Fusion between large phagocytic vesicles: targeting of yeast and other particulates to phagolysosomes that shelter the bacterium Coxiella burnetii or the protozoan Leishmania amazonensis in Chinese hamster ovary cells

Abstract

This report examines the fusion of phagocytic vesicles with the large phagolysosome-like vacuoles induced in Chinese hamster ovary cells by the bacterium Coxiella burnetti or the Protozoan flagellate Leishmania amazonensis. Infection by these organisms is compatible with cell survival and multiplication. Fusion was inferred from the transfer of microscopically identifiable particles from donor to target vesicles. Donor vesicles contained heat-killed yeast, zymosan, beta-glucan or latex beads taken up by the host cells. Yeast and zymosan were also coated with Concanavalin A to increase their uptake by the cells (Goldman, R., Exp. Cell Res. 104, 325–334, 1977). Particle localization, routinely ascertained by phase-contrast microscopy, was confirmed by confocal laser fluorescence and by transmission electron microscopy. Coxiella vacuoles admitted all the particles tested and transfer took place whether the particles were given to the cells prior to or after infection. Transfer of uncoated or Concanavalin-A-coated yeast or zymosan was dependent on the number of particles ingested and on the incubation period (between 2 and 24 hours). Furthermore, the transfer step was quite efficient, since over 85% of the particles ingested entered Coxiella vacuoles at all particle to cell ratios examined. The fraction of uncoated or Concanavalin-A-coated yeast or zymosan transferred to Leishmania vacuoles was consistently lower and diminished at higher particle loads. In addition, only rarely did latex beads enter these vacuoles. The models proposed may be useful for the delineation of biochemical and molecular mechanisms involved in the fusion of large phagocytic vesicles and the modulation of the latter by cellular and pathogen-derived signals.