PHAGOCYTOSED LIVE LISTERIA-MONOCYTOGENES INFLUENCES RAB5-REGULATED IN-VITRO PHAGOSOME-ENDOSOME FUSION

Survival or destruction of a pathogen following phagocytosis depends, in part, on fusion events between the phagosome and the endosomal or l ysosomal compartments. Here we use an in vitro assay to show that phag osome-endosome fusion is regulated by the small GTPase rab5 and that f usion events are influenced by an internalized live organism, Listeria monocytogenes (LM), We compare the in vitro fusion of phagosomes cont aining heat-killed organisms (dead LM) with that of phagosomes contain ing a Live nonhemolytic mutant (live LM(hly-)). Unlike the wild-type o rganism, LM(hly-) remains trapped inside the phagosome. Phagosome-endo some fusion was reconstituted using biotinylated organisms and endosom es containing horseradish peroxidase conjugated with avidin, With both Five LM(hly-) and dead LM preparations, in vitro phagosome-endosome f usion was time-, temperature-, and cytosol-dependent. Live LM(hly-) ph agosomes exhibited a faster rate of fusion, Fusion in both preparation s was regulated by rab5 and possibly by other GTPases. Anti-rab5 antib odies and immunodepletion of cytostolic rab5 inhibited fusion. Additio n of glutatione S-transferase-rab5 in the GTP form stimulated phagosom e-endosome fusion, whereas addition of a dominant negative mutant of r ab5 blocked fusion. Purified live LM(hly-) phagosomal membranes were e nriched in rab5 as revealed by Western blotting, compared with dead LM phagosomes. Fusion of endosomes with dead LM containing phagosomes re quired ATP and was inhibited by ATP depletion and by N-ethylmaleimide (NEM) and anti-NEM-sensitive factor (NSF) antibodies, Unexpectedly, ph agosome-endosome fusion with live LM(hly-)-containing phagosomes was n ot inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Weste rn blot analysis revealed that live LM(hly-)-containing phagosomes wer e enriched for membrane-bound NSF, while dead LM containing phagosomes contained low or undetectable quantities, Washing live LM(hly-)-conta ining phagosomes with 0.5 M KCl removed NSF associated with the membra nes and rendered them NEM, ATP, anti-NSF antibody sensitive for fusion . We conclude that rab5 regulates phagosome-endosome fusion and that l ive microorganisms can up-regulate this process by recruiting rab5 to the membrane.