Article Figures & Data
Figures
- FIG. 1.
Distinction between phagocytosis and active invasion. Panels a to d show the same view, but panels a and b show LAMP1 (a) or spore wall protein SWP1 (b), while panel c was visualized by phase-contrast microscopy, and panel d is a merged picture of panels a to c. The association of SWP1-positive vacuoles (arrowheads) with the lysosomal marker LAMP1 is indicative of phagocytic uptake of spores in HFF. (e) MAb 6G2 detected with Cy2-conjugated donkey anti-mouse IgG. (f) Merged picture of panel e and phase contrast. MAb 6G2 specifically detects meronts. (g) Double-immunofluorescence staining of a sample fixed immediately after infection (∼1 min). MAb 6G2 was detected with a Cy3-conjugated anti-mouse IgG. The spore wall and polar filament were stained with a polyclonal rabbit anti-E. cuniculi antibody and detected with a Cy2-conjugated donkey anti-rabbit IgG. 4′,6′-Diamidino-2-phenylindole (DAPI) staining was used to detect nuclei. The arrowhead shows the extruded sporoplasm (red) with the nucleus (blue) at the tip of the polar filament. (h) Double-immunofluorescence staining of a sample fixed 2 h p.i. MAb 6G2 was detected with a Cy3-conjugated donkey anti-mouse IgG. A polyclonal rabbit antibody against the spore wall protein SWP1 was detected with a Cy2-conjugated donkey anti-rabbit IgG, and DAPI staining was used to detect nuclei. Extra- or intracellularly located spores (green) can be clearly distinguished from MAb 6G2-positive meronts (red), which entered host cells by active invasion (arrowhead).
- FIG. 2.
The E. cuniculi PV membrane does not contain markers for endosomes, lysosomes, and the ER. L929 murine fibroblasts were immunostained at 1 min, 5 min, 1 h, and 24 h p.i. with the following antibodies: rabbit anti-TfR (1:100), goat anti-EEA1 (1:500), goat anti-calnexin (1:500), or goat anti-LAMP1 (1:500). Primary antibodies were obtained from Santa Cruz Biotechnology and detected with a Cy3-conjugated anti-goat or anti-rabbit IgG. The E. cuniculi PV was stained with MAb 6G2 and detected by the Cy2 conjugate. Note that Cy2 and Cy3 labeling is reversed for LAMP1. Samples were analyzed with a Leica confocal laser-scanning microscope (model TCS SP2) or with a conventional fluorescence microscope (model DM R; Leica) in combination with digital camera equipment (AxioCam; Zeiss). Merged pictures (pictures using Cy2- and Cy3-conjugated antibodies plus the picture from phase-contrast microscopy) from conventional microscopy are presented for the 1-min and 24-h samples. Merged pictures (pictures using Cy2- and Cy3-conjugated antibodies) from confocal microscopy are shown for the 5-min and 1-h samples.
