Figures
- Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi

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Fig. 1: determination of intracellular Trypanosoma cruzi infective forms inside and outside the parasitophorous vacuole. After centrifugation onto Vero cells, metacyclic trypomastigotes (A) or amastigotes (B) could be localized within LAMP-1 positive parasitophorous vacuoles (labeled in red, white arrowheads) or free in the cytoplasm (not labeled, black arrowheads). Differential interference contrast (green) and DNA labeling with DAPI (blue) are overlaid with LAMP-1 (red) images obtained by confocal microscopy. Magnification bar = 10 µm

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Fig. 2: chloroquine increases the time of residence of Trypanosoma cruzi metacyclic trypomastigotes in the parasitophorous vacuole. A: kinetics of T. cruzi parasitophorous vacuole formation in Vero cells determined by the percentage of anti-LAMP-1 positive parasites at different times. The sequential steps of the process are indicated: formation, residence and escape. The half-lives of the formation and escape steps are also indicated; B: data obtained in the presence of 100 µM chloroquine are represented by open circles: metacyclic trypomastigotes remain up to 6 h more in the parasitophorous vacuole when compared to the controls, represented by closed triangles; C: the overall kinetics of amastigote para-sitophorous vacuole formation, residence and escape, are not affected by chloroquine (controls represented by closed triangles, 100 µM chloroquine by open circles).

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Fig. 3: cytoplasmic alkalinization of Vero cells with chloroquine only inhibits metacyclic trypomastigote invasion. Vero cells were incubated with 100 µM chloroquine (represented by hatched bars) and exposed to Trypanosoma cruzi infective forms. Invasion indexes were determined as indicated in the text. Bars denote mean three experiments with standard deviations and *indicates that p 0.05 relative to the controls (represented by open bars).

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Fig. 4: reduced expression of sialic acid on host cells affects parasitophorous vacuole escape but not invasion by Trypanosoma cruzi infective forms. A, B: CHO and sialic acid-deficient Lec-2 mutant cells were infected with metacyclic trypomastigotes (A) or amastigotes (B) and invasion indexes determined as described in Materials and Methods; C, D: the percentages of remaining LAMP-1 labeled parasites were determined after 3 and 6 h. Bars denote mean three experiments with standard deviations and *indicates that p 0.05 relative to the CHO cell controls. Note that the reduction in anti-LAMP-1 labeling, compatible with parasitophorous vacuole escape, is significant for both infective forms in Lec-2 cells.

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Fig. 5: drugs that affect host cell free calcium concentration only inhibit metacyclic trypomastigote invasion. Vero cells were incubated for 30 min with 10mM calcium ionophore A23187 or 1 mM thapsigargin (TPS), and subjected to invasion by metacyclic trypomastigotes (A) or amastigotes (B). Bars denote mean three experiments with standard deviations and *indicates p 0.05 relative to the controls. C is the control for A, and in B, C1 and C2 are the controls carried out for each drug.