Alvarez-Dominguez C, Barbieri A M, Berón W, Wandinger-Ness A, Stahl P D
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1996 Jun 7;271(23):13834-43. doi: 10.1074/jbc.271.23.13834.
Survival or destruction of a pathogen following phagocytosis depends, in part, on fusion events between the phagosome and the endosomal or lysosomal compartments. Here we use an in vitro assay to show that phagosome-endosome fusion is regulated by the small GTPase rab5 and that fusion events are influenced by an internalized live organism, Listeria monocytogenes (LM). We compare the in vitro fusion of phagosomes containing heat-killed organisms (dead LM) with that of phagosomes containing a live nonhemolytic mutant (live LMhly-). Unlike the wild-type organism, LMhly- remains trapped inside the phagosome. Phagosome-endosome fusion was reconstituted using biotinylated organisms and endosomes containing horseradish peroxidase conjugated with avidin. With both live LMhly- and dead LM preparations, in vitro phagosome-endosome fusion was time-, temperature-, and cytosol-dependent. Live LMhly- phagosomes exhibited a faster rate of fusion. Fusion in both preparations was regulated by rab5 and possibly by other GTPases. Anti-rab5 antibodies and immunodepletion of cytosolic rab5 inhibited fusion. Addition of glutatione S-transferase-rab5 in the GTP form stimulated phagosome-endosome fusion, whereas addition of a dominant negative mutant of rab5 blocked fusion. Purified live LMhly- phagosomal membranes were enriched in rab5 as revealed by Western blotting, compared with dead LM phagosomes. Fusion of endosomes with dead LM containing phagosomes required ATP and was inhibited by ATP depletion and by N-ethylmaleimide (NEM) and anti-NEM-sensitive factor (NSF) antibodies. Unexpectedly, phagosome-endosome fusion with live LMhly--containing phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Western blot analysis revealed that live LMhly--containing phagosomes were enriched for membrane-bound NSF, while dead LM containing phagosomes contained low or undetectable quantities. Washing live LMhly--containing phagosomes with 0.5 M KCl removed NSF associated with the membranes and rendered them NEM, ATP, anti-NSF antibody sensitive for fusion. We conclude that rab5 regulates phagosome-endosome fusion and that live microorganisms can up-regulate this process by recruiting rab5 to the membrane.
病原体在被吞噬作用后是存活还是被破坏，部分取决于吞噬体与内体或溶酶体区室之间的融合事件。在此，我们使用体外试验表明，吞噬体 - 内体融合受小GTP酶rab5调控，并且融合事件受内化的活生物体——单核细胞增生李斯特菌（LM）影响。我们比较了含有热杀死生物体（死LM）的吞噬体与含有活的非溶血突变体（活LMhly -）的吞噬体的体外融合情况。与野生型生物体不同，LMhly - 仍被困在吞噬体内。使用生物素化的生物体和含有与抗生物素蛋白偶联的辣根过氧化物酶的内体来重建吞噬体 - 内体融合。对于活的LMhly - 和死的LM制剂，体外吞噬体 - 内体融合都具有时间、温度和胞质溶胶依赖性。活的LMhly - 吞噬体表现出更快的融合速率。两种制剂中的融合均受rab5调控，可能还受其他GTP酶调控。抗rab5抗体和胞质rab5的免疫耗竭抑制融合。添加GTP形式的谷胱甘肽S - 转移酶 - rab5刺激吞噬体 - 内体融合，而添加rab5的显性负突变体则阻断融合。与死LM吞噬体相比，蛋白质印迹显示纯化的活LMhly - 吞噬体膜中rab5富集。内体与含有死LM的吞噬体的融合需要ATP，并且受ATP消耗以及N - 乙基马来酰亚胺（NEM）和抗NEM敏感因子（NSF）抗体抑制。出乎意料的是，含有活LMhly - 的吞噬体的吞噬体 - 内体融合不受ATP消耗、NEM或抗NSF抗体抑制。蛋白质印迹分析显示，含有活LMhly - 的吞噬体富含膜结合的NSF，而含有死LM的吞噬体含有少量或无法检测到的量。用0.5 M KCl洗涤含有活LMhly - 的吞噬体可去除与膜相关的NSF，并使其对融合具有NEM、ATP、抗NSF抗体敏感性。我们得出结论，rab5调节吞噬体 - 内体融合，并且活微生物可以通过将rab5募集到膜上来上调这一过程。
