FIG. 4.

Colocalization of O. tsutsugamushi and early endosome antigen 1 or LAMP2 in the presence of NH₄Cl (20 mM). Representative immunofluorescence as seen in confocal microscopic images of ECV304 cells infected with O. tsutsugamushi for the indicated time periods. Infected cells were washed, fixed, permeabilized, and double labeled with human serum against O. tsutsugamushi (goat anti-human rhodamine conjugate) together with anti-clathrin, anti-EEA1, or anti-LAMP2 (Alexa Fluor 488). (A) Colocalization of EEA1 (green) with O. tsutsugamushi (red) at 30 min, 1 h, and 2 h. (B) Colocalization of LAMP2 (green) with O. tsutsugamushi (red) at 30 min, 1 h, and 2 h. Ratios are the number of O. tsutsugamushi organisms that colocalized with EEA1 (A) or LAMP2 (B) to the total number of infecting O. tsutsugamushi organisms. The colocalization of O. tsutsugamushi with EEA1 or LAMP2 is indicated by arrows. Bars, 10 μm.