Brucella suis-impaired specific recognition of phagosomes by lysosomes dueto phagosomal membrane modifications

Brucella species are gram-negative, facultatively intracellular bacteria th at infect humans and animals. These organisms can survive and replicate wit hin a membrane-bound compartment in phagocytic and nonprofessional phagocyt ic cells. Inhibition of phagosome-lysosome fusion has been proposed as a me chanism for intracellular survival in both types of cells. However, the bio chemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence m icroscopy analysis of general intracellular trafficking on whole cells in t he presence of Brucella and (li) a flow cytometry analysis of in vitro reco nstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We conclud ed that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Bru cella in the cell. By in vitro reconstitution experiments, we observed a sp ecific association between killed B. suis-containing phagosomes and lysosom es, which was dependent on exogenously supplied cytosol, energy, and temper ature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restric ted to the pathogen phagosomal membrane, as noted in the in vivo experiment s.