The LmjF-LUC-SAT and LmjF-PHLEO strains show comparable virulence in infections of resistant mice.

<p>C57BL/6 mice (5 per group) were infected with 10⁵ metacyclic stage LmjF-LUC-SAT or LmjF-PHLEO <i>L</i>. <i>major</i>. (A) Measurements of lesion pathology (increase in footpad thickness). Error bars show the standard deviation. (B) Persistent parasites numbers were determined by limiting dilution assay from footpad tissue 130 days post infection. Horizontal bars show the geometric mean. †, <i>P</i> > 0.05.</p

Retention of both primary and secondary infecting parasites following secondary challenge despite protection from disease pathology.

<p>The graph plots the number of persistent parasites present in sites of primary and secondary <i>Leishmania</i> infections >10 weeks post secondary challenge as assessed by limiting dilution analysis in unselective (white bar), nourseothricin-containing (gray bars; resistance mediated by <i>SAT</i> marker) or phleomycin-containing (black bars; resistance mediated by <i>PHLEO</i> marker) as described in the methods. The number of parasites in the primary infection site (LmjF-LUC-SAT inocula) is displayed in the top graph, and the number of parasites in secondary infection site (LmjF-PHLEO inocula) foot is displayed in the bottom graph. The numbers between the two graphs represent the mouse identification number (experiment number-mouse number). “Avg.” represents the mean for all mice.</p

Mice persistently infected with LmjF-LUC-SAT show protection from disease pathology by secondary challenge with LmjF-PHLEO parasites.

<p>Mice (4-5/group) were inoculated with 10⁵ metacyclic LmjF-LUC-SAT parasites in the left hind footpad (primary site), after which they developed lesions and then went on to heal (similar to that shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004811#pntd.0004811.g001" target="_blank">Fig 1A</a>). (A) At least one month after resolution of the primary lesions, each mouse was inoculated in the right hind footpad (secondary site) with 10⁵ metacyclic LmjF-PHLEO parasites, and lesion progression is shown in the figure. The dashed line represents the average of the data presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004811#pntd.0004811.g001" target="_blank">Fig 1A</a> for infections of naïve mice with LmjF LUC-SAT and LmjF-PHLEO for comparison. In these experiments “time 0” is when the secondary inoculation was performed unless otherwise indicated. For all plots, error bars show the standard deviation (n = 4 or 5 in expt. 1 or 2 respectively). (B) Footpad thickness at the primary injection site (left foot). (C) Monitoring of reactivation of the primary LmjF-LUC-SAT parasites at the primary (♦,■) or secondary (◊,□) infection sites site by bioluminescent imaging of luciferase expression <i>in vivo</i>; experiment 1 (♦,◊); experiment 2 (■,□). The gray circle (upper right) shows the luminescence profile of LmjF-LUC-SAT parasites infecting naïve mice at the peak of infection, included for comparison only. Error bars depict the standard deviation.</p

Cytological markers differentiate metacyclic promastigote from amastigote stage <i>Leishmania major</i>.

Comparison of marker expression on metacyclic promastigotes (left) or parasites 72 hours post infection of PEMs (i.e. amastigotes, right). (i) Parasite nuclei are detected with antisera against L. major histone proteins (green), and PFR is shown in red. (ii) Parasite histones, red. mAB T17, green. (iii) Parasite histones, red. mAB T18, green. (iv) YFP fluorescence (yellow) overlaid onto DIC image. Scale bar represents 5 μm.</p

Developmental regulation of YFP reporter.

(A) Flow cytometric analysis of YFP signal from WT L. major and L. major expressing YFP from the small ribosomal subunit locus under log-phase (procyclic promastigotes) or stationary-phase (includes metacyclic promastigotes) conditions. (B) Western blot analysis of YFP expression in log phase and stationary phase parasites stably transfected with YFP transgene. L. major histone H2A levels were assessed as a protein loading control. (C) The abundance of YFP mRNA in log and stationary phase parasites was determined by q-RT-PCR. *, P (PDF)</p

Timing of developmental changes associated with amastigote differentiation in PEMs.

(A) Parasite flagella were identified at different time points following infection of PEMs with anti-LPG (≤ 8 h post infection) or mAb T17 at longer time points. The fraction of parasites retaining long flagella (≥ 5 μm; arrows) was determined at the indicated time points and plotted in (B). Scale bar represents 5 μm. N > 100 parasites analyzed per time point. (C) Quantitative analysis of parasite flagellar length of metacyclics or parasites at the indicated time points post infection of PEMs. Each data point represents one parasite. (D) The kinetics of L. major metacyclic-stage parasites acquiring amastigote-like shape characteristics. Metacyclic-stage parasites, parasites at the indicated time points post infection of PEMs, or amastigotes from infected mouse footpad tissue 2 weeks post infection were stained to detect the parasite cell body and imaged by confocal microscopy. Image analysis was then performed to determine the length and width of each parasite, with cell shape determined as the ratio between parasite length and width. Metacyclic parasites and parasites within PEMs were stained with anti-LPG (time points ≤ 8 h or mAb T18 (time points > 8 h). Tissue amastigotes were detected by YFP fluorescence. N > 50 parasites. (E) Confocal micrograph of BrdU-labeled parasites. PEMs were infected with metacyclic-stage L. major for 72 h in the presence of 0.1 mM BrdU prior to fixation and immunolabeling to detect BrdU (green) and Leishmania histones (red). Arrows indicate parasite nuclei showing BrdU-labeling. Arrowhead, BrdU-negative parasite nucleus. Boxed region is magnified in the images on the right. k, BrdU-positive kinetoplast. Scale bar, 5 μm. (F) The percentage of parasites showing an “amastigote-like” phenotype for the various markers is plotted as a function of time after infection of PEMs. N > 200 parasites analyzed per marker per time point. (G) Schematic summary of the data in (F) showing the timing of amastigote development culminating in new DNA synthesis. Data, means ± S.E.M for 3 independent experiments; *, P < 0.05; **, P < 0.001 by ANOVA.</p

mAb T17 and T18 reactivity precedes YFP expression during metacyclic-to-amastigote transition.

(A) C57B6 mice were injected subcutaneously in footpad tissue with metacyclic stage parasites stably transfected with YFP transgene. 16 d later, mice were sacrificed and infected footpad tissue sectioned and stained to detect parasite histones (blue) and YFP. YFP-negative parasites are indicated by arrows. Scale bars, 5 μm. (B, C) PEMs were fixed 10 h after infection and stained with T17 (B) or T18 (C) antibodies (red). Parasites were identified based on nuclear staining with antibodies against parasite histones (blue). The percent of parasites positive for T17 or T18 reactivity and YFP fluorescence (green) was determined by analysis of confocal micrographs. Arrowheads indicate T17/T18 positive, YFP-negative parasites, and arrows indicate double-negative parasites. Scale bars, 5 μm. (PDF)</p

LPG down-regulation is coincident with loss of metacyclic-specific arabinose-capped LPG but independent of YFP induction.

(A) PEMs were infected with L. major and harvested 2, 24, and 72 hours after infection. Ara-capped LPG is detected with mAB 3F12 (green), and parasite histones are shown in red. Data indicate the percent of parasites positive for 3F12 labeling out of the total number of parasites analyzed (N). Scale bar, 5 μm. (B) Representative image of parasites within BMM fixed 72 h post infection showing LPG and/or YFP positivity. Parasite nuclei are shown in blue. Arrows indicate LPG and YFP double-negative parasites. Scale bar, 5 μm. Analysis of images like that in (B) was performed to determine the LPG-positivity (C) or YFP-positivity (D) of total parasites or within the indicated parasite sup-populations. N = 362 parasites. N.S., not specific (Chi-square). (PDF)</p

Acquisition of amastigote traits by <i>L</i>. <i>major</i> cultured at 37°C in the absence of host cells.

L. major promastigotes were cultured until the third day of stationary phase at 26°C, then placed in fresh media and cultured at 37°C for 24 hours prior to harvest and analysis. (A) Parasites were fixed and stained with the indicated markers prior to confocal microscopy. Micrographs were visually scored for PFR negativity or T17/T18 positivity. N > 200 parasites. (B) Flow cytometric analysis of YFP fluorescence of the starting culture of stationary phase parasites (green, 26°C) and parasites following 24 hours of culture at 37°C (purple). (C) Representative electron micrograph of a parasite following culture at 37°C that has typical amastigote morphology including a round shape and a spacious flagellar pocket. (PDF)</p

Metacyclic-to-amastigote transition is similar in different host cell types.

(A-D) Comparison of metacyclic-to-amastigote transition in PEM (black diamonds), BMM (gray squares) and BMDC (open triangles) for the percent of parasites positive for PFR labeling (A), mAb T18 labeling (B), YFP signal (C), and mAb T17 labeling (D). (E) Comparison of parasite cell shape 24 h after infection of the indicated cell types with metacyclic-stage L. major. Data, means ± S.E.M for 3 independent experiments; *, P 350 parasites per marker/time point.</p