Figure 9.

Iron depletion induces differentiation of noninfective promastigotes into virulent amastigotes. The infectivity for macrophages (BMDMs) and mice was compared between WT promastigotes grown in regular medium for 4 d, WT promastigotes grown in iron-depleted medium for 4 d, mid-log phase promastigotes treated with the ROS-generating agent menadione for 18 h [(+) menadione], and axenic amastigotes induced by reduced pH/elevated temperature (axenic amastigotes). (A) Immunofluorescence images of BMDMs derived from BALB/c mice after infections with the indicated parasite populations for 1 or 48 h. Red indicates anti-Leishmania antibodies, green indicates anti-Lamp1, and blue indicates DAPI DNA stain. Bars, 5 µm. (B) BMDMs were infected for 1 h and either fixed immediately (1 h) or further incubated for 24, 48, or 72 h, and the number of intracellular parasites was determined microscopically. The data represent the mean ± SD of triplicate determinations and are representative of more than three independent experiments. (C–E) BALB/c mice were inoculated in the left hind footpad with regular, iron-depleted, or menadione-treated WT parasites. Axenic amastigotes induced by reduced pH/elevated temperature were injected as positive control. (C) Lesion development was determined by weekly caliper measurements. The data correspond to the mean ± SD of values obtained from five individual mice in each group. (D) Representative images showing the extent of lesion formation in each mouse group at 8 wk after challenge. (E) Parasite load in the footpad was determined at 8 wk after challenge. The results represent parasite loads per footpad of individual mice with geometric means and p-values between respective groups (Student’s two-tailed t test).