Fig 1.
Time-course of promastigote-to-amastigote differentiation of L. infantum LEM 5700 parasites hosted in macrophage-like RAW 264.7 cells.
(A) Confocal micrographs showing intracellular promastigotes (left image) and amastigotes (right image) in macrophage-like cells. (B) 3D-reconstruction surface rendering confocal micrographs of the parasite body shape transformation during an infection time-course. 1—A promastigote form expressing one long flagellum positioned at the anterior pole of the spindle-shaped body. 2—Fusiform-like form with a shortened flagellum. 3—Condensed fusiform-like form with an enlarged anterior appendage. 4—Condensed fusiform-like form engulfing the enlarged appendage. 5—Non-flagellated condensed ellipsoidal form. 6—Non-flagellated highly condensed ovoid form. 7—Non-flagellated round amastigote form. (C) Quantification of the number of promastigotes, intermediate differentiated forms (summation of parasitic forms viewed in Fig 2 to 6), and amastigotes during an infection time-course. (D) 3D-reconstruction surface rendering confocal micrograph showing elongation of an L. infantum-containing vacuole preceding fission into two new parasite-containing vacuoles. The white dashed box shows the elongated L. infantum-containing vacuole viewed at high magnification in the adjacent image (Right panel). The white dashed line delineates the area of the elongated vacuole. Confocal micrographs are representative of two independent experiments in duplicate. The percentage of parasite forms was determined by examining at least 30 cells at each time-point for each condition. Data are presented as averages ± SEM. Data are from two or three independent experiments in duplicate.
Fig 2.
Decoration of hosted L. infantum LEM 5700 parasites by the GTPase Rab7, Qb-SNAREs Vti1a and Vti1b, Lamp-2, hydrolase cathepsin D, hexose transporter GLUT8, and polypeptide transporter TAPL during an infection time-course in macrophage-like RAW 264.7 cells.
(A) Graph showing the kinetics of decoration of parasites by Rab7, Lamp-2, cadherin D (CatD), GLUT8, TAPL, Vti1a, and Vti1b during an infection time-course. (B) 3D-reconstruction surface rendering confocal micrographs of a fusiform-like parasite and an amastigote decorated with Rab7-positive patches. (C) 3D-reconstruction surface rendering confocal micrographs of fusiform-like and ellipsoidal parasite forms and amastigotes densely decorated with elongated Lamp-2-positive lamellae and Lamp-2-positive patches. See also S1 Video. (D) 3D-reconstruction surface rendering confocal micrographs of a fusiform-like parasite decorated by large Vti1a-positive patches and an amastigote sparsely decorated with large Vti1a-positive patches. (E) 3D-reconstruction surface rendering confocal micrographs of a fusiform-like parasite decorated by small Vti1b-positive patches and an amastigote sparsely decorated with small Vti1b-positive patches. (F) 3D-reconstruction surface rendering confocal micrographs of a fusiform-like parasite highly decorated with GLUT8-positive large patches and an amastigote sparsely decorated with small GLUT8-positive patches. (G) 3D-reconstruction surface rendering confocal micrographs of a fusiform-like parasite decorated with TAPL-positive patches and an amastigote sparsely decorated with small TAPL-positive patches. (H) z-stack and 3D-reconstruction surface rendering confocal micrographs of fusiform-like and ellipsoidal parasite forms, and amastigotes almost completely enwrapped by large and continuous CatD-positive patches. See also S2 Video. (I) z-stack and 3D-reconstruction surface rendering confocal micrographs showing strong Lysotracker Red positivity for ellipsoidal, ovoid, and round parasite forms. Confocal micrographs are representative of three independent experiments. The percentage of positive parasites for each endolysosomal marker was determined by counting parasites hosted in at least 30 cells for each time-point. Data are represented as the average ± SEM.
Fig 3.
Decrease or lack of decoration of L. infantum LEM 5700 parasites with GTPase Rab7, Lamp-2, and Qb-SNAREs Vti1a and Vti1b in macrophage-like RAW 264.7 cells infected in the continuous presence of Retro-2.
(A) z-stack and 3D-reconstruction surface rendering confocal micrographs showing decreased decoration of fusiform-like and ellipsoidal parasites hosted in Retro-2 (1 μM)-treated cells with Rab7-positive patches. See Fig 2B for control decoration of parasites in untreated cells. (B) z-stack and 3D-reconstruction surface rendering confocal micrographs showing decreased decoration of fusiform-like and ellipsoidal parasite forms in Retro-2 (1 μM)-treated infected cells with Lamp-2, characterized by the presence of dispersed Lamp-2-positive small patches. See also S3 Video. See Fig 2C for control decoration of parasites in untreated cells. (C) 3D-reconstruction surface rendering confocal micrograph showing the absence of decoration of fusiform-like parasites with Vti1a patches in a Retro-2 (1 μM)-treated infected cell. See Fig 2D for control decoration of parasites in untreated cells. (D) Bar graph showing a decrease in the percentage of parasites decorated with Rab7-, Lamp-2-, or Vti1a- or Vti1b-positive patches during an infection time-course in cells infected in the continuous presence of Retro-2 (1 μM). (E) Bar graph showing the average percentage of parasites decorated with Lamp-2 in untreated infected cells and cells infected in the continuous presence of increasing concentrations of Retro-2. Confocal micrographs are representative of three independent experiments. High-magnification images correspond to the white dashed boxes. The percentage of parasites decorated with Rab7-, Lamp-2-, Vti1a-, and Vti1b-positive patches was determined by counting parasites hosted in at least 30 cells at each time-point for each condition. Data are represented as the average ± SEM and were analyzed using the unpaired Student t test. *p < 0.01.
Fig 4.
Lack of decoration of L. infantum LEM 5700 parasites with lysosome-associated hydrolase cathepsin, hexose transporter GLUT8, and polypeptide transporter TAPL in macrophage-like RAW 264.7 cells infected in the continuous presence of Retro-2.
(A) 3D-reconstruction surface rendering confocal micrograph showing the absence of decoration of fusiform-like and ellipsoidal parasite forms with GLUT8-positive patches in a Retro-2 (1 μM)-treated cell. See Fig 2F for control decoration of parasites in untreated cells. (B) 3D-reconstruction surface rendering confocal micrograph showing the absence of decoration of fusiform-like and ellipsoidal parasite forms with TAPL-positive patches in a Retro-2 (1 μM)-treated cell. See Fig 2G for control decoration of parasites in untreated cells. (C) z-stack and 3D-reconstruction surface rendering micrograph showing the absence of decoration with CatD-positive patches of fusiform-like and ellipsoidal parasite forms hosted in Retro-2 (1 μM)-treated cells. See also S4 Video. See Fig 2H for control decoration of parasites in untreated cells. (D) Bar graph showing the decrease in the percentage of parasites decorated with GLUT8-, TAPL- and CatD-positive patches during an infection time-course in cells infected in the continuous presence, or not, of Retro-2 (1 μM). (E) Bar graph showing the average percentage of parasites positive for CatD in cells infected in the continuous presence, or not, of increasing concentrations of Retro-2. (F) Confocal micrographs (z-stack projection) showing green (parasite) and red immunofluorescence labeling (cathepsin D, CatD) in a delineated fusiform-like parasite hosted in an untreated infected cell. Profile showing the scanning analysis of the relative fluorescence intensity (RFI) of green and red fluorescence signals in the delineated fusiform-like parasite. (G) A representative profile showing the scanning analysis of RFIs of green and red fluorescence signals measured in a fusiform-like parasite hosted in a Retro-2-treated infected cell. (H) Bar graph showing the RFIs of CatD measured in fusiform-like parasites hosted in untreated infected cells and Retro-2 (1 μM)-treated infected cells. Confocal micrographs are representative of three independent experiments. High-magnification images correspond to the white dashed boxes. The percentage of parasites associated with TAPL, GLUT8, or CatD were determined by counting parasites hosted in at least 30 cells at each time-point for each condition. The RFIs were measured by analyzing 8 to 10 parasites for each condition. Data are represented as the average ± SEM. Data are from two independent experiments in duplicate and were analyzed by the unpaired Student t test. *p < 0.01.
Fig 5.
Lack of acquisition of LysoTracker Red positivity by L. infantum LEM 5700 parasites during an infection time-course in macrophage-like RAW 264.7 cells infected in the continuous presence of Retro-2.
(A) z-stack and 3D-reconstruction surface rendering confocal micrograph showing the absence of acquisition of Lysotracker Red by fusiform-like and ellipsoidal parasites hosted in Retro-2 (1 μM)-treated infected cells during an infection time-course. See Fig 2I for control LysoTracker Red positivity of parasites in untreated cells. (B) Graph showing an increase in the percentage of parasites positive for LysoTracker Red in untreated infected cells and the absence of an increase of parasite LysoTracker Red-positivity in Retro-2 (1 μM)-treated infected cells during an infection time-course. Confocal micrographs are representative of two independent experiments. The high-magnification image corresponds to the white dashed box. The percentage of positive parasites was determined by counting parasites hosted in at least 30 cells at each time-point for each condition. Data are presented as the average ± SEM.
Fig 6.
L. infantum LEM 5700 parasites are morphologically altered and die when hosted in Retro-2-treated macrophage-like RAW 264.7 cells.
(A) Graph showing the percentage of intermediate parasite forms and amastigotes during an infection time-course in cells infected in the continuous presence, or not, of Retro-2 (1 μM). (B) 3D-reconstruction surface rendering confocal micrographs showing representative altered fusiform-like and ellipsoidal forms of parasites with a wrinkled cell surface, forming deep undulations when hosted in 18 h-Retro-2-treated infected cells. For comparison, see the smooth surface of parasite forms hosted in untreated infected cells in Fig 1. (C) Bar graph showing the percentage of intermediate parasite forms and amastigotes exhibiting a smooth or wrinkled body surface in cells infected for 18 h in the continuous presence, or not, of Retro-2. (D) Graph showing the parasite (left) and amastigote (right) loads determined by RT-qPCR in cells infected in the continuous presence, or not, of Retro-2 during an infection time-course. Confocal micrographs are representative of two independent experiments. The percentage of intermediate parasite forms and amastigotes was determined by analyzing at least 40 cells for each time-point for each condition. The percentage of parasites with a smooth or wrinkled surface was determined by analyzing parasites hosted in at least 30 cells for each time-point for each condition. Parasite and amastigote levels per culture well were quantified by RT-qPCR in two independent experiments in duplicate. Data are presented as averages ± SEM and were analyzed using the unpaired Student t test. *p < 0.01.
Fig 7.
Retro-2 treatment results in disassembly the cell microtubule network.
(A) 3D-reconstruction surface rendering confocal micrograph showing the well-ordered perinuclear distribution of tubulin immunolabeling in an untreated infected macrophage-like cell. (B) 3D-reconstruction surface rendering confocal micrograph showing the dispersion of tubulin immunolabeling in a cell infected for 24 h in the continuous presence of Retro-2 (1 μM). (C) Bar graph showing the percentage of macrophage-like cells infected in the continuous presence, or not, of Retro-2, with homogeneous perinuclear or dispersed tubulin immunolabeling. (D) x-y confocal micrograph showing the MT network in an untreated epithelial HeLa cell. (E) x-y confocal micrograph showing marked disorganization of the MT network in 24 h-Retro-2-treated HeLa cells. Note the appearance of short tubulin-positive bar-like (1 μM) and vesicle/aggregate (5 μM) structures distributed throughout the cell cytoplasm. (F) Graph showing quantification of cells expressing well-organized tubulin-positive fibers, dispersed bar-like structures, or vesicle-like aggregates in untreated cells and cells treated for 24 h with DMSO or increasing concentrations of Retro-2. (G) Graph showing the effect of increasing concentrations of Retro-2 on GTP-dependent tubulin polymerization in vitro. Confocal micrographs are representative of two independent experiments in duplicate. High-magnification images correspond to the white dashed boxes. Tubulin in the reaction buffer was incubated at 37°C in the presence of vehicle (DMSO) or Retro-2 and polymerization measured by spectrophotometry. Data were obtained from two independent experiments. Data are presented as the mean ± SEM.
Fig 8.
The microtubule-disassembly agent nocodazole abolishes the maturation of L. infantum LEM 5700-containing phagosomes, delays parasite differentiation, and promotes parasite death in macrophage-like Raw 264.7 cells.
(A) 3D-reconstruction surface rendering confocal micrographs showing limited association of cathepsin D (CatD) with parasites hosted in nocodazole-treated infected cells. For comparison, see the CatD decoration of parasites hosted in untreated cells in Fig 1G. (B) Bar graph showing the percentage of parasites decorated with CatD in cells infected in the continuous presence, or not, of nocodazole during a time-course of infection. (C) Graph showing the time-dependent evolution of the percentage of amastigotes in cells infected in the continuous presence, or not, of nocodazole (1 μM) during a time-course of infection. (D) Bar graph showing the percentage of intermediate parasite forms and amastigotes exhibiting a smooth or wrinkled body surface when hosted in cells infected in the continuous presence, or not, of nocodazole. Confocal micrographs are representative of two independent experiments. The percentage of parasites was determined by analyzing at least 30 cells for each time-point for each condition. Data are presented as averages ± SEM and were analyzed using the unpaired Student t test. *p < 0.01.
Fig 9.
Silencing of the dynein gene in macrophage-like Raw 264.7 cells abolishes the maturation of L. infantum LEM 5700-containing phagosomes/PVs, delays parasite differentiation, and promotes parasite death.
(A) Bar graph showing dynein mRNA levels relative to those of gapdh mRNA measured by RT-qPCR in mock and dynein siRNA-transfected cells. (B) 3D-reconstruction surface rendering confocal micrograph showing limited association of cathepsin D (CatD) with fusiform-like parasites hosted in an infected dynein siRNA-transfected cell, and the highly altered morphology of these parasites. For comparison, see the CatD decoration of parasites hosted in untreated cells in Fig 1G. (C) Bar graph showing the percentage of parasites associated with CatD, the percentage of intermediate parasite forms and amastigotes, and the percentage of parasites with a wrinkled cell surface in mock-transfected and siRNA dynein-transfected cells. (D) Bar graph showing the amastigote load determined by RT-qPCR in mock-infected cells and dynein siRNA-transfected infected cells. The confocal micrograph is representative of two or three independent experiments. The high-magnification view corresponds to the white dashed box. The percentage of parasites was determined by analyzing at least 30 cells for each condition. The percentage of parasites with a smooth or wrinkled cell surface was determined by analyzing parasites hosted in at least 20 cells for each condition. Dynein mRNA levels were quantified by RT-qPCR of over three independent biological samples for each time-point. Amastigote levels per culture well were quantified by RT-qPCR in two independent experiments in duplicate. Data are presented as averages ± SEM and were analyzed by the unpaired Student t test. *p < 0.01.
Fig 10.
Scheme summarizing the abolition of maturation of tight-fitting L. infantum-containing PVs by structurally and functionally altering the MT network of macrophage-like cells.
Such alteration of the MT network results in most of the phagocytosed L. infantum promastigotes remaining permanently insulated in non-mature phagosomes, in which they do not undergo normal promastigote-to-amastigote differentiation and finally die.