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Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells.

Seveau S, Bierne H, Giroux S, Prévost MC, Cossart P - J. Cell Biol. (2004)

Bottom Line: We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry.In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent.Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

View Article: PubMed Central - PubMed

Affiliation: Unité des Interactions Bactéries-Cellules, INSERM U604, Institut Pasteur, 75015 Paris Cedex 15, France.

ABSTRACT
Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

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Recruitment of raft markers during InlB-dependent entry. (A) Non-transfected Vero cells (A) and Vero cells transfected with CFP-MyrPalm (B) or with CFP-GerGer (C), were incubated with L. monocytogenes (BUG 1641) for 10 min, washed, and fixed. GM1 was labeled with the fluorescent B subunit of cholera toxin (A) and F-actin was labeled with Alexa Fluor 647 (A′). The images B and C represent the CFP fluorescence, and A′′, B′′, and C′′ correspond to the phase contrast images. Bacteria were labeled with DAPI (B′ and C′). (B) Non-transfected Vero cells (A and B), Vero cells transfected with CFP-MyrPalm (C), with CFP-GerGer (D), or with GFP-GPI (E), were incubated with InlB-coated beads for 10 min, washed, and fixed. Cells were then labeled for GM1 (A and B), for HGF-R (A′, C′, D′, and E′), for transferrin receptor (B′), and for F-actin (E′′). A′′, B′′, C′′, and D′′ correspond to phase contrast images. Bar, 10 μm.
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fig4: Recruitment of raft markers during InlB-dependent entry. (A) Non-transfected Vero cells (A) and Vero cells transfected with CFP-MyrPalm (B) or with CFP-GerGer (C), were incubated with L. monocytogenes (BUG 1641) for 10 min, washed, and fixed. GM1 was labeled with the fluorescent B subunit of cholera toxin (A) and F-actin was labeled with Alexa Fluor 647 (A′). The images B and C represent the CFP fluorescence, and A′′, B′′, and C′′ correspond to the phase contrast images. Bacteria were labeled with DAPI (B′ and C′). (B) Non-transfected Vero cells (A and B), Vero cells transfected with CFP-MyrPalm (C), with CFP-GerGer (D), or with GFP-GPI (E), were incubated with InlB-coated beads for 10 min, washed, and fixed. Cells were then labeled for GM1 (A and B), for HGF-R (A′, C′, D′, and E′), for transferrin receptor (B′), and for F-actin (E′′). A′′, B′′, C′′, and D′′ correspond to phase contrast images. Bar, 10 μm.

Mentions: To study the InlB pathway, we infected Vero epithelial cells with L. monocytogenes overexpressing an InlB protein covalently anchored at the bacterial surface or incubated the cells with InlB-coated beads. As presented in Fig. 4 A, L. monocytogenes corecruited F-actin and the lipid raft marker GM1. The CFP-MyrPalm, but not the CFP-GerGer, was recruited at the bacterial entry site. We repeated these experiments with InlB-coated beads, and as shown in Fig. 4 B, GM1 and HGF-R were corecruited, whereas the transferrin receptor was not. HGF-R colocalized with the CFP-MyrPalm but not with CFP-GerGer, and finally the GFP-GPI chimera was corecruited with the HGF-R and F-actin. These results clearly showed that lipid raft markers are recruited at both internalin and InlB entry site.


Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells.

Seveau S, Bierne H, Giroux S, Prévost MC, Cossart P - J. Cell Biol. (2004)

Recruitment of raft markers during InlB-dependent entry. (A) Non-transfected Vero cells (A) and Vero cells transfected with CFP-MyrPalm (B) or with CFP-GerGer (C), were incubated with L. monocytogenes (BUG 1641) for 10 min, washed, and fixed. GM1 was labeled with the fluorescent B subunit of cholera toxin (A) and F-actin was labeled with Alexa Fluor 647 (A′). The images B and C represent the CFP fluorescence, and A′′, B′′, and C′′ correspond to the phase contrast images. Bacteria were labeled with DAPI (B′ and C′). (B) Non-transfected Vero cells (A and B), Vero cells transfected with CFP-MyrPalm (C), with CFP-GerGer (D), or with GFP-GPI (E), were incubated with InlB-coated beads for 10 min, washed, and fixed. Cells were then labeled for GM1 (A and B), for HGF-R (A′, C′, D′, and E′), for transferrin receptor (B′), and for F-actin (E′′). A′′, B′′, C′′, and D′′ correspond to phase contrast images. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172418&req=5

fig4: Recruitment of raft markers during InlB-dependent entry. (A) Non-transfected Vero cells (A) and Vero cells transfected with CFP-MyrPalm (B) or with CFP-GerGer (C), were incubated with L. monocytogenes (BUG 1641) for 10 min, washed, and fixed. GM1 was labeled with the fluorescent B subunit of cholera toxin (A) and F-actin was labeled with Alexa Fluor 647 (A′). The images B and C represent the CFP fluorescence, and A′′, B′′, and C′′ correspond to the phase contrast images. Bacteria were labeled with DAPI (B′ and C′). (B) Non-transfected Vero cells (A and B), Vero cells transfected with CFP-MyrPalm (C), with CFP-GerGer (D), or with GFP-GPI (E), were incubated with InlB-coated beads for 10 min, washed, and fixed. Cells were then labeled for GM1 (A and B), for HGF-R (A′, C′, D′, and E′), for transferrin receptor (B′), and for F-actin (E′′). A′′, B′′, C′′, and D′′ correspond to phase contrast images. Bar, 10 μm.
Mentions: To study the InlB pathway, we infected Vero epithelial cells with L. monocytogenes overexpressing an InlB protein covalently anchored at the bacterial surface or incubated the cells with InlB-coated beads. As presented in Fig. 4 A, L. monocytogenes corecruited F-actin and the lipid raft marker GM1. The CFP-MyrPalm, but not the CFP-GerGer, was recruited at the bacterial entry site. We repeated these experiments with InlB-coated beads, and as shown in Fig. 4 B, GM1 and HGF-R were corecruited, whereas the transferrin receptor was not. HGF-R colocalized with the CFP-MyrPalm but not with CFP-GerGer, and finally the GFP-GPI chimera was corecruited with the HGF-R and F-actin. These results clearly showed that lipid raft markers are recruited at both internalin and InlB entry site.

Bottom Line: We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry.In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent.Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

View Article: PubMed Central - PubMed

Affiliation: Unité des Interactions Bactéries-Cellules, INSERM U604, Institut Pasteur, 75015 Paris Cedex 15, France.

ABSTRACT
Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

Show MeSH
Related in: MedlinePlus