There are many similarities between the interactions of environmental protozoa with pathogenic bacterial species and those observed in mammalian macrophages. Since single-celled protozoa predate mammalian hosts, it is likely that interactions in environmental biofilms have selected for many of the bacterial virulence mechanisms responsible for human disease. In order to better understand bacterial-phagocyte interactions, we developed a selection for Acanthamoeba castellanii variants that are more resistant to killing by bacterial pathogens. We identified four amoebal clones that display decreased phagocytosis of bacteria but no difference in uptake of latex beads compared to wild-type amoebae. These amoebal variants display differences in cellular morphology, partial resistance to killing by bacteria, more bactericidal activity, and higher frequencies of lysosome fusion with the bacterial vacuole. Three proteins are present at lower levels in these variants than in wild-type amoebae, and matrix-assisted laser desorption ionization-time of flight mass spectrometry allowed identification of two of them as actin and hsp90. We found that specific inhibitors of hsp90 produce a similar phenotypic effect in macrophages. These data suggest that hsp90 plays a role in phagocytic and, possibly, bactericidal pathways that affect interactions of phagocytic cells with bacteria.Uptake and degradation of bacterial particles are key functions of phagocytes in the mammalian immune system. Phagocytic environmental protozoa within aquatic biofilms carry out analogous activities in complex ecosystems that contain numerous prokaryotic organisms. Recent data suggest that environmental protozoa, particularly amoebae such as Acanthamoeba castellanii, have played an important role in the evolution of bacterial pathogens of mammals and may represent natural hosts for virulent bacteria (4, 9). A number of different pathogenic species, including Mycobacterium spp. (12), Vibrio cholerae (69), Burkholderia cepacia, Chlamydia pneumoniae, Edwardsiella tarda, Legionella pneumophila (32, 59), Cryptococcus neoformans (66), Listeria monocytogenes, and Francisella tularensis (1), have the ability to survive and replicate in environmental protozoa. Environmental amoebae, including A. castellanii, can undergo a respiratory burst (19), produce oxygen radicals (20), express cell surface receptors (2), and have phagocytic mechanisms (3,8,39) that are similar to those of human macrophages.There are several important steps during the interactions of phagocytic cells with bacterial pathogens, including adherence, phagocytosis, signal transduction, trafficking, and intracellular replication. It is likely that a large number of host cell components are involved in these processes, yet few have been described in great detail. Protozoal models for bacterial-host cell interactions, such as the amoeba A. castellanii, should be very useful for identification of the host cell components involved. As opposed to that of mammalian hosts, the unicellular nature of A. c...