FIG 1.

Activity of occidiofungin against the growth of Cryptosporidium parvum in vitro. (A) Covalent structure of occidiofungin, a glycolipopeptide. (B) In vitro efficacy of occidiofungin against C. parvum cultured in two host cell lines (HCT-8 and Caco-2) and cytotoxicity. Paromomycin (PRM) at 140 μM was used as a positive control. (C) Effect of occidiofungin on the growth of C. parvum in vitro with various lengths of treatment. Compound was added to the medium along with oocyst inoculation for the specified durations of treatment, and the parasite loads were assayed at the end of each treatment. (D) Drug withdrawal assay. Occidiofungin was added at 3 hpi after the removal of uninvaded parasites. After treatment of infected cells for specified durations, compound was removed, and the parasites were allowed to grow and recover in the absence of compound for up to 44 hpi before the parasite loads were determined. PRM at 140 μM was used as a positive control. (E) Effect of pretreating sporozoites with occidiofungin on parasite infection. Free sporozoites were treated with occidiofungin at 200 and 400 nM for 40 min in culture medium and then allowed to invade host cells for 2 and 6 h. The parasite loads were determined at 2 and 6 hpi time points. (F) Effect of pretreating host cells with occidiofungin on parasite infection. HCT-8 cells were treated with occidiofungin at 200 nM for 24 h. After removal of compound, host cells were incubated with parasite oocysts and assayed at 3, 9, and 18 hpi. Parasite loads were determined by qRT-PCR. Statistical significance was evaluated by two-way ANOVA and Sidak’s multiple-comparison test. Error bars represent standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.