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© The Rockefeller University Press, 0021-9525/2000//741 $5.00
The Journal of Cell Biology, Volume 148, Number 4, , 2000 741-754

Original Article

Live Salmonella Recruits N-Ethylmaleimide–Sensitive Fusion Protein on Phagosomal Membrane and Promotes Fusion with Early Endosome



Konark Mukherjeea, Shadab A. Siddiqia, Shehla Hashima, Manoj Rajeb, Sandip K. Basua, and Amitabha Mukhopadhyaya

a National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
b Institute of Microbial Technology, Sector 39A, Chandigarh 160014, India
Cell Biology Lab, National Institute of Immunology, New Delhi 110067, India.91-11-6109433 or 91-11-616212591-11-6162281 or 91-11-6183004

amitabha{at}nii.res.in

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. GTP{gamma}S and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, {alpha}-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, ATP{gamma}S 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 {alpha}-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.

Key Words: phagocytosis • reconstitution • fusion • phagosome • Rab

© 2000 The Rockefeller University Press

Abbreviations used in this paper: ATP{gamma}S, adenosine 5'-0-thiotriphosphate; DSP, dead Salmonella-containing phagosome; GDI, GDP dissociation inhibitor; GTP{gamma}S, 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.


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