Quantitative single-cell analysis of Leishmania major amastigote differentiation demonstrates variably extended expression of the lipophosphoglycan (LPG) virulence factor in different host cell types
Fig 2
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.