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.</p

<i>LMIT1</i> single knockout metacyclic forms are strongly impaired in virulence for mice.

<p>(A) C57BL/6 female mice were inoculated with 1x10⁶ wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) or complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) purified metacyclic promastigotes in the left hind footpad and lesion development was measured weekly. The data represent the mean ± SD of 5 mice. (B) The parasite load in footpad tissues was determined 11 weeks after infection (n = 5). **, <i>p</i> = 0.009; *, <i>p</i> = 0.167 (Student’s t test). Relative levels of episomally expressed LMIT1-HA protein was determined in western blots using 10 μg of whole cell extracts prepared from <i>LMIT1/Δlmit1+LMIT1</i> parasites prior to footpad injection (injected) or recovered from lesions post-sacrifice (recovered) and detected with anti-HA antibody. (C) Balb/c female mice were inoculated with single knockout (<i>LMIT1/Δlmit1</i>) or complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) purified metacyclic promastigotes in the left hind footpad and lesion development was measured weekly. The data represent the mean ± SD of 5 mice. (D) Parasite load in footpad tissues was determined 12 weeks after infection (n = 5). **, <i>p</i> = 0.007 (Student’s t test).</p

Metacyclogenesis is not affected in <i>LMIT1</i> single knockout, but infectivity for macrophages is markedly reduced.

<p>(A) A total of 2.5 x 10⁸ wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) or complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) parasites from 7-day stationary phase were used to isolate metacyclic forms by selective agglutination of promastigotes with the 3A.1 mAb. The data represents the mean ± SD of the percentage of metacyclic forms recovered in triplicate determinations, and are representative of three independent experiments. (B) SEM images of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) parasites from 7-day stationary phase cultures. (C) Mitochondrial membrane potential (ΔΨ_m) was estimated in wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) parasites from stationary phase cultures based on JC-1 uptake, with and without addition of the respiratory chain inhibitor Antimycin A, or 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). (D) Aconitase activity was measured in mitochondrial fractions from wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) parasites. (E) TEM of WT (top panels) and <i>LMIT1/Δlmit1</i> (lower panels) parasites from 7-day stationary phase cultures. m, mitochondria; k, kinetoplast; white arrows, normal mitochondrial cristae. Bars = 1 μm. (F) BMMs were infected with wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) purified metacyclics and fixed immediately or incubated further for indicated time points, and the number of intracellular parasites was determined microscopically. The data represent the mean ± SD of the results of three independent experiments. The asterisks indicate significant differences in infectivity between WT and <i>LMIT1/Δlmit1</i> parasites (Student’s <i>t</i> test 48 h, <i>p</i> = 0.017; 72 h, <i>p</i> = 0.008).</p

<i>LMIT1</i> single knockout parasites induced to differentiate axenically into amastigotes show reduced FeSOD activity and mitochondrial abnormalities.

<p>(A) Log phase (~2x10⁷/ml) wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) parasites grown in regular promastigote medium (pH 7.4 / 26°C) were transferred to amastigote media (pH 4.5) and incubated at an elevated temperature (32°C) for 72 h, followed by determination of SOD activity in whole cell extracts. The data represents the mean ± SD of triplicate determinations and are representative of three independent experiments. (Student’s <i>t</i> test compared to <i>WT</i>: 24 h **<i>p</i> = 0.013, 48 h ***<i>p</i> = 0.0097 and *<i>p</i> = 0.093, 72 h ***<i>p</i> = .0.008 and **<i>p</i> = 0.026). (B) TEM micrographs of wild type (WT) and single knockout (<i>LMIT1/Δlmit1</i>) parasites incubated in amastigote media at 32°C for 48 h. (a-c) WT; (d-h) <i>LMIT1/Δlmit1</i>. m, mitochondria; k, kinetoplast; white arrows, normal mitochondrial cristae; black arrows, aggregates inside mitochondria; black arrowheads, enlarged mitochondria. Bars = 1 μm.</p

Identification of <i>LMIT1</i>, a functional <i>Leishmania</i> mitochondrial iron transporter.

<p>(A) Multiple sequence alignment showing conservation of functional residues, including the canonical signature sequence motif Px(D/E)xxK/R)x(K/R) (marked with red asterisks), in mitoferrin homologs from <i>L</i>. <i>amazonensis</i>, <i>L</i>. <i>mexicana</i> (<i>LmxM</i>.<i>08_29</i>.<i>2780</i>), <i>T</i>. <i>brucei</i> (<i>Tb927</i>.<i>3</i>.<i>2980</i>) <i>S</i>. <i>cerevisiae</i> (mrs3) and <i>H</i>. <i>sapiens (</i>mitoferrin-1) as predicted by ClustalW analysis. Identical and conserved residues highlighted in black, dark and light gray represent 100%, 80% or 60% conservation, respectively. Critical functional residues in substrate contact points are indicated in bold. (B,C) The <i>Leishmania</i> LMIT1 protein localizes to mitochondria. (B) Promastigotes expressing LMIT1-3xFLAG were subjected to cellular fractionation following treatment with increasing concentrations of digitonin (as indicated). Antibodies against cytoplasmic adenosuccinate lyase (ASL) and mitochondrial Ldp27 were used to assess separation of cytosolic and mitochondrial proteins in soluble (S) and pellet (P) fractions. The LMIT1-3xFLAG protein was detected using a monoclonal antibody against the FLAG tag. (C) <i>Leishmania</i> wild type (WT) and LMIT1-3xFLAG promastigotes were treated with anti-FLAG antibodies followed by a fluorescent secondary antibody (green). Mitochondria were visualized by staining with MitoTracker Red (red), and the localization of LMIT1-3xFLAG tag to mitochondria was assessed by overlaying the images (yellow). Bars = 5 μm. (D) The <i>Leishmania LMIT1</i> gene functionally compensates for lack of the mitochondrial iron transporter mrs3 in yeast. <i>S</i>. <i>sacharomyces Δmrs3Δmrs4</i> was transformed with empty vector pYES2 (EV) or with yeast <i>mrs3</i> or <i>Leishmania LMIT1</i> constructs. Serial spot growth assays were carried out with transformed yeast cells plated on regular or iron depleted (BPS treated) media in the presence of galactose.</p

Deletion of one <i>LMIT1</i> allele impairs <i>L</i>. <i>amazonensis</i> promastigote growth, enhances sensitivity to ROS and inhibits amastigote generation triggered by iron deprivation.

<p>(A) Growth curves of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1)</i> promastigotes in regular growth medium. (B) The iron content in whole cells and mitochondrial fractions was determined in 4 day-old cultures of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) promastigotes. The data represent the mean ± SD of three independent experiments. (C) Effect of menadione, an inducer of superoxide generation, on the survival of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) promastigotes. The parasites were cultured for 48 h in the presence increasing concentrations of mitochondrial superoxide generator menadione and the number of viable cells was determined after staining with FDA. The data expressed as percentage of the number of viable cells in cultures without menadione, represent the mean ± SD of triplicate determinations and are representative of three independent experiments. (D) Growth curves of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1)</i> promastigotes in iron-depleted medium. (E) Fraction of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1)</i> parasites with a rounded morphology and short flagellum after 48 h of culture in iron-depleted medium.</p