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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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Cholesterol depletion does not affect HGF-R recruitment or initial membrane recruitment at the InlB entry site but subsequent closure of the phagocytic cup. (A) Control and cholesterol-depleted Vero cells were incubated for 30 min with InlB-coated beads. Cells were washed three times, fixed, permeabilized, and labeled for HGF-R (clone DL21; Alexa Fluor 546–conjugated secondary antibodies) and for F-actin (Alexa Fluor 488 phalloidin). Phase-contrast and fluorescent images were acquired with a 100× objective. Arrows indicate the InlB-coated beads that recruited HGF-R. Arrowheads indicate the InlB-coated beads that recruited HGF-R and F-actin. Bar, 10 μm. (B) Percentage of InlB-coated beads which have recruited HGF-R. (C) Control and cholesterol-depleted Vero cells were incubated for 5, 15, and 30 min with InlB-coated beads. Cells were washed three times and processed for scanning EM as described in Materials and methods. Extracellular portion of the beads were colorized using the Adobe photoshop software. Bar, 1 μm.
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fig8: Cholesterol depletion does not affect HGF-R recruitment or initial membrane recruitment at the InlB entry site but subsequent closure of the phagocytic cup. (A) Control and cholesterol-depleted Vero cells were incubated for 30 min with InlB-coated beads. Cells were washed three times, fixed, permeabilized, and labeled for HGF-R (clone DL21; Alexa Fluor 546–conjugated secondary antibodies) and for F-actin (Alexa Fluor 488 phalloidin). Phase-contrast and fluorescent images were acquired with a 100× objective. Arrows indicate the InlB-coated beads that recruited HGF-R. Arrowheads indicate the InlB-coated beads that recruited HGF-R and F-actin. Bar, 10 μm. (B) Percentage of InlB-coated beads which have recruited HGF-R. (C) Control and cholesterol-depleted Vero cells were incubated for 5, 15, and 30 min with InlB-coated beads. Cells were washed three times and processed for scanning EM as described in Materials and methods. Extracellular portion of the beads were colorized using the Adobe photoshop software. Bar, 1 μm.

Mentions: It has been shown that InlB-coated beads or bacteria induce HGF-R recruitment at the entry site, tyrosine phosphorylation of HGF-R, and the activation of signaling cascades leading to F-actin polymerization (Bierne and Cossart, 2002). We first analyzed the ability of InlB-coated beads to trigger HGF-R recruitment after cholesterol depletion. Control and cholesterol-depleted Vero cells were incubated for 30 min with InlB-coated beads, fixed, permeabilized, and labeled for HGF-R. As shown in Fig. 8 A, HGF-R recruitment around the InlB-coated beads could be observed for control as well as for cholesterol-depleted cells. We quantified this result by calculating the percentage of InlB-coated beads which had recruited detectable amounts of HGF-R. No significant difference between control and cholesterol-depleted cells was observed (Fig. 8 B).


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)

Cholesterol depletion does not affect HGF-R recruitment or initial membrane recruitment at the InlB entry site but subsequent closure of the phagocytic cup. (A) Control and cholesterol-depleted Vero cells were incubated for 30 min with InlB-coated beads. Cells were washed three times, fixed, permeabilized, and labeled for HGF-R (clone DL21; Alexa Fluor 546–conjugated secondary antibodies) and for F-actin (Alexa Fluor 488 phalloidin). Phase-contrast and fluorescent images were acquired with a 100× objective. Arrows indicate the InlB-coated beads that recruited HGF-R. Arrowheads indicate the InlB-coated beads that recruited HGF-R and F-actin. Bar, 10 μm. (B) Percentage of InlB-coated beads which have recruited HGF-R. (C) Control and cholesterol-depleted Vero cells were incubated for 5, 15, and 30 min with InlB-coated beads. Cells were washed three times and processed for scanning EM as described in Materials and methods. Extracellular portion of the beads were colorized using the Adobe photoshop software. Bar, 1 μm.
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fig8: Cholesterol depletion does not affect HGF-R recruitment or initial membrane recruitment at the InlB entry site but subsequent closure of the phagocytic cup. (A) Control and cholesterol-depleted Vero cells were incubated for 30 min with InlB-coated beads. Cells were washed three times, fixed, permeabilized, and labeled for HGF-R (clone DL21; Alexa Fluor 546–conjugated secondary antibodies) and for F-actin (Alexa Fluor 488 phalloidin). Phase-contrast and fluorescent images were acquired with a 100× objective. Arrows indicate the InlB-coated beads that recruited HGF-R. Arrowheads indicate the InlB-coated beads that recruited HGF-R and F-actin. Bar, 10 μm. (B) Percentage of InlB-coated beads which have recruited HGF-R. (C) Control and cholesterol-depleted Vero cells were incubated for 5, 15, and 30 min with InlB-coated beads. Cells were washed three times and processed for scanning EM as described in Materials and methods. Extracellular portion of the beads were colorized using the Adobe photoshop software. Bar, 1 μm.
Mentions: It has been shown that InlB-coated beads or bacteria induce HGF-R recruitment at the entry site, tyrosine phosphorylation of HGF-R, and the activation of signaling cascades leading to F-actin polymerization (Bierne and Cossart, 2002). We first analyzed the ability of InlB-coated beads to trigger HGF-R recruitment after cholesterol depletion. Control and cholesterol-depleted Vero cells were incubated for 30 min with InlB-coated beads, fixed, permeabilized, and labeled for HGF-R. As shown in Fig. 8 A, HGF-R recruitment around the InlB-coated beads could be observed for control as well as for cholesterol-depleted cells. We quantified this result by calculating the percentage of InlB-coated beads which had recruited detectable amounts of HGF-R. No significant difference between control and cholesterol-depleted cells was observed (Fig. 8 B).

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