Afipia felis induces uptake by macrophages directly into a nonendocytic compartment

Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. A. felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A. felis-containing phagosomes is unknown. Here, we demonstrate that, in murine macrophages. most A. felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection. Established Afipia-containing phagosomes possess neither early endosomal marker proteins [early endosome antigen 1 (EEA1), Rab5, transferrin receptor. trytophane aspartate containing coat protein (TACO)] nor late endosomal or lysosomal proteins [cathepsin D. P-glucuronidase, vacuolar proton-pumping ATPase. rab7, mannose-g-phosphate receptor. vesicle-associated membrane protein 8. lysosome-associated membrane proteins LAMP-1 and LAMP-2]. Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment. which remains negative for LAMP-1. The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment. which also subsequently acquires LAMP-1. Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A. felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing A. felis are disconnected from the endosome-lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.