Figure 10. ASP5 is an important virulence factor. .
(A) Four groups of six C57BL/6 mice were intraperitoneally injected with a live dose of 15 ± 3 tachyzoites or PBS alone and survival measured over a 20-day period. Mice infected with wild type (RHΔhx) and Δasp5CRISPR:ASP5WT-HA3 all succumbed to infection within 8 days, whereas all PBS-injected mice and those infected with Δasp5CRISPR parasites survived the 20 day experiment. At day 14 (#), all mice were bled and tested for antibodies against tachyzoites. Animals were weighed daily throughout the course of the experiment (lower panel) and bodyweights were compared for statistical analysis while all animals were alive. Mice injected with PBS alone maintained a stable body weight, while those infected with wild type and Δasp5CRISPR:ASP5WT-HA3 parasites lost weight beginning at day 6 and day 4, respectively, with significant weight loss evident in comparison to those injected with Dasp5CRISPR parasites by day 7. (B) At 24 days post infection, surviving mice (from A) that were injected with PBS or Δasp5CRISPR tachyzoites were re-challenged with 50 live RHΔhx parasites. The naïve PBS-injected mice all succumbed to infection by day 10, whereas those that had been injected with Δasp5CRISPR parasites were protected from death. Bodyweight was also monitored daily (lower panel) where mice previously injected with Δasp5CRISPR parasites maintained a stable bodyweight, while the naïve PBS mice began losing weight on approximately day 6. (C) A separate cohort of C57/BL6 mice was also injected with 50 live parasites to assess the effect of parasite number on virulence during infection. All mice infected with WT or and Δasp5CRISPR:ASP5WT-HA3 parasites again succumbed to infection by days 8-10, whereas there was a delay in death for the and Δasp5CRISPR-infected mice. One of these mice survived the 15-day experiment and was seropositive for antibodies against Toxoplasma (serum collected at day 14, #). Bodyweights were measured daily (lower panel). Log-rank (Mantel-Cox) testing was used to derive statistical significance for survival curves while two-way analysis of variance testing was used for bodyweight data. Values are mean ± SD. * P < 0.05, ** P < 0.005, **** P <0.0001. ASP5, Aspartyl Protease 5; WT, Wild Type.
Figure 10—figure supplement 1. Seroconversion of Δasp5 parasites.
