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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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Internalin- and InlB-mediated entry require membrane cholesterol independently of the presence of the cholesterol-binding toxin LLO. (A) Control (−) and cholesterol-depleted (+) L2071hEcad or Vero cells were incubated at 37°C with the EGD isogenic mutant strains (EGDΔInlB, EGDΔInlA, or EGDhly::Tn917) for 30 min (L2071hEcad) or 1 h (Vero). The number of intracellular bacteria (EGD strain) per cell was quantified by the gentamicin assay and results were expressed relative to control nondepleted cells (mean of at least three independent experiments). (B) Control (−) and cholesterol-depleted (+) cells were incubated for 30 min at 37°C with InlB- or internalin-coated latex beads. After three washes, cells were fixed and extracellular beads were fluorescently labeled. The percent of intracellular beads per cell was quantified by microscopy. Results of at least three independent experiments were expressed relative to control nondepleted cells.
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fig2: Internalin- and InlB-mediated entry require membrane cholesterol independently of the presence of the cholesterol-binding toxin LLO. (A) Control (−) and cholesterol-depleted (+) L2071hEcad or Vero cells were incubated at 37°C with the EGD isogenic mutant strains (EGDΔInlB, EGDΔInlA, or EGDhly::Tn917) for 30 min (L2071hEcad) or 1 h (Vero). The number of intracellular bacteria (EGD strain) per cell was quantified by the gentamicin assay and results were expressed relative to control nondepleted cells (mean of at least three independent experiments). (B) Control (−) and cholesterol-depleted (+) cells were incubated for 30 min at 37°C with InlB- or internalin-coated latex beads. After three washes, cells were fixed and extracellular beads were fluorescently labeled. The percent of intracellular beads per cell was quantified by microscopy. Results of at least three independent experiments were expressed relative to control nondepleted cells.

Mentions: To analyze further the effect of cholesterol depletion on internalin- and InlB-dependent uptake, we used two isogenic mutant strains, EGDΔInlB and EGDΔInlA that only express internalin or InlB, respectively. As shown in Fig. 2 A, internalin- and InlB-mediated entry into L2071hEcad cells were cholesterol dependent.


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)

Internalin- and InlB-mediated entry require membrane cholesterol independently of the presence of the cholesterol-binding toxin LLO. (A) Control (−) and cholesterol-depleted (+) L2071hEcad or Vero cells were incubated at 37°C with the EGD isogenic mutant strains (EGDΔInlB, EGDΔInlA, or EGDhly::Tn917) for 30 min (L2071hEcad) or 1 h (Vero). The number of intracellular bacteria (EGD strain) per cell was quantified by the gentamicin assay and results were expressed relative to control nondepleted cells (mean of at least three independent experiments). (B) Control (−) and cholesterol-depleted (+) cells were incubated for 30 min at 37°C with InlB- or internalin-coated latex beads. After three washes, cells were fixed and extracellular beads were fluorescently labeled. The percent of intracellular beads per cell was quantified by microscopy. Results of at least three independent experiments were expressed relative to control nondepleted cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172418&req=5

fig2: Internalin- and InlB-mediated entry require membrane cholesterol independently of the presence of the cholesterol-binding toxin LLO. (A) Control (−) and cholesterol-depleted (+) L2071hEcad or Vero cells were incubated at 37°C with the EGD isogenic mutant strains (EGDΔInlB, EGDΔInlA, or EGDhly::Tn917) for 30 min (L2071hEcad) or 1 h (Vero). The number of intracellular bacteria (EGD strain) per cell was quantified by the gentamicin assay and results were expressed relative to control nondepleted cells (mean of at least three independent experiments). (B) Control (−) and cholesterol-depleted (+) cells were incubated for 30 min at 37°C with InlB- or internalin-coated latex beads. After three washes, cells were fixed and extracellular beads were fluorescently labeled. The percent of intracellular beads per cell was quantified by microscopy. Results of at least three independent experiments were expressed relative to control nondepleted cells.
Mentions: To analyze further the effect of cholesterol depletion on internalin- and InlB-dependent uptake, we used two isogenic mutant strains, EGDΔInlB and EGDΔInlA that only express internalin or InlB, respectively. As shown in Fig. 2 A, internalin- and InlB-mediated entry into L2071hEcad cells were cholesterol dependent.

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