Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils

Abstract

Biogenesis of phagolysosomes proceeds through a sequential series of interactions with endocytic organelles, a process known to be regulated by Rab and SNARE proteins. The molecular mechanisms underlying phagosome maturation in neutrophils are, however, not clearly understood. We investigated fusion between phagosomes containing the intracellular pathogen Mycobacterium tuberculosis versus the extracellular pathogen Staphylococcus aureus (designated MCP for mycobacteria-containing phagosome and SCP for S. aureus-containing phagosome) and cytoplasmic compartments in human neutrophils. Western blot analysis of phagosomes isolated after internalisation revealed that lactoferrin (a constituent of secondary granules) and LAMP-1 were incorporated into both SCP and MCP, whereas hck (marker of azurophil granules) interacted solely with SCP. The subcellular distribution of the proteins Rab5a and syntaxin-4 suggested a role in docking of granules and/or endosomes to the target membrane in the neutrophil. We observed that during phagocytosis, Rab5a in GTP-bound form interacted with syntaxin-4 on the membrane of MCP and were retained for up to 90 minutes, whereas the complex was recruited to the SCP within 5 minutes but was selectively depleted from these vacuoles after 30 minutes of phagocytosis. Downregulation of Rab5a by antisense oligonucleotides efficiently reduced the synthesis of Rab5a, the binding of syntaxin-4 to MCP and SCP and the capacity for fusion exhibited by the pathogen-containing phagosomes, but it had no effect on bacteria internalisation. These data indicate that the difference in granule fusion is correlated with a difference in the association of Rab5a and syntaxin-4 with the phagosomes. Intracellular pathogen-containing phagosomes retain Rab5a and syntaxin-4, whereas extracellular pathogen-containing phagosomes bind briefly to this complex. These results also identified Rab5a as a key regulator of phagolysosome maturation in human neutrophils.