Fig. 4.

SJ733 arrests parasite development and induces eryptosis selectively in infected erythrocytes. (A) (+)-SJ733 causes a significant proportion of infected erythrocytes to shrink and expose PS. Uninfected erythrocytes, whether treated with (+)-SJ733 or not (Bottom Left and Bottom Right), remain normally sized, do not have exposed PS, and do not possess substantial DNA, as evidenced by examining the cell population for forward scatter, Annexin V binding, and SYBR green binding in FACS. Cells are shown as a population contour plot that compared scatter with SYBR green binding and are color-coded to indicate Annexin binding. After infection, magnetically purified trophozoite/schizont-infected erythrocytes can be detected in three populations (large, with little exposed PS; small, with moderate levels of exposed PS; and intermediate-sized, with high levels of exposed PS), with the majority being in the small- and large-sized populations. Treatment with (+)-SJ733 causes the population distribution to shift strongly toward the intermediate-sized population with high amounts of exposed PS—consistent with strong induction of eryptosis. This shift is not seen with either control drugs or DMSO treatment. (B) When the eryptosis induction effect is examined for (+)-SJ733 by using a concentration-response experiment, the maximal effect is seen at a concentration of ∼40 nM and the EC₅₀ at 30 nM—congruent with the doses causing similar levels of response for proliferation inhibition and parasite Na⁺ levels. Artesunate has no such effect. (C) (+)-SJ733 causes a significant increase in rigidity of infected, treated erythrocytes. As previously reported, infection of erythrocytes (iRBC) causes a significant increase in their rigidity (P < 0.001; Jonckheere–Terpstra test). After treatment with (+)-SJ733, these infected erythrocytes become significantly more rigid, with the degree of rigidity peaking at ∼7 h after treatment. *P < 0.001 (Jonckheere–Terpstra test). There are no detectable effects on unparasitized erythrocytes at any time point. (D) (+)-SJ733 induces a rapid arrest of parasite motility inside infected erythrocytes that is maintained for the period of treatment as shown by time-lapse microscopy of the treated and untreated cells. The first row of images shows typical motility and growth of a ring-stage parasite over 18 h, which is in stark contrast to the treated parasite in the second row of images that immediately arrests both motility and growth. In some cases the parasite can be observed to lyse within the erythrocyte as shown in the third row. In additional cases the infected, treated erythrocyte itself will lyse after lysis of the parasite as shown in the fourth row. No changes are noted to erythrocyte morphology after treatment of uninfected erythrocytes.