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First published online 22 July 2003
doi: 10.1242/jcs.00666

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Novel PI 3-kinase-dependent mechanisms of trypanosome invasion and vacuole maturation

Aaron M. Woolsey1, Lisa Sunwoo1, Christine A. Petersen1, Saskia M. Brachmann2,3, Lewis C. Cantley2 and Barbara A. Burleigh1,*

1 Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA
2 Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA
3 Institut fuer Biochemie, Freie Universitaet Berlin, Berlin, Germany



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  		Fig. 2. Time-lapse fluorescence imaging of Akt-PH-GFP recruitment to T. cruzi-associated cell membranes. Transfected L6E9 myoblasts on 25 mm coverslips were placed in a heated chamber (at 37°C) on an inverted fluorescence microscope. Akt-PH-GFP-expressing cells were located and images acquired at 5-second intervals before (0 minutes) and for 17 minutes following addition of T. cruzi trypomastigotes (2.5x107 in Ringers/BSA). At 3.75 minutes post-addition, a cell-associated extracellular trypomastigote is seen as a shadow over the cell (arrow) to which Akt-PH-GFP is slowly recruited (5-9.6 minutes) and then gradually dissipates (~10-17 minutes). A second cell-associated parasite, first observed at 9.6 minutes, associates with Akt-PH-GFP-enriched membrane ruffles (arrowheads) at 10.5-12.25 minutes that appear to envelope the parasite (12.8-16.3 minutes). Scale bar: 10 µm.

 


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  		Fig. 1. Rapid accumulation of PtdIns(3,4,5)P3/PtdIns (3,4)P2 at the T. cruzi invasion site. Fluorescence images of (A) CHO cells or (B) primary rat cardiomyocytes transiently expressing Akt-PH-GFP following incubation with infective T. cruzi trypomastigotes for 15 minutes. (C) T. cruzi invasion of CHO cells expressing Akt-PHGFPR25C which fails to bind to PtdIns(3,4,5)P3/PtdIns(3,4)P2. Extracellular parasites were stained with T. cruzi-specific antibodies in non-permeabilized cells. Host cell and parasite DNA were visualized with DAPI (A-C, blue). (D) Recruitment of Akt-PH-GFP to membranes surrounding invading T. cruzi trypomastigotes is sensitive to wortmannin. The relative number of Akt-PH-GFP-associated parasites was determined in CHO cells infected for 15 minutes following treatment with 20 nM wortmannin or vehicle control for 30 minutes. Data are represented as means ± s.d. n=3.

 


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  		Fig. 3. Lamp-1 association with the Akt-PH-GFP-enriched T. cruzi vacuole occurs after parasite entry. (A,B) Immunofluorescence staining of lamp-1 (red) in T. cruzi-infected CHO cells at 15 minutes post-infection showing diffuse (arrows) and punctuate (arrowheads) staining patterns outlining intracellular parasites. External parasites stained with T. cruzi-specific antibodies (green); parasite and mammalian DNA stained with DAPI (blue). (C-F) L6E9 myoblasts expressing Akt-PH-GFP (green), were infected with T. cruzi for 15 minutes (C,D) or 60 minutes (E,F) and stained with anti-lamp-1 (red) and DAPI (blue). No overlap of Akt-PH-GFP and lamp-1 staining is observed (C-F, merge).

 


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  		Fig. 4. T. cruzi associates with host cell plasma membrane markers in an actin-independent entry process. (A,B) CHO cells transfected with myr-GFP were infected with T. cruzi trypomastigotes for 15 minutes. The extracellular region of partially internalized parasites was stained with a T. cruzi-specific antibody followed by TR-conjugated secondary antibody (arrowheads) highlighting the ability of T. cruzi to enter cells either (A) posterior end (DAPI-stained, blue) or (B) anterior end first. (C) Fully internalized trypomastigote in CHO cells expressing PLC{delta}-PH-GFP 15 minutes after infection. (D) Akt-PH-GFP-expressing L6E9 infected with T. cruzi for 15 minutes were stained with TR-phalloidin (E; arrowhead). (F) Partial co-localization of T. cruzi-associated Akt-PHGFP and F-actin is observed (merged image). (G) Phagocytic uptake of IgG-coated 3 µm latex beads by FCR{gamma}III-CHO cells stained with TR-phalloidin (arrowheads). (H,I) CHO cells transfected with PLC{delta}-PH-GFP were pre-treated with 10 µM cytochalasin D or vehicle control for 10 minutes, infected with trypomastigotes for 15 minutes and the number of intracellular parasites (H) and the percentage of intracellular parasites associating with PLC{delta}-PH-GFP (I) was determined. Data are represented as means ± s.d. for 3 coverslips. Scale bars: 5 µm.

 


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  		Fig. 5. Early association of T. cruzi with early endosomes is minimal and precedes lamp-1 acquisition. Immunofluorescence staining of extracellular T. cruzi (arrowheads) following infection of CHO cells transiently expressing (A-C) Rab5-GFP or (D,E) 2xFYVE-GFP for 7.5 minutes. Host cell and parasite DNA is visualized with DAPI (blue). (F) Immunostaining of endogenous EEA1 in T. cruzi-infected CHO cells (15 minutes post-infection). (G) T. cruzi trypomastigotes or (H) fibronectin-coated 3 µm latex beads, incubated with L6E9 myoblasts for 10 minutes were washed extensively to remove uninternalized beads or parasites and further incubated for a total of 60 minutes. Three samples from each condition were fixed at 10, 20, 30 45 and 60 minutes post-incubation and immunostained for EEA1 or lamp-1. Data are represented as means ± s.d.; n=3. Scale bars: 3 µm.

 


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  		Fig. 6. Inhibition of host cell PI 3-kinases during T. cruzi invasion blocks the lysosome-mediated entry pathway. (A,B) CHO cells expressing PLC{delta}-PH-GFP were treated with 40 nM wortmannin or vehicle control for 30 minutes prior to incubation with trypomastigotes for 15 minutes. Fixed permeabilized cells were stained with antibodies to lamp-1. The relative number of (A) intracellular parasites and (B) lamp-1 or PLC{delta}-PH-GFP-positive intracellular parasites was determined. (C) The relative number of intracellular parasites determined following T. cruzi infection of p85{alpha}+/-ß-/- ({alpha}HET) and p85{alpha}-/-ß-/- (KO) MEFs 15 minutes. (D) Kinetics of lamp-2 association with intracellular T. cruzi in {alpha}HET and KO cells at 10, 20, 30, 45, and 60 minutes post-infection. Data are represented as means ± s.d.; n=3.

 


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  		Fig. 7. T. cruzi vacuole maturation involves an early PI 3-kinase-dependent step. CHO cells were pulsed with (A,B) T. cruzi or (C) IgG-coated latex beads for 10 minutes, washed extensively and incubated in the absence or presence of 40 nM wortmannin for a total of 10, 20, 30, 45 and 60 minutes. (D) Kinetics of lamp-1 association with the T. cruzi vacuole in untreated CHO cells (control), cells pre-treated with 40 nM wortmannin for 30 minutes (wortmannin-pre) or in wortmannin pretreated cells to which drug was added after 10 minutes of infection (wortmannin pre+post). Data are represented as means ± s.d.; n=3.

 




© The Company of Biologists Ltd 2003