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First published online 2 August 2005
doi: 10.1242/jcs.02514

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Invasion of endothelial cells by Neisseria meningitidis requires cortactin recruitment by a phosphoinositide-3-kinase/Rac1 signalling pathway triggered by the lipo-oligosaccharide

¹ Département de Biologie Cellulaire, Institut Cochin, INSERM U567, CNRS UMR8104, Université Paris 5 – René Descartes, 22 rue Méchain, 75014 Paris, France
² Laboratoire de Microbiologie, INSERM U570, Faculté de Médecine Necker-Enfants, Malades, 156 rue de Vaugirard, 75015 Paris, France

View larger version (22K): [in a new window]   Fig. 1. LOS is required for the efficient internalization of N. meningitidis into human endothelial cells. (A) LOS structures of the wild-type, rfaC, lgtA and lgtE mutant strains. The inner core structure of the LOS molecule of N. meningitidis consists of two molecules of 3-deoxy-D-manno-2-octulosonic acid (Kdo) attached to a lipid A. The oligosaccharide core consists of two heptose residues attached to the Kdo by the heptosyltransferase I RfaC and a lacto-N-neotetraose -chain added by glycosyl transferases such as LgtA or LgtE. LOS can be further modified by the addition of terminal sialic-acid (NeuNAc) moieties. By inactivating rfaC, the rfaC mutant strains produced contain only the lipid A moiety and the two Kdos of the LOS structure. lgtA and lgtE mutants express a truncated oligosaccharide structure. (B) HBMECs were infected with the wild-type (WT) or the isogenic rfaC (rfaC), lgtA (lgtA), lgtE (lgtE) or mtrC (mtrC) defective mutants of the 2C43 strain of N. meningitidis. After 30 minutes of contact, the number of adherent bacteria in relation to the number of bacteria contained in the inocula was determined (left). After 3 hours, the number of internalized bacteria in relation to the number of adherent bacteria was determined (right). Average values (± s.e.m.) from one representative experiment out of four independent experiments performed in triplicate are presented.

View larger version (40K): [in a new window]   Fig. 2. LOS is required for the formation of actin-rich cell projections promoting bacterial uptake and for cortactin recruitment. HBMECs infected for 3 hours with the 2C43 wild-type strain of N. meningitidis (WT) or the isogenic rfaC-defective mutant strains (rfaC) were double stained for actin (red) and ezrin (green) (A), for ErbB2 (green) and bacteria (red) (B) or for cortactin (green) and bacteria (red) (C), and were analysed by confocal microscopy. Merged images of the same fields are presented on the right-hand panels (overlay). (D) The frequency of formation of actin-rich cell projections or of bundle of actin filaments and of cortactin recruitment at the entry site of WT, rfaC or lgtA bacterial strains was determined by counting 50 cells. Average values (± s.e.m.) are presented from four independent experiments.

View larger version (29K): [in a new window]   Fig. 3. LOS is required for cortactin tyrosine phosphorylation but not for ErbB2 or Src kinase activation. HBMECs (starved for 24 hours) were either not infected (–) or infected for 3 hours with the wild-type (WT) or isogenic rfaC (rfaC), lgtA (lgtA), lgtE (lgtE) or mtrC (mtrC) defective mutants of the 2C43 or ROU strains of N. meningitidis, as indicated. Inocula were adjusted to obtain similar adhesion events between wild-type and mutant bacteria. After lysis, ErbB2 receptor was immunoprecipitated and immunoblotted with an anti-phosphotyrosine antibody (PY) (A, top), Src was immunoprecipitated and subjected to an in-vitro kinase assay using acid-denatured enolase as a substrate (A, middle), or cortactin was immunoprecipitated and immunoblotted with an anti-phosphotyrosine antibody and the blot was reprobed with an anti-cortactin antibody to confirm that similar protein levels were immunoprecipitated (A, bottom, B). (C) Quantification by densitometry analysis (using NIH Image software) of cortactin phosphorylation induced by the wild-type and mutants of the 2C43 strain. Average values (± s.e.m.) are presented from four independent experiments.

View larger version (78K): [in a new window]   Fig. 4. Cortactin tyrosine phosphorylation induced by N. meningitidis is required for the formation of actin-rich cell projections promoting bacterial uptake. HBMECs transiently transfected with GFP alone, wild-type GFP-cortactin or a GFP-tagged cortactin mutant deficient in tyrosine phosphorylation (GFP-cortactinY/F), as indicated, were infected for 3 hours with the 2C43 wild-type strain of N. meningitidis. (A) Cells were then double stained for actin and ezrin, and analysed by confocal microscopy. (right) Higher magnification of the inset in the middle panels. (B) The frequency of formation of either actin-rich cell projections or bundles of actin filaments at bacterial entry sites in cells expressing high levels of both GFP-cortactin and GFP-cortactinY/F was determined by counting 40 cells. Average values (± s.e.m.) are presented from four independent experiments.

View larger version (43K): [in a new window]   Fig. 5. LOS induces the activation of Rac1 GTPase and PI3K. HBMECs, transiently transfected with wild-type Rac1 coupled to GFP (GFP-Rac1), with the GTPase-binding domain of PAK1 coupled to GFP (GFP-PAK1-PBD) or with the PH domain of Akt coupled to GFP (GFP-Akt-PH) were infected for 3 hours with either the 2C43 wild type (WT) or the isogenic rfaC (rfaC) or lgtA (lgtA) defective mutants of N. meningitidis. When indicated (WT + wortmannin), HBMECs were pretreated for 2 hours with 100 ng ml–1 wortmannin and then infected with the 2C43 wild-type strain of N. meningitidis in the presence of the inhibitor. (A-C) Cells were then stained for bacteria or ezrin (red) and recruitment of the different GFP-tagged proteins at bacterial entry sites was analysed by confocal microscopy. (right) Merged images (overlay) of the same fields. (D) The frequency of recruitment of the different GFP-tagged proteins at the entry site of WT, rfaC or lgtA bacterial strains was determined by counting 50 cells. Average values (± s.e.m.) are presented from three independent experiments.

View larger version (65K): [in a new window]   Fig. 6. The activation of Rac1 GTPase is required for cortactin recruitment and the formation of actin-rich cell projections promoting bacterial uptake. (A) The upper row (a) shows a cell expressing the GFP-Rac1-DN and the lower row (b) shows a nontransfected control cell. Both were infected with the bacterial wild-type strain. HBMECs transiently transfected with the dominant-negative form of Rac1 coupled to GFP (GFP-Rac1DN) were infected for 3 hours with the 2C43 wild-type strain of N. meningitidis. Cells were then double stained for actin (red) and cortactin (blue), and were analysed by confocal microscopy. (right) Merged images (overlay) of the same fields. (B) The frequency of formation of either actin-rich cell projections or bundle of actin filaments at bacterial entry sites in both control and Rac1-DN-expressing cells was determined by counting 40 cells. Average values (± s.e.m.) are presented from three independent experiments.

View larger version (57K): [in a new window]   Fig. 7. Activation of PI3K is required for the recruitment and phosphorylation of cortactin and bacterial internalisation. (A,B) HBMECs were pretreated or not for 2 hours with 100 ng ml–1 wortmannin and then infected for 3 hours with the 2C43 wild-type strain of N. meningitidis. (A) Cells were triple stained for ezrin (green), cortactin (blue) and actin (red), and were analysed by confocal microscopy. (right) Merged images (overlay) of the same fields. (B) Cortactin was immunoprecipitated and immunoblotted with an anti-phosphotyrosine antibody (PY). (C) HBMECs were either left untreated (–) or pretreated for 2 hours with 100 ng ml–1 wortmannin (+) before infection with the 2C43 wild-type strain of N. meningitidis (WT) or with the isogenic rfaC-defective mutant strain (rfaC) in the presence or absence of the inhibitor. After 3 hours, the number of internalized bacteria in relation to the number of adherent bacteria was determined. Average values (± s.e.m.) are presented from one representative experiment out of four independent experiments performed in triplicate.

View larger version (26K): [in a new window]   Fig. 8. Schematic representation of the signalling pathways activated by N. meningitidis and involved in bacterial entry into endothelial cells. Type-IV pili initiate the interaction of virulent, encapsulated N. meningitidis with human endothelial cells by interacting with a cellular receptor, possibly CD46 (Kallstrom et al., 1997). This pilus-dependent adhesion induces the recruitment of ezrin and the clustering of several transmembrane proteins: the ErbB2 tyrosine-kinase receptor and the ezrin-binding proteins CD44 and ICAM-1. The activation of both Rho and Cdc42 GTPases induces a local polymerization of cortical actin. ErbB2 clustering leads to the activation of Src tyrosine kinase. In parallel, LOS of N. meningitidis, by a mechanism which remains to be identified, provides a co-stimulatory signal leading to PI3K and Rac1 activation, and the subsequent translocation of cortactin to site of cortical actin rearrangements. When localized to the cell plasma membrane, cortactin is tyrosine phosphorylated by Src kinase and contributes to the formation of dynamic actin structures, leading to the formation of membrane projections that surround bacteria and provoke their internalization within endothelial intracellular vacuoles

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