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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 internalin-dependent entry. Non-transfected Lovo cells (A), Lovo cells transfected with CFP-MyrPalm (B), with CFP-GerGer (C), or with GFP-GPI (D), and epithelial Rov9 cells stably expressing the PrP (E), were incubated with L. innocua expressing internalin (BUG 1489) for 10 min, washed and fixed. The images B–D represent the CFP or GFP fluorescence, and the images A′, B′, C′, D′′, and E′′ correspond to phase contrast images. GM1 was labeled with fluorescent B subunit of cholera toxin (A). F-actin was labeled with Alexa Fluor 546 (D′). PrP (E) and internalin (E′) were labeled with mAbs (clone L7.7 and clone 4F2) and secondary fluorescent antibodies. Note that L. innocua expressing internalin, in some cases, appears as chains. Bars, 10 μm.
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fig3: Recruitment of raft markers during internalin-dependent entry. Non-transfected Lovo cells (A), Lovo cells transfected with CFP-MyrPalm (B), with CFP-GerGer (C), or with GFP-GPI (D), and epithelial Rov9 cells stably expressing the PrP (E), were incubated with L. innocua expressing internalin (BUG 1489) for 10 min, washed and fixed. The images B–D represent the CFP or GFP fluorescence, and the images A′, B′, C′, D′′, and E′′ correspond to phase contrast images. GM1 was labeled with fluorescent B subunit of cholera toxin (A). F-actin was labeled with Alexa Fluor 546 (D′). PrP (E) and internalin (E′) were labeled with mAbs (clone L7.7 and clone 4F2) and secondary fluorescent antibodies. Note that L. innocua expressing internalin, in some cases, appears as chains. Bars, 10 μm.

Mentions: To study the internalin pathway, we infected Lovo epithelial cells with L. innocua, which expresses internalin. Lovo cells are permissive for both internalin- and InlB-mediated entry (Pizarro-Cerda et al., 2004), are more easily transfected, and more efficiently labeled by the B subunit of cholera toxin than the L2071hEcad cells. Our results show that L. innocua expressing internalin recruited at the plasma membrane the raft markers GM1 and CFP-MyrPalm, but not the nonraft marker CFP-GerGer (Fig. 3). The raft markers GFP-GPI, and PrP were also recruited at the bacterial entry site (Fig. 3).


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 internalin-dependent entry. Non-transfected Lovo cells (A), Lovo cells transfected with CFP-MyrPalm (B), with CFP-GerGer (C), or with GFP-GPI (D), and epithelial Rov9 cells stably expressing the PrP (E), were incubated with L. innocua expressing internalin (BUG 1489) for 10 min, washed and fixed. The images B–D represent the CFP or GFP fluorescence, and the images A′, B′, C′, D′′, and E′′ correspond to phase contrast images. GM1 was labeled with fluorescent B subunit of cholera toxin (A). F-actin was labeled with Alexa Fluor 546 (D′). PrP (E) and internalin (E′) were labeled with mAbs (clone L7.7 and clone 4F2) and secondary fluorescent antibodies. Note that L. innocua expressing internalin, in some cases, appears as chains. Bars, 10 μm.
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Related In: Results  -  Collection

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fig3: Recruitment of raft markers during internalin-dependent entry. Non-transfected Lovo cells (A), Lovo cells transfected with CFP-MyrPalm (B), with CFP-GerGer (C), or with GFP-GPI (D), and epithelial Rov9 cells stably expressing the PrP (E), were incubated with L. innocua expressing internalin (BUG 1489) for 10 min, washed and fixed. The images B–D represent the CFP or GFP fluorescence, and the images A′, B′, C′, D′′, and E′′ correspond to phase contrast images. GM1 was labeled with fluorescent B subunit of cholera toxin (A). F-actin was labeled with Alexa Fluor 546 (D′). PrP (E) and internalin (E′) were labeled with mAbs (clone L7.7 and clone 4F2) and secondary fluorescent antibodies. Note that L. innocua expressing internalin, in some cases, appears as chains. Bars, 10 μm.
Mentions: To study the internalin pathway, we infected Lovo epithelial cells with L. innocua, which expresses internalin. Lovo cells are permissive for both internalin- and InlB-mediated entry (Pizarro-Cerda et al., 2004), are more easily transfected, and more efficiently labeled by the B subunit of cholera toxin than the L2071hEcad cells. Our results show that L. innocua expressing internalin recruited at the plasma membrane the raft markers GM1 and CFP-MyrPalm, but not the nonraft marker CFP-GerGer (Fig. 3). The raft markers GFP-GPI, and PrP were also recruited at the bacterial entry site (Fig. 3).

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