Deletion of one <i>LMIT1</i> allele impairs temperature/pH-induced differentiation of promastigotes into axenic amastigotes.

<p>Late-log phase promastigotes were washed, resuspended in pH 4.5 at 2x10⁵ parasites/ml, and cultured at 32°C. (A) Numbers of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1)</i> parasites following shift to amastigote medium. The data represent the mean +/- SD of triplicate determinations and are representative of four independent experiments. (B–D) wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) parasites incubated for 48 h in amastigote medium at 32°C were subjected to: (B) Quantification of viable rounded forms with a short flagellum. At least 400 FDA-stained parasites were counted microscopically in each sample. The data represent the mean ± SD of quadruplicate determinations. (C) SEM analysis of the morphology of parasites. Bars = 2 μm. (D) Determination of cell viability and membrane integrity by staining with FDA (green) and PI (red). Bars = 27 μm. (E) The iron content of whole cells and mitochondrial fractions was determined in parasites collected 24 h after induction of axenic differentiation (pH 4.5/ 32°C). Data represents the mean ± SD of three independent experiments (Student’s <i>t</i> test *p = 0.037, **<i>p</i> = 0.004). (F) Aconitase activity was determined in mitochondrial fractions from parasites collected 24 h after induction of axenic differentiation (pH 4.5/ 32°C). The data represent the mean ± SD of three independent experiments (**p≤ 0.008). (G) Determination of mitochondrial membrane potential (ΔΨ_m) with JC-1 with and without prior treatment with the mitochondrial uncoupler CCCP. The data represents the mean ± SD of three independent experiments (Student’s <i>t</i> test * <i>p</i> = 0.01; **<i>p</i> = 0.002). ((H) Viable amastigotes obtained by temperature/pH- induced axenic differentiation in wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) cultures were tested for their ability to infect BMMs. BMMs were infected and either fixed immediately or after further incubation 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.