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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 affects differently the internalin–E-cadherin and the InlB–HGF-R interactions. Control (−) and cholesterol-depleted (+) cells were incubated for 30 min at 37°C with internalin- or with InlB-coated beads. After three washes cells were fixed and extracellular beads were immunolabeled. The total number of beads (extracellular plus intracellular) associated with the cells, and the number of extracellular beads associated to the cell surface, were assessed by microscopy. Results of at least three independent experiments were expressed relative to control nondepleted cells.
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fig5: Cholesterol depletion affects differently the internalin–E-cadherin and the InlB–HGF-R interactions. Control (−) and cholesterol-depleted (+) cells were incubated for 30 min at 37°C with internalin- or with InlB-coated beads. After three washes cells were fixed and extracellular beads were immunolabeled. The total number of beads (extracellular plus intracellular) associated with the cells, and the number of extracellular beads associated to the cell surface, were assessed by microscopy. Results of at least three independent experiments were expressed relative to control nondepleted cells.

Mentions: We analyzed which steps (adherence and/or internalization) of internalin–E-cadherin- and InlB–HGF-R–mediated uptake were affected by cholesterol depletion. To study the internalin- and InlB-mediated entry pathways independently and specifically, control and cholesterol-depleted cells were incubated with internalin- or InlB-coated beads for 30 min and then fixed. The total number of beads associated with the cells (adherent extracellular beads plus intracellular beads) was quantified by phase-contrast microscopy and the number of extracellular and intracellular beads was assessed by fluorescence microscopy (see Materials and methods). As seen in Fig. 5, after cholesterol depletion the total number of internalin-coated beads associated with L2071hEcad cells decreased, whereas the total number of InlB-coated beads associated with L2071hEcad or with Vero cells was not significantly affected. The number of extracellular internalin-coated beads interacting with L2071hEcad cells was reduced after cholesterol depletion, whereas cholesterol depletion of Vero cells had no effect on InlB-coated bead adherence. In conclusion, initial internalin-mediated attachment of the beads to the cell surface was strongly affected after cholesterol depletion. In contrast, InlB-mediated attachment at the cell surface did not significantly decrease after cholesterol depletion, but downstream events necessary for the phagocytic uptake were abrogated.


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 affects differently the internalin–E-cadherin and the InlB–HGF-R interactions. Control (−) and cholesterol-depleted (+) cells were incubated for 30 min at 37°C with internalin- or with InlB-coated beads. After three washes cells were fixed and extracellular beads were immunolabeled. The total number of beads (extracellular plus intracellular) associated with the cells, and the number of extracellular beads associated to the cell surface, were assessed 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

fig5: Cholesterol depletion affects differently the internalin–E-cadherin and the InlB–HGF-R interactions. Control (−) and cholesterol-depleted (+) cells were incubated for 30 min at 37°C with internalin- or with InlB-coated beads. After three washes cells were fixed and extracellular beads were immunolabeled. The total number of beads (extracellular plus intracellular) associated with the cells, and the number of extracellular beads associated to the cell surface, were assessed by microscopy. Results of at least three independent experiments were expressed relative to control nondepleted cells.
Mentions: We analyzed which steps (adherence and/or internalization) of internalin–E-cadherin- and InlB–HGF-R–mediated uptake were affected by cholesterol depletion. To study the internalin- and InlB-mediated entry pathways independently and specifically, control and cholesterol-depleted cells were incubated with internalin- or InlB-coated beads for 30 min and then fixed. The total number of beads associated with the cells (adherent extracellular beads plus intracellular beads) was quantified by phase-contrast microscopy and the number of extracellular and intracellular beads was assessed by fluorescence microscopy (see Materials and methods). As seen in Fig. 5, after cholesterol depletion the total number of internalin-coated beads associated with L2071hEcad cells decreased, whereas the total number of InlB-coated beads associated with L2071hEcad or with Vero cells was not significantly affected. The number of extracellular internalin-coated beads interacting with L2071hEcad cells was reduced after cholesterol depletion, whereas cholesterol depletion of Vero cells had no effect on InlB-coated bead adherence. In conclusion, initial internalin-mediated attachment of the beads to the cell surface was strongly affected after cholesterol depletion. In contrast, InlB-mediated attachment at the cell surface did not significantly decrease after cholesterol depletion, but downstream events necessary for the phagocytic uptake were abrogated.

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