Figure 4
PfATP4 inhibitors induce the lysis of erythocytes infected with nanoluciferase reporter parasites. (A) Diagram of the nanoluciferase reporter N-terminally appended to the export signal region from the parasite PEXEL protein Hyp1 (PF3D7_0113300) that was used to transfect wildtype 3D7 reporter parasites. The RLL-TE PEXEL cleavage site is shown to indicate the mature form of the reporter protein. (B) Hyp1-Nluc expressing trophozoite stage infected erythrocyte following 0.04% v/v DMSO treatment showing export of the reporter protein into the host erythrocyte compartment. The cells were probed with rabbit anti-nanoluciferase and a mouse monoclonal antibody for parasitophorous vacuole marker protein EXP2. DNA stained with DAPI (4,6-diamidino-2-phenylindole). (C) Lysis of parasitized erythrocytes was ascertained following a treatment time course with MB14 (5, 10, 20 µM), cipargamin (5, 10, 20 nM) and control compound artemisinin (25, 50, 100 nM). Compound dependent cell lysis (%) was estimated by measuring the amount of Hyp1-Nluc released into the media relative to that in the whole culture minus the DMSO vehicle (~1% at 8 h). The data are from a single experiment, and are representative of those obtained in three independent experiments, each performed in triplicate.
