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

Retention of LPG expression following infection of BMMs and BMDCs.

(A) Anti-LPG fluorescence intensity on a per-parasite basis. Anti-LPG intensity of WT metacyclic-stage parasites as well as at various time points after infection of PEMs was measured as described in Methods. As a negative control, the anti-LPG intensity of L. major lpg1- (open circles) was measured 0.3 hr after infection of PEMs. The gray line shows the mean anti-LPG intensity of ‘LPG-negative’ WT parasites plus two standard deviations, and any parasite with anti-LPG values below that line would be considered “LPG-negative”. Black bars represent geometric mean of the data for each sample. Data shown is pooled from at least two independent experiments. ***, P (B) Representative images comparing the LPG-positivity of purified metacyclics and parasites within PEMs, DCs, and BMMs at 72 hours post-infection. Parasite nuclei are shown in green, LPG is shown in red. Scale bar represents 5 μm. (C) Percent of parasites within the three host cell types that are LPG+ as determined by the ‘qualitative’ assay. PEM data, black diamonds, BMM data, gray squares, DC data, open triangles. N > 200 parasites per time point/cell type. (D) Quantitation of LPG on the surface of parasites within PEMs, BMMs, and DCs at 24 and 72 hours post infection. For comparison, anti-LPG intensity data for metacyclic-stage parasites is also shown. The gray line shows the cut-off for LPG-positivity. Black bars represent geometric mean of the data for each sample. n ≥ 3 experiments. N.S, not significant, *, P P P < 0.0001 (ANOVA).</p

Localization of T17 antibody labeling in amastigotes.

(A) Epifluorescent microscopy of an amastigote (72 h post metacyclic infection) within a PEM labeled with T17 antibody along with antibodies recognizing parasite histones and DNA stain Hoechst 33342. Scale bar, 2 μm. (B) Electron micrograph of an L. major amastigote within a PEM following immuno-gold labeling with T17 antibody. Scale bar, 0.5 μm. Arrowhead indicates concentration of T17 immuno-labeling at the distal tip of the amastigote flagellum. (C) T17 immuno-labeling around the perimeter of the parasite flagellum. Scale bar, 0.5 μm. (D) T17 immuno-labeling is concentrated at the phagolysosome-parasite flagellum contact site, with some signal on the host cell-side of the phagolysosome. Scale bar, 0.5 μm. (E) Confocal micrograph of PEM 3 d after L. major or mock infection. Samples were stained with T17, antibodies reactive to Leishmania histones, and a DNA stain. Zoomed in images of the boxed regions are shown to the right. Scale bar, 10 μm. (F) ImmunoEM labeling with T17 of vesicular structures in L. major-infected PEM. Images from three different cells shown. Arrows indicate membranous structures containing the parasite antigen recognized by T17 within the PEM cytoplasm. Scale bar, 0.5 μm. Abbreviations: p, parasite posterior; n, nucleus; k, kinetoplast; pf, parasite flagellum.</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

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