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. 2024 Sep 30;7:1226. doi: 10.1038/s42003-024-06716-2

Fig. 1. Leishmania infection triggers host ATP7A trafficking and copper accumulation.

Fig. 1

A Representative immunofluorescence image of CF4 dye or Ctrl-S2-CF4 dye (magenta) in J774A.1 macrophages with (INF) and without (BASAL) L.major infection (12 h). Both macrophage and Leishmania nuclei were stained with DAPI (blue). The merged images represent colocalization of the dyes with Leishmania nuclei. White arrows indicate intracellular parasites in infected cells (smaller nuclei). The scale bar represents 5 μm. The magnified inset corresponds to the region of the merged image marked by arrows indicating the association of CF4 with Leishmania-positive endosomes. B Fraction of dye colocalized with Leishmania nuclei from the above mentioned conditions demonstrated by a box plot with jitter points. The box represents the 25th to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5× interquartile range from the first and third quartiles. Asterisks indicate values that are significantly different between CF4 and Ctrl-S2-CF4 treated sample. Sample size of macrophage (n): 12, ∗∗∗∗p ≤ 0.0001 (Wilcoxon rank-sum test). C Measurement of intracellular copper level in J774A.1 macrophages with (Inf 12 h) or without (Uninf) L.major infection using ICP-MS. Error bars represent mean ± SD of values calculated from three independent experiments. ns; (Student’s t test). D Immunofluorescence image of ATP7A (green) in J774A.1 macrophage with FluoSpheres™ Carboxylate-modified Microsphere beads (magenta) confirming absence ATP7A trafficking to those beads due to general phagoscytosis. Macrophage nuclei is stained with DAPI (blue). E Representative immunofluorescence image of ATP7A (green), co-stained with endo-lysosomal marker Lamp1 (magenta), in J774A.1 macrophages with and without L.major infection (12 h) followed by basal, high copper (100 μM Cu) and copper chelated conditions (25 μM TTM) treatment for 2 h. The merged images represent colocalization of ATP7A with Lamp1. Both macrophage and Leishmania nuclei were stained with DAPI (blue). White arrows indicate intracellular parasites in infected cells (smaller nuclei). White arrowheads indicate vesicularised ATP7A. The scale bar represents 5 μm. The magnified inset corresponds to the region of the merged image marked by arrows indicating the association of ATP7A and Lamp1 with Leishmania-positive endosomes. F Fraction of ATP7A colocalization with Lamp1 from the above mentioned conditions demonstrated by a box plot with jitter points. The box represents the 25th to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5× interquartile range from the first and third quartiles. Asterisks indicate values that are significantly different from Uninf samples. Sample size of macrophage (n): 15. ∗∗∗∗p ≤ 0.0001, ns; (Wilcoxon rank-sum test). G Fraction of ATP7A colocalization with L.major compartments from the above mentioned conditions demonstrated by a box plot with jitter points. The box represents the 25th to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5× interquartile range from the first and third quartiles. ns indicates values that are not significantly different from Inf samples. Sample size of macrophage (n): 15. ns non significant; (Wilcoxon rank-sum test). H Number of ATP7A vesicles from the above mentioned conditions are plotted. Asterisks indicate values that are significantly different from Uninf samples. I Comparison of copper levels (in ppb/million cells) in Leishmania infective uninfected cells under different treatments. Error bars represent mean ± SD of values calculated from three independent experiments. Asterisks indicate values that are significantly different from Uninf samples. ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, ns; (Student’s t test).