Lipid microdomain-dependent macropinocytosis determines compartmentation of Afipia felis

Phagocytic compartments are specialized endocytic organelles and usually mature along the degradative pathway into phagolysosomes. The rare human pathogen Afipia felis localizes to a compartment that is different from canonical phagocytic compartments. Here, we present evidence that internalization of Afipia by macrophages and unusual phagosome development are considerably decreased by attachment of cholera toxin B subunit to macrophage ganglioside GM1 or by extraction or oxidation of plasma membrane cholesterol. Amiloride (an inhibitor of Na+/H+ exchanger and macropinocytosis) strongly inhibited uptake of A. felis at a late step, i.e. the closure of macropinocytic structures rather than the production of membrane ruffles. Ultrastructural evidence showed that A. felis was taken up by macrophages via macropinocytosis. In contrast, A. felis opsonized with a monoclonal IgG antibody was ingested by a zipper-like mechanism, resulting in normal phagosome maturation. Hence, while the preferred path of A. felis uptake is dependent on the integrity of lipid microdomains and on macropinocytosis, and while this uptake leads to an unusual phagosome and to intracellular survival of A. felis, those bacteria that enter using Fc gamma receptors are delivered to a late endocytic compartment.