The rat Toxo1 locus directs toxoplasmosis outcome and controls parasite proliferation and spreading by macrophage-dependent mechanisms

Abstract

Toxoplasmosis is a healthcare problem in pregnant women and immunocompromised patients. Like humans, rats usually develop a subclinical chronic infection. LEW rats exhibit total resistance to Toxoplasma gondii infection, which is expressed in a dominant mode. A genome-wide search carried out in a cohort of F(2) progeny of susceptible BN and resistant LEW rats led to identify on chromosome 10 a major locus of control, which we called Toxo1. Using reciprocal BN and LEW lines congenic for chromosome 10 genomic regions from the other strain, Toxo1 was found to govern the issue of T. gondii infection whatever the remaining genome. Analyzes of rats characterized by genomic recombination within Toxo1, reduced the interval down to a 1.7-cM region syntenic to human 17p13. In vitro studies showed that the Toxo1-mediated refractoriness to T. gondii infection is associated with the ability of the macrophage to impede the proliferation of the parasite within the parasitophorous vacuole. In contrast, proliferation was observed in fibroblasts whatever the genomic origin of Toxo1. Furthermore, ex vivo studies indicate that macrophage controls parasitic infection spreading by a Toxo1-mediated mechanism. This forward genetics approach should ultimately unravel a major pathway of innate resistance to toxoplasmosis and possibly to other apicomplexan parasitic diseases.

Fig. 1.

Susceptibility to T. gondii infection in (LEW × BN) F₂ rats according to their genotype at the D10Rat116 microsatellite marker. A total of 107 rats were studied, of which 22 were homozygous LEW (ll), 50 were heterozygous BN/LEW (nl), and 35 were homozygous BN (nn). (A) Anti-T. gondii Ab titers measured by immunofluorescence. Titers represent the last dilution of the serum at which positive results were observed. Titers >1/100 were considered as negative; titers ≤1/20.000 as strongly positive. ll vs. nl, nl vs. nn, nn vs. ll: P < 0.001. (B) Number of brain cysts. Framed numbers represent the rats that develop no brain cyst. ll vs. nn and nl vs. nn, P < 0.001.

Fig. 2.

Toxo1 controls the proliferation of T. gondii within the macrophages. (A) BN or LEW MΦ were mixed with T. gondii for 1 h, washed, and cultured for 20 h. The figure represents the repartition of infected MΦ according to the number of parasites per parasitophorous vacuole. The columns and the bars show the mean result and the standard deviation of three independent experiments. (B) The intracellular growth of T. gondii on macrophage and fibroblast monolayers from BN, LEW, and congenic lines (BN.LEWc10-E, BN.LEWc10-Cc, LEW.BNc10-F, LEW.BNc10-C) was measured by monitoring [³H]uracil incorporation into T. gondii RNA. From the two different LEW.BNc10 lines of the same BN genotype at Toxo1, the -F line was used for macrophage studies and the -C line was used for fibroblast studies. The columns and the bars show the mean result and the standard deviation of triplicates in one rat. These results are representative of two (fibroblasts) and three (macrophages) independent experiments. As a whole, studies on macrophages were performed on six BN, four LEW, and four rats of each congenic line with similar results. Dotted lines indicate the limits of Toxo1 (boundary markers: D10Rat116 and D10Rat80). N, homozygous BN; L, homozygous LEW.