Parasite-Mediated Remodeling of the Host Microfilament Cytoskeleton Enables Rapid Egress of Trypanosoma cruzi following Membrane Rupture

Figures

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  FIG 1. No host cell plasma membrane degradation is observed immediately prior to egress. Cells were first observed by confocal microscopy and later imaged by SEM. (A, upper panel) DIC. Size bar, 15 μm. (Middle panel) The CMFDA marker shows that there is no cytoplasmic content leaking prior to egress. (Lower panel) Same cell observed by SEM (correlative microscopy). (B) Enlarged portion of the same cell, showing no ruptures or pores at the membrane. Size bar, 10 μm. In this image, it is possible to observe the trypomastigotes in low relief beneath the host cell plasma membrane.

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  FIG 2. T. cruzi egress is a rapid event. (A) Vero cells, infected with T. cruzi trypomastigote forms, were visualized by confocal live-cell imaging up to parasite egress. −20 s, pre-egress; 0 s, moment of egress; 20 s, after egress. Images were acquired with a capture interval of 20 s. Cells were preincubated with CMFDA. Blue arrowheads indicate the site of membrane rupture. The image is representative of 80 observed events. Size bar, 10 μm. (B) Amastigotes can be released during T. cruzi egress. Arrowheads point to some of the released amastigotes, and several other amastigotes can be found in the field of view. −20 s, pre-egress time point; 0 s, moment of egress; 100 s, time point that better shows released amastigotes. Time interval between frames, 20 s. Size bar, 20 μm.

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  FIG 3. Infected cells stain for propidium iodide only after parasite egress. Infected cells were submitted to confocal live-cell imaging and monitored up to parasite egress. The image shows that prior to egress (−40 s), there is no propidium iodide stain of the nucleus. We observed PI staining of the nucleus only 100 s after parasite egress. The image is representative of 60 observed cells. CMFDA was used to monitor membrane rupture. White arrowheads indicate the nucleus. As indicated by blue arrowheads, it is possible to observe the site of membrane rupture. Size bar, 10 μm.

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  FIG 4. Actin cage-shaped structures observed in cells before and after parasite egress. (A) Vero cells infected with trypomastigotes prior to egress show F-actin cages. Images were obtained by confocal microscopy. A three-dimensional (3D) topological rendering of the actin cytoskeleton provides a detailed model of the actin cages observed (see Videos S5 and S6 in the supplemental material). (B) Infected Vero cells monitored by live-cell imaging up to trypomastigote egress were fixed and incubated with phalloidin-TRITC. (To observe the same cell after fixing, we used a grid coverslip [see Fig. 5 and Video S4].) Three-dimensional topological micrographs of the actin structures were generated using Imaris software (Surface tool). Size bar, 15 μm. Arrowheads indicate the entire portion of membrane rupture, seen also in Fig. 5 and Video S4.

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  FIG 5. Host cell remains after parasite egress. (A) DIC and SEM showing correlative images of the same cell at the moment of parasite egress (DIC; size bar, 15 μm). (B, left panel) CMFDA staining used to assist egress precise moment. (Right panel) Magnified SEM image of the host cell. (C, right and left panels) SEM higher magnification. It is possible to observe the remains of the host cell cytoskeleton (green arrowhead) and nucleus (blue arrowhead). The red arrowhead maps to the membrane rupture region.

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  FIG 6. Cytoskeleton is gradually disrupted during parasite infection. Shown are scanning electron microscopy (SEM) images of Vero cells treated with Triton X-100-based solution containing phalloidin and paclitaxel (to stabilize cytoskeleton filaments). Images represent the gradual cytoskeleton disruption during the parasite lytic cycle. (A) Noninfected cell with dense meshwork of cytoskeleton. (B) Infected cell at the amastigote stage showing a less dense meshwork. (C) Infected cell with parasites at the trypomastigote stage showing the lack of an overlaying meshwork. It is possible to visualize actin filaments forming a peripheral cage surrounding the trypomastigotes. Zoom images (right panels) show loss of cytoskeleton of infected cells. Blue arrowheads show parasite sites with less or no cytoskeleton, and white arrowheads show preserved cytoskeleton filaments.

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  FIG 7. Cytoskeleton disruption at intracellular trypomastigote sites. The SEM image shows that in cells infected with T. cruzi at the trypomastigote stage, it is still possible to see cytoskeleton filaments (white arrowheads). At the trypomastigote site (blue arrowheads), we observe less or no cytoskeleton structures.

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  FIG 8. Actin cage structures are formed in cells infected with amastigotes. Infected Vero cells during the intracellular amastigote stage were fixed and incubated with phalloidin-TRITC for actin cytoskeleton observations. (A) Noninfected cell. (B and C) Cells containing amastigotes (arrowheads). Actin cages are clearly visible (Arrows). Images were obtained by confocal microscopy and reconstructed using Imaris software. Size bar, 15 μm.

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  FIG 9. T. cruzi intracellular development promotes actin cytoskeleton disassembly, likely to be mediated by parasite proteases. (A to C) Actin cytoskeleton changes from a dense actin meshwork to almost cortical actin only in late stages of the T. cruzi intracellular cycle. (D) T. cruzi amastigote- and trypomastigote-expressed proteases participate in degradation of host cell actin cytoskeleton. (A) Infected cells were fixed and stained with phalloidin-TRITC, and actin patterns were observed and photographically recorded each day. Representative images of an infected cell from each day are shown. On day 0, there is no infection (N.I.). At 24 to 48 h postinfection (P.I.), there is a dense actin meshwork. At 72 to 96 h postinfection, actin spikes appear on the cytoplasm. At 120 to 144 h postinfection, there is spheroidal actin cage formation. By days 7 to 9 (168 to 216 h postinfection), actin cytoskeleton cages are completely formed. Most of the actin stress fibers are absent. Size bar, 15 μm. (B) Actin morphologies of infected cells were evaluated under epifluorescence microscopy during intracellular parasite development. To precisely determine the actin cytoskeleton pattern during…