Figure 1.
A fraction of metacyclic promastigotes exposes phosphatidylserine in vivo and in vitro.
Characterization of small size (R2-lower left gate) and large size (R1-lower right gate) sub-populations of (A) logarithmic, (B) total stationary, and (C) purified metacyclic promastigotes, showed by dot-plots of forward light scatter (FSC) vs. side angle light scatter (SSC) obtained by flow cytometric analysis. PS exposure, assessed by AnV staining in (D) logarithmic, (E) total stationary, and (F) infective metacyclic populations. Figure displays one representative experiment out of five with similar results. PS exposure in promastigotes purified from the gut of Lutzomya longipalpis at (H) 5 days and (I) 9 days after artificial feeding with J774 cells infected with La and (G) the appropriate control parasites stained with PI only. Figure displays one experiment out of two with similar results.
Figure 2.
Purified PSPOS promastigotes are required for the in vitro infectivity of PSNEG forms.
(A) Rates of internalization (2 h p. i.) and of intracellular proliferation (72 h p. i.) of magnetic purified PSNEG and PSPOS metacyclic promastigotes measured by their infectivity index in macrophage cultures. (B) Inhibition of NO synthesis by PSPOS and PSNEG populations. Macrophages activated with IFNγ and LPS were infected with magnetic purified PSNEG or PSPOS metacyclic promastigotes. After 48 h of infection, NO production by cultured macrophages were quantified by Griess reaction in the supernatants. Figure displays one experiment out of three with similar results. *p<0.001, **p<0.05.
Figure 3.
Purified PSPOS promastigotes are required for the in vitro infectivity of PSNEG forms.
Differential interference contrast (left panels) and fluorescence microscopy (right panels) at 2 h and 72 h p. i. of macrophage monolayers infected with CFSE-treated purified PSPOS, PSNEG and PSPOS+PSNEG (reconstituted at a 1∶1 ratio) population of promastigotes. Arrowheads indicate PSPOS CFSE-unlabeled parasites stained with DAPI (panels J and L). Arrows indicate PSNEG CSFE-unlabeled parasites stained with DAPI (panels N and P). Nuclei are stained with DAPI. Bars represent 20 µm. Figure displays one experiment out of two with similar results.
Figure 4.
Purified PSPOS promastigotes are required for the in vivo infectivity of PSNEG forms.
Footpad swelling in BALB/c mice inoculated with 105 metacyclic promastigotes as well as the same number of sorted PSPOS, PSNEG and PSPOS+PSNEG (reconstituted at a 1∶1 ratio) population of promastigotes. Each dot represents a single animal and the horizontal bar represents the median lesion size of each group.
Figure 5.
PSPOS parasites are apoptotic cells.
(A) Oligonucleosomal DNA cleavage in logarithmic phase (lane 1), stationary phase (lane 2) and purified metacyclic (lane 3) promastigote populations. (B) Differential interference contrast (left panels) and confocal microscopy (right panels) Confocal image of purified metacyclics stained with AnV-Alexa 488 and DAPI. (C) TUNEL staining of stationary-phase promastigotes. (D) Quantification of TUNELPOS and round shaped parasites by counting at least 200 parasites per slide under the microscope. (E) PS exposure by PINEG parasites treated with a pan-caspase inhibitor Z-VAD-FMK. (F) Proliferation of parasites treated with 10 µM of Z-VAD-FMK at day 3, 6 and 9 of culture. Ultrastructural analysis of (G) metacyclic promastigote and (H and I) PSPOS metacyclics showing morphology of nucleus (n), mitochondrion (m), kinetoplast (k), flagellar pocket (fp) and flagellum (f). Bars represent 1 µm.
Figure 6.
Ultrastructural analysis of metacyclic promastigotes within infected sand flies.
Lutzomyia longipalpis sand flies were fed with heparinized mice blood containing infected macrophages. After 10 days female insects were pooled and guts were harvested and fixed for transmission electron microscopy. (A) Morphological heterogeneity of promastigotes in the intestinal lumen of a sand fly, displaying normal nucleai, kinetoplasts and mitochondria. (B) As in A with a higher magnification. (C) Promastigote showing intense mitochondrial swelling (white arrowheads) and loss of kinetoplast organization in addition to vacuole-like structures, formed by mitochondrial or endoplasmic-reticulum membranes containing cytoplasmic material. (D) Promastigote attached to intestinal microvilli showing highly condensed chromatin clumped to protrusions in nuclear lobes (white arrowheads). (E) Promastigotes with condensed chromatin and mitochondrial membrane expansions eventually containing cytoplasmic material. Bars represent 1 µm; m, mitochondrion; k, kinetoplast; n, nucleus; mv, microvili.
Figure 7.
TUNELPOS promastigotes are present in the intestinal tract of infected sand flies.
Lutzomyia longipalpis sand flies were fed with mice blood containing infected macrophages. After 10 days female insects were fixed and histochemical analysis for TUNEL labeling was performed. (A) Hematoxilin-eosin stain showing overview of sand fly section. (B) Parasite concentration at the bulbous cardia region of the foregut and isolated and clumped TUNELPOS promastigotes in the foregut (fg). Most of the stained parasites are already acquiring a round-shaped morphology (arrows). (C) and (D) elongated TUNELNEG promastigotes in respectively the anterior midgut (amg) and posterior midgut (pmg) of infected sand flies (arrowheads). Bars represent 200 µm (A) and 20 µm (B, C and D). Asterisks on panels A and B indicate the bulbous cardia region of the foregut.