Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome.
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. GTPgammaS 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, ATPgammaS 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.
Mukherjee
K
K
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Siddiqi
S A
SA
Hashim
S
S
Raje
M
M
Basu
S K
SK
Mukhopadhyay
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002939:Ciprofloxacin / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects / Q000382:microbiology; D008561:Membrane Fusion / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009154:Mutation / Q000235:genetics; D052066:N-Ethylmaleimide-Sensitive Proteins; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012475:Salmonella / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013696:Temperature; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D008565:Membrane Proteins; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; C103987:SopE protein, Salmonella; D033921:Vesicular Transport Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002939:Ciprofloxacin; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins; C498733:Nsf protein, mouse; D020696:rab5 GTP-Binding Proteins; D005033:Ethylmaleimide