on the left of chromosome XII explains 90.9% of the variance in the virulence phenotype. Markers are plotted on the x axis along the assembled genomic DNA sequence scaffolds of the 14 chromosomes (http://ToxoDB.org). Chromosomes are listed above the plot; UNK denotes markers on unassembled contigs. Dotted lines indicate marginal (lower line) and strongly significant (upper line) associations. (B) The matrix of two-locus associations shows a complete lack of nonadditive interactions or epistasis between pairs of loci. In A and B, Fisher's exact probability (P) was used for computing phenotype–genotype independence; results from other statistical strategies (t test statistic, Wilcoxon nonparametric, and mutual information) revealed the same QTL (SI Materials and Methods and SI Results). Chromosomes are listed at the top and right side. (C) The virulence QTL on XII is bound by SFP markers 145.m00749_at9 and 38.m01072_at10, which spans a region of ∼ 400 Kb containing 51 genes (blue).

Fig. 3.

Gene expression and CNV within the virulence QTL. (A) Gene expression (Exp) and CNV in type I and type II tachyzoites for the 51 genes within the XII locus. Heat map was created using the expression values, and colors represent fold expression of normalized intensities. Black boxes identify three genes with greater than or equal to twofold expression differences and one gene with a greater than or equal to twofold CNV difference between types I and II strains. (B and C) The number of GT-1 and ME49 trace reads (expect value = 0.0) per 2-kb segments across chromosome XII. The red dot denotes the fragment containing ROP5, and values are fold above the median number of trace reads across XII (gray line is the median and blue line is the mean).

Fig. 4.

Composition of the ROP5 locus in type I and type II lineages. (A) Tandem array of 6 copies of ROP5 in types I and III vs. 11 copies in type II. Alignment of trace reads determined that types I and III have a four-copy major allele, M (red), and two single-copy minor alleles, m1 (orange) and m2 (white), and that type II ME49 has eight copies of a major allele, M (yellow), a single-copy minor allele, m1 (green), and two copies of a minor allele that encodes a pseudogene, m2 (blue). Alleles with a single nonsynonymous SNP have an asterisk. Pseudogenes are indicated by ψ. Copy numbers and sequences of individual alleles were reconstructed from the trace archive reads (Fig. S6), although the arrangement shown here is arbitrary. (B) ClustalX alignment of types I and II ROP5 alleles with active ROP kinases from T. gondii and host kinases (Fig. S7). Residues essential for catalysis (263K and 407D) are conserved in all ROP5 alleles. The catalytic aspartic residue (389D) is divergent in all ROP5 alleles, where instead, all type II ME49 alleles contain 389R and the majority of type I GT-1 alleles have 389H, except ROP5 type I m2, which has 389R (Fig. 4A). (C) Phylogenetic network of ROP5 sequences. The major allele in type I and III as well as m1 alleles are more closely related, whereas type II alleles form a second major group. The minor alleles m2 in types I and III show a closer ancestry to type II. Table of synonymous (dS) and nonsynonymous (dN) changes in ROP5 alleles. Alleles are as in A, with the suffix indicating copy number.

Fig. 5.

Deletion of ROP5 greatly reduces virulence. (A) Schematic of the approach used to create the RHΔku80Δrop5 parasite. Regions surrounding the ROP5 locus were used to replace the region with the selectable marker HXGPRT (Hx). (B) PCR analysis to confirm the knockout of ROP5 in RHΔku80Δrop5 by detecting of flanking regions (flanks) using the primer pairs depicted in A. Ladder is a 1-kb ladder (New England Biolabs). (C) Virulence of RHΔku80Δrop5 parasites in female CD-1 mice inoculated with 10², 10⁴, or 10⁶ parasites vs. 10¹ RHΔku80 strain parasites. (D) Infection of CD-1 mice with Δrop5 complement, RHCmpl34, restores virulence comparable with WT severity. Mice were inoculated with 10² RHΔku80 or 10², 10³, or 10⁴ RHCmpl34 parasites.