FIG 3.

CSE disrupts initial contact of the merozoite with the RBC but not heparin binding to MSP1-42. (A, B) Flow cytometry of late-stage parasite cultures, in which parasite stages were differentiated on the basis of ethidium bromide staining, was used to track parasite rupture (in which the results are given as percent schizonts) (A) and merozoite invasion (in which the results are given as percent ring forms) (B) in cultures treated with CSE at 100 μg/ml and uninhibited cultures treated with PBS. After 3 h of incubation, there were increased frequencies of schizonts and decreased ring forms in cultures incubated with CSE. Data are the means ± SEMs from two assays performed in duplicate. *, P < 0.05. (C) Live video microscopy of merozoite invasion in the presence of CSE. Merozoites were able to make initial contact with the RBC, but the contact was not sustained and the merozoites disassociated from the RBC surface. Times (in seconds) are indicated in the lower right corner, and the white arrows highlight a single merozoite that attached to and then disassociated from the RBC. (D) Heparin bead binding assays with P. falciparum protein extract. The protein extract was incubated with heparin beads along with soluble inhibitors, as indicated. Unbound and bead-bound fractions were probed for MSP1-42 binding via Western blotting. MSP1-42 was found in the unbound fraction when it was incubated with heparin as a soluble inhibitor, indicating that soluble heparin was able to outcompete MSP1-42 for binding. However, MSP1-42 was found in the bound fraction when it was incubated with soluble de-6-OS-heparin, CSE, or CSC, indicating that these compounds were not able to compete with heparin binding. (E) MSP1-42 coated on ELISA plates was incubated with heparin-BSA along with soluble heparin, CSC, and CSE at increasing concentrations. The binding of heparin-BSA was detected with anti-BSA antibodies. Soluble heparin, but not CSE or CSC, inhibited the binding of heparin-BSA to MSP1-42.