Pyrazoleamide and spiroindolone induced saponin sensitivity is due to cholesterol incorporation into the parasite.

<p>(A and B) Cholesterol extraction with MβCD eliminates saponin sensitivity induced by the drugs, whereas cholesterol-loaded MβCD does not. Freed 30–34 h post-infection trophozoites were treated with either PA21A050 (A) or KAE609 (B). Parasites were treated with the drugs (lanes 1 and 2), or were exposed to MβCD to extract cholesterol and then treated with the drugs (lanes 3 and 4). Freed parasites were exposed to MβCD loaded with cholesterol followed by the drug treatment (lanes 5). Freed parasites were first treated with the drugs and then exposed to either MβCD (lanes 6 and 7) or MβCD loaded with cholesterol (lanes 8). After the indicated time, parasites were subjected to a brief exposure to saponin, centrifuged and subjected to SDS-PAGE followed by immunobloting with antibodies to aldolase and Exp2. (C and D) Cholesterol loaded MβCD can donate cholesterol to cholesterol-depleted freed parasites, restoring drug-induced saponin sensitivity. Experiments with PA21A050 treatment (C) and KAE609 treatment (D) are shown. Inclusion of just 0.625 mM cholesterol-loaded MβCD in cholesterol-depleted parasites restored saponin sensitivity to parasites when treated with the drugs. However, in absence of the drugs inclusion of cholesterol-loaded MβCD did not impart saponin sensitivity, suggesting an active process of cholesterol exclusion from the parasite.