To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPγS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, α-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)–depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPγS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of α-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.
Live Salmonella Recruits N-Ethylmaleimide–Sensitive Fusion Protein on Phagosomal Membrane and Promotes Fusion with Early Endosome
Abbreviations used in this paper: ATPγS, adenosine 5′-0-thiotriphosphate; DSP, dead Salmonella-containing phagosome; GDI, GDP dissociation inhibitor; GTPγS, guanine 5′-3-0-(thio) triphosphate; HB, homogenization buffer; LSP, live Salmonella-containing phagosome; NEM, N-ethylmaleimide; NSF, NEM-sensitive fusion protein; PNS, postnuclear supernatant; SNAP, soluble NSF attachment protein; WT, wild-type.
Konark Mukherjee, Shadab A. Siddiqi, Shehla Hashim, Manoj Raje, Sandip K. Basu, Amitabha Mukhopadhyay; Live Salmonella Recruits N-Ethylmaleimide–Sensitive Fusion Protein on Phagosomal Membrane and Promotes Fusion with Early Endosome. J Cell Biol 21 February 2000; 148 (4): 741–754. doi: https://doi.org/10.1083/jcb.148.4.741
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