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Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome.

Mukherjee K, Siddiqi SA, Hashim S, Raje M, Basu SK, Mukhopadhyay A - J. Cell Biol. (2000)

Bottom Line: LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7.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.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.

ABSTRACT
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.

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Detection of rab5-binding protein from Salmonella. (a) To detect the rab5-binding protein, GST pull-out assay was carried out with GST-Rab5 (middle), GST-Rab7 (top), or GST alone (bottom) in the presence of concentrated Salmonella spent medium as described in Materials and Methods. The Salmonella proteins associated with respective beads were detected by Western blot analysis using antibodies against SopE, SopB, and SipC. (b) J774E macrophages were infected with live Salmonella as described in Materials and Methods. Cells were washed and incubated for 10 h, and subsequently, cytosol was purified from the infected cells. Western blot analysis was carried out to determine the presence of Sop E in the respective cytosol using specific antibody.
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Figure 8: Detection of rab5-binding protein from Salmonella. (a) To detect the rab5-binding protein, GST pull-out assay was carried out with GST-Rab5 (middle), GST-Rab7 (top), or GST alone (bottom) in the presence of concentrated Salmonella spent medium as described in Materials and Methods. The Salmonella proteins associated with respective beads were detected by Western blot analysis using antibodies against SopE, SopB, and SipC. (b) J774E macrophages were infected with live Salmonella as described in Materials and Methods. Cells were washed and incubated for 10 h, and subsequently, cytosol was purified from the infected cells. Western blot analysis was carried out to determine the presence of Sop E in the respective cytosol using specific antibody.

Mentions: Salmonella have evolved a complex protein secretion system termed type III to deliver the bacterial effector proteins into the host cells that modulate the host cellular function (Galan and Collmer 1999; Uchiya et al. 1999). To detect the rab5-binding protein from Salmonella, immobilized GST-Rab5, GST-Rab7, or GST alone was incubated in the presence of concentrated spent medium for 10 h at 4°C. Subsequently, Salmonella proteins bound to respective beads were detected by Western blot analysis using antibodies against SopE, SopB and SipC. The results presented in Fig. 8 a show that rab5 specifically binds with SopE but not with SopB and SipC (middle panel). In contrast, rab7 (upper panel) and GST (lower panel) alone were unable to bind any of these secretory proteins from the Salmonella extract. Moreover, when immobilized GST-Rab5 was incubated with a Salmonella lysate obtained after growing the cells in the presence of 35S-methionine, specifically a 30-kD protein associated with immobilized GST-Rab5 was detected by autoradiography (data not shown). Western blot analysis of cytosols from uninfected or Salmonella-infected macrophages with anti-SopE antibody revealed the presence of SopE only in the infected cytosol (Fig. 8 b).


Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome.

Mukherjee K, Siddiqi SA, Hashim S, Raje M, Basu SK, Mukhopadhyay A - J. Cell Biol. (2000)

Detection of rab5-binding protein from Salmonella. (a) To detect the rab5-binding protein, GST pull-out assay was carried out with GST-Rab5 (middle), GST-Rab7 (top), or GST alone (bottom) in the presence of concentrated Salmonella spent medium as described in Materials and Methods. The Salmonella proteins associated with respective beads were detected by Western blot analysis using antibodies against SopE, SopB, and SipC. (b) J774E macrophages were infected with live Salmonella as described in Materials and Methods. Cells were washed and incubated for 10 h, and subsequently, cytosol was purified from the infected cells. Western blot analysis was carried out to determine the presence of Sop E in the respective cytosol using specific antibody.
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Related In: Results  -  Collection

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Figure 8: Detection of rab5-binding protein from Salmonella. (a) To detect the rab5-binding protein, GST pull-out assay was carried out with GST-Rab5 (middle), GST-Rab7 (top), or GST alone (bottom) in the presence of concentrated Salmonella spent medium as described in Materials and Methods. The Salmonella proteins associated with respective beads were detected by Western blot analysis using antibodies against SopE, SopB, and SipC. (b) J774E macrophages were infected with live Salmonella as described in Materials and Methods. Cells were washed and incubated for 10 h, and subsequently, cytosol was purified from the infected cells. Western blot analysis was carried out to determine the presence of Sop E in the respective cytosol using specific antibody.
Mentions: Salmonella have evolved a complex protein secretion system termed type III to deliver the bacterial effector proteins into the host cells that modulate the host cellular function (Galan and Collmer 1999; Uchiya et al. 1999). To detect the rab5-binding protein from Salmonella, immobilized GST-Rab5, GST-Rab7, or GST alone was incubated in the presence of concentrated spent medium for 10 h at 4°C. Subsequently, Salmonella proteins bound to respective beads were detected by Western blot analysis using antibodies against SopE, SopB and SipC. The results presented in Fig. 8 a show that rab5 specifically binds with SopE but not with SopB and SipC (middle panel). In contrast, rab7 (upper panel) and GST (lower panel) alone were unable to bind any of these secretory proteins from the Salmonella extract. Moreover, when immobilized GST-Rab5 was incubated with a Salmonella lysate obtained after growing the cells in the presence of 35S-methionine, specifically a 30-kD protein associated with immobilized GST-Rab5 was detected by autoradiography (data not shown). Western blot analysis of cytosols from uninfected or Salmonella-infected macrophages with anti-SopE antibody revealed the presence of SopE only in the infected cytosol (Fig. 8 b).

Bottom Line: LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7.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.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.

ABSTRACT
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.

Show MeSH