Identification of quantitative trait loci controlling acute virulence in Toxoplasma gondii

Abstract

Strains of Toxoplasma gondii can be grouped into three predominant clonal lineages with members of the type I group being uniformly lethal in mice. To elucidate the basis of this extreme virulence, a genetic cross was performed between a highly virulent type I strain (GT-1) and a less-virulent type III strain (CTG), and the phenotypes of resulting progeny were analyzed by genetic linkage mapping. Analysis of independent recombinant progeny identified several quantitative trait loci that contributed to acute virulence. A major quantitative trait locus located on chromosome VII accounted for approximately 50% of the virulence phenotype, whereas a minor locus on chromosome IV, linked to the ROP1 gene, accounted for approximately 10%. These loci are conserved in other type I strains, indicating that acute virulence is controlled by discrete genes common to the type I lineage.

Fig 1.

(A) Isolation of recombinant progeny for genetic mapping and virulence linkage analysis. Clonal isolates of the type I strain GT-1 resistant to 5-fluoro-2-deoxyuridine (FUDR^R) and type III strain CTG resistant to adenine arabinoside and sinefungin (AraA^R/SNF^R) were crossed, and recombinant progeny clones were selected with drug selections (AraA^R/FUDR^R or SNF^R/FUDR^R combination). Alternatively, progeny clones were chosen at random and genotyped to obtain recombinant ones. (B) An example of genotyping using PCR/RFLP analysis for the SAG3 gene amplified from the parental strains (indicated as I, III) and the progeny (indicated as 1–9). PCR products were digested with restriction enzyme NciI and resolved in 3% agarose gel in the presence of ethidium bromide. DNA marker = φ174 DNA digested with HaeIII.

Fig 2.

Mortality of mice inoculated with parental strains or one of three representative phenotypes observed in recombinant progeny. Recombinant progeny C7SF has a virulent phenotype similar to GT-1 FUDR^R, whereas A9SF has a nonvirulent phenotype similar to CTG AraA^R/SNF^R. The cloneC285–1 has an intermediate phenotype (Int, 10³ ≈LD₅₀ >1, surviving mice sera positive). Mice were infected by i.p. of 10, 100, or 1,000 of tachyzoites (five animals/dose), and mortality was recorded for 30 days postinfection. Days post-i.p. = days after i.p. injection.

Fig 3.

Interval mapping of a virulence-associated QTLs. (A) A major QTL is located between the markers M95 and cS10-A6 on chromosome VII. The dotted line represents the highly significant linkage threshold (P = 0.001) for a genome-wide analysis. (B) A second QTL observed on chromosome IV lies near ROP1. The dotted line represents the significance level (P = 0.05) for a genome-wide analysis. Markers are indicated at the base of map and are separated by distance proportional to map units.