Membrane sorting during swimming internalization of Brucella is required for phagosome trafficking decisions.

Brucella infects macrophages by swimming internalization, after which it is enclosed in macropinosomes. We investigated the role of the uptake pathway in phagosome trafficking, which remains unclear. This study found membrane sorting during swimming internalization and is essential in intracellular replication of Brucella. The B. abortus virB mutant replicated intracellularly when it was in the macropinosome established by wild-type B. abortus that retained its ability to alter phagosome trafficking. Lipid rafts-associated molecules, such as GM1 ganglioside, were selectively included into macropinosomes, but Rab5 effector early endosome autoantigen (EEA1) and lysosomal glycoprotein LAMP-1 were excluded from macropinosomes containing B. abortus induced by swimming internalization. In contrast, when the swimming internalization was bypassed by phorbol myristate acetate (PMA)-induced macropinocytosis, lipid raft-associated molecules were excluded, and EEA1 and LAMP-1 were included into macropinosomes containing bacteria. The phosphatidylinositol 3-kinase inhibitor wortmannin that inhibits PMA-induced macropinocytosis blocked internalization of virB mutant, but not of wild-type of B. abortus and wortmannin treatment did not affect intracellular replication. Our results suggest that membrane sorting requires swimming internalization of B. abortus and decides the intracellular fate of the bacterium, and that Brucella -induced macropinosome formation is a different mechanism from PMA-induced macropinocytosis.