Limits...
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.

Show MeSH

Related in: MedlinePlus

Cholesterol-dependent entry of L. monocytogenes in mammalian cells. Control, cholesterol-depleted, and cholesterol-repleted L2071hEcad or Vero cells were incubated at 37°C for 30 min (L2071hEcad) or 1 h (Vero) with bacteria at a multiplicity of infection = 50. 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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172418&req=5

fig1: Cholesterol-dependent entry of L. monocytogenes in mammalian cells. Control, cholesterol-depleted, and cholesterol-repleted L2071hEcad or Vero cells were incubated at 37°C for 30 min (L2071hEcad) or 1 h (Vero) with bacteria at a multiplicity of infection = 50. 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).

Mentions: We investigated if cholesterol-enriched membrane microdomains could be implicated in L. monocytogenes entry into nonphagocytic mammalian cells. We used methyl-β-cyclodextrin (MβCD), a water-soluble cyclic oligosaccharide (Kilsdonk et al., 1995) to deplete selectively membrane cholesterol and disrupt lipid domains in the fibroblastic cell line L2071hEcad, and in the Vero epithelial cell line (two cell lines that we routinely use in our invasion studies). The L2071hEcad cells express E-cadherin and HGF-R and consequently allow internalin- and InlB-mediated entry. The Vero cell line expresses HGF-R but not E-cadherin limiting entry to InlB-mediated mechanism. Cells were depleted with 10 mM MβCD for 1 h (L2071hEcad) or for 30 min (Vero cells) and were assayed for bacterial entry by the gentamicin assay. As shown in Fig. 1, for wild-type L. monocytogenes (EGD strain), internalin- and InlB-mediated entry into L2071hEcad cells and InlB-mediated entry into Vero cells were strongly inhibited after cholesterol depletion. After cholesterol depletion cellular viability, permeability to gentamicin, and cellular adherence were not affected (unpublished data). The use of lower concentration of MβCD (5 mM) also decreased bacterial entry by 50% in both cell lines (unpublished data). To further demonstrate that inhibition of bacterial entry was specifically due to cholesterol depletion and not to a secondary effect of the MβCD, cells were first depleted with MβCD and cholesterol was then added back to the membrane with cholesterol-chelated MβCD. As shown in Fig. 1, cholesterol repletion restored bacterial uptake.


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-dependent entry of L. monocytogenes in mammalian cells. Control, cholesterol-depleted, and cholesterol-repleted L2071hEcad or Vero cells were incubated at 37°C for 30 min (L2071hEcad) or 1 h (Vero) with bacteria at a multiplicity of infection = 50. 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).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Cholesterol-dependent entry of L. monocytogenes in mammalian cells. Control, cholesterol-depleted, and cholesterol-repleted L2071hEcad or Vero cells were incubated at 37°C for 30 min (L2071hEcad) or 1 h (Vero) with bacteria at a multiplicity of infection = 50. 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).
Mentions: We investigated if cholesterol-enriched membrane microdomains could be implicated in L. monocytogenes entry into nonphagocytic mammalian cells. We used methyl-β-cyclodextrin (MβCD), a water-soluble cyclic oligosaccharide (Kilsdonk et al., 1995) to deplete selectively membrane cholesterol and disrupt lipid domains in the fibroblastic cell line L2071hEcad, and in the Vero epithelial cell line (two cell lines that we routinely use in our invasion studies). The L2071hEcad cells express E-cadherin and HGF-R and consequently allow internalin- and InlB-mediated entry. The Vero cell line expresses HGF-R but not E-cadherin limiting entry to InlB-mediated mechanism. Cells were depleted with 10 mM MβCD for 1 h (L2071hEcad) or for 30 min (Vero cells) and were assayed for bacterial entry by the gentamicin assay. As shown in Fig. 1, for wild-type L. monocytogenes (EGD strain), internalin- and InlB-mediated entry into L2071hEcad cells and InlB-mediated entry into Vero cells were strongly inhibited after cholesterol depletion. After cholesterol depletion cellular viability, permeability to gentamicin, and cellular adherence were not affected (unpublished data). The use of lower concentration of MβCD (5 mM) also decreased bacterial entry by 50% in both cell lines (unpublished data). To further demonstrate that inhibition of bacterial entry was specifically due to cholesterol depletion and not to a secondary effect of the MβCD, cells were first depleted with MβCD and cholesterol was then added back to the membrane with cholesterol-chelated MβCD. As shown in Fig. 1, cholesterol repletion restored bacterial uptake.

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