Phagocytosed live Listeria monocytogenes influences rab5-regulated in vitro phagosome-endosome fusion
Phagocytosed live Listeria monocytogenes influences rab5-regulated in vitro phagosome-endosome fusion
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DOI：
10.1074/jbc.271.23.13834
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发表时间：
1996-06-07
影响因子：
4.8
通讯作者：
Stahl, PD
中科院分区：
文献类型：
--
作者：
AlvarezDominguez, C;Barbieri, AM;Stahl, PD
病原体在吞噬作用后的存活或破坏部分取决于吞噬体与内体或溶酶体隔室之间的融合事件。在这里，我们使用体外试验表明，吞噬体-内体融合是由小GTcirab 5和融合事件的影响，内化的活的生物体，单核细胞增生李斯特菌（LM），我们比较了在体外融合的吞噬体含有热杀死的生物体（死LM）与吞噬体含有活的非溶血性突变体（活LM（hly-））。与野生型生物不同，LM（hly-）仍然被困在吞噬体内。用生物素标记的生物体和含有辣根过氧化物酶与抗生物素蛋白缀合的内体重建吞噬体-内体融合。用五个LM（hly-）和死LM制备物，体外吞噬体-内体融合是时间、温度和胞质依赖性的。活的LM（hly-）吞噬体表现出更快的融合速率。两种制备物中的融合受rab 5调节，并且可能受其他GTP酶调节。抗rab 5抗体和免疫耗竭的cytokerab 5抑制融合。此外，谷胱甘肽S-转移酶-rab 5的GTP形式刺激吞噬体-内体融合，而除了一个显性负突变体的rab 5阻断融合。纯化的活LM（hly-）吞噬体膜富集rab 5所揭示的蛋白质印迹，与死LM吞噬体相比。内体与含有吞噬体的死LM的融合需要ATP，并且被ATP消耗以及被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，与含有活LM（hly-）的吞噬体的吞噬体-内体融合不被ATP消耗抑制，也不被NEM或抗NSF抗体抑制。Western印迹分析显示，活的含LM（hly-）的吞噬体富含膜结合的NSF，而死的含LM的吞噬体含有低的或不可检测的量。用0.5M KCl洗涤活的含LM（hly-）的吞噬体去除与膜结合的NSF，并使它们对NEM、ATP、抗NSF抗体融合敏感。我们的结论是，rab 5调节吞噬体-内体融合，活微生物可以通过将rab 5募集到膜上来上调这一过程。
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 LM(hly-)). Unlike the wild-type organism, LM(hly-) remains trapped inside the phagosome. Phagosome-endosome fusion was reconstituted using biotinylated organisms and endosomes containing horseradish peroxidase conjugated with avidin, With both Five LM(hly-) and dead LM preparations, in vitro phagosome-endosome fusion was time-, temperature-, and cytosol-dependent. Live LM(hly-) 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 cytostolic 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 LM(hly-) 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 LM(hly-)-containing phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Western blot analysis revealed that live LM(hly-)-containing phagosomes were enriched for membrane-bound NSF, while dead LM containing phagosomes contained low or undetectable quantities, Washing live LM(hly-)-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.