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Microvilli-like structures are associated with the internalization of virulent capsulated Neisseria meningitidis into vascular endothelial cells

Emmanuel Eugène1, Isabelle Hoffmann2, Céline Pujol1, Pierre-Olivier Couraud2, Sandrine Bourdoulous2 and Xavier Nassif1,*

1 INSERM U411, Faculté de Médecine Necker-Enfants Malades, Université René Descartes, Paris, France
2 CNRS UPR 415, Institut Cochin de Génétique Moléculaire, Paris, France



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  		Fig. 1. Neisseria meningitidis induces localized cellular membrane protrusions. SEM examination of HUVEC monolayers infected with the ROU strain of N. meningitidis. Bars represent 1µm. (A) After 4 hours of infection, adhering bacteria proliferate and form small colonies at their site of attachment (localized adhesion). During this step, small protrusions of the cellular membrane are induced underneath bacterial colonies. (B) Detail showing cellular protrusions engulfing a bacterium during the localized adhesion step of the infection. (C) After 6 hours, meningococci start to spread over the apical cell surface (diffuse adhesion). (D) Higher magnification showing that the membrane protrusions have disappeared at the late stage of infection.

 


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  		Fig. 2. Cellular membrane protrusions induced by N. meningitidis lead to bacteria engulfment within host cells. (A-D) TEM photographs of a HUVEC monolayer infected by N. meningitidis for 4 hours (localized adhesion). A,B,C show cellular protrusions engulfing bacteria. Bars represent 1 µm.

 


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  		Fig. 3. Adhesion versus internalization of N. meningitidis. Approximately 107 bacteria (ROU strain of N. meningitidis) in culture medium were added to the HUVECs and allowed to adhere for 15-30 minutes. The medium was changed every hour to avoid reinfection from the supernatant. At the indicated times (4 hours or 8 hours), monolayers were harvested and adherent bacteria counted. The number of internalized bacteria was determined by gentamicin protection assay, as described in Materials and Methods. Results are in Log CFU/ml.

 


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  		Fig. 4. Cellular protrusions induced by N. meningitidis are associated with ezrin recruitment and actin polymerization. Monolayers of HUVECs were infected for 4 hours with the ROU strain of N. meningitidis. A,B,C: xy sections performed by confocal microscopy of double fluorescence labeling ((A) bacteria and F-actin) or triple fluorescence labeling ((B) bacteria, F-actin and paxillin staining; (C) F-actin, ezrin and CD44) in the same fields. (D) xz section of a double fluorescence labeling of F-actin and ezrin. (A,C,D): x200; (B) x63.

 


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  		Fig. 5. Ezrin recruitment is responsible for actin polymerization. (A) 2 hours prior to infection, HUVECs were microinjected with pCB6-Nter-Ezrin-VSVG, encoding the VSVG-tagged truncated N-terminal domain of ezrin, as a dominant-negative form of ezrin. Cells were infected with the ROU strain of N. meningitidis for 4 hours and then labeled for F-actin, endogenous ezrin and VSVG-tag (Nter-ezrin). In the transfected cells a large amount of VSVG-labeled truncated ezrin was recruited below the bacterial colonies (top right) without actin polymerization (top left). Endogenous ezrin (bottom right) was recruited underneath bacterial colonies in non-transfected cells. Transfection with the dominant-negative form almost completely prevented the recruitment of endogenous ezrin. (B) Bacterial colonies with polymerized actin and/or recruited endogenous ezrin were counted by immunofluorescence analysis in non-transfected cells or cells transfected with a plasmid encoding the VSVG-tagged truncated amino-terminal domain of ezrin (pCB6-ezrin-Nter). These data correspond to the results of at least six independent experiments. Results are expressed in percent of adherent colonies. (C) Bacterial colonies with polymerized actin were counted by immunofluorescence analysis in HUVECs that were transfected with a plasmid encoding full length ezrin (pCB6-ezrin) or the VSVG-tagged truncated amino-terminal domain of ezrin (pCB6-ezrin-Nter). Results are expressed in percent of adherent colonies.

 


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  		Fig. 6. Treatment by C. difficile toxin B prevents actin polymerization but not ezrin recruitment induced by N. meningitidis. (A,B) Confluent monolayers of HUVECs were either left untreated (control) or treated with 1 ng/ml of C. difficile toxin B (ToxB) in starvation medium for 16 hours prior to the infection. Control and Tox-B-treated cells were then infected with the ROU strain of N. meningitidis for 4 hours. Tox B treatment was maintained during infection. Cells were double stained for F-actin and ezrin and the percentage of bacterial colonies recruiting ezrin (A) or F-actin-positive (B) was determined by immunofluorescence analysis. Results correspond to counts of 300 bacterial colonies in three distinct experiments. (C) xy section of double fluorescence labeling of F-actin (middle panel) and ezrin (right panel) in the same field. Analysis was performed by confocal microscopy at magnification x63. The localization of the bacterial colony in the same field is shown by phase contrast (left panel).

 


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  		Fig. 7. Effect of GTPase inhibition on actin polymerization induced by N. meningitidis. (A) HUVECs were infected for 4 hours with the ROU strain of N. meningitidis. Cells were labeled for bacteria, F-actin and RhoA, and xz section analysis was performed by confocal microscopy at magnification x300.

(B) HUVECs, pretreated with the Rho kinase inhibitor Y27632 at 30 µM in starvation medium for 1 hour, were infected for 4 hours in the presence of the inhibitor and stained for actin (right panel, xy section at magnification x63). The localization of the bacterial colony in the same field is shown by Nomarsky (left panel).

(C) HUVECs were microinjected with the cDNA encoding the dominant-negative form of Cdc42 (Cdc42N17) coupled to a myc tag, 5 hours prior to cell infection. Cells were then labeled for bacteria, F-actin and the myc tag. xy sections were performed by confocal microscopy at magnification x100. xz sections showed in the lower panels were performed along the indicated lines of the transfected cell and the non transfected cell as control at magnification x100.

(D) HUVECs were microinjected with the cDNA encoding the dominant-negative form of Rac1 (Rac1N17) coupled to a myc tag, 18 hours prior to cell infection. Cells were then labeled for bacteria, actin and the myc tag. xy sections were performed by confocal microscopy at magnification x63. The arrows in D localize the polymerized actin visible either on control cell or microinjected cell.

 


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  		Fig. 8. Rho and Cdc42, but not Rac 1, are involved in actin polymerization induced by N. meningitidis. Confluent monolayers of HUVECs were either left untreated (control), treated with 30 µM Y27632 prior to the infection and during the infection (Y27632), microinjected with the recombinant exoenzyme C3 of C. botulinum 2 hours before cell infection (C3) or microinjected with the cDNA encoding a dominant-negative form of Rac (RacN17) or Cdc42 (Cdc42N17) 18 hours or 5 hours prior to infection of the monolayer, respectively. Cells were then infected for 4 hours and labeled for bacteria, F-actin and myc tag. The percentages of bacterial colonies associated with polymerized actin were determined by immunofluorescence analysis. Each condition was compared with non-treated cells infected in the same conditions. Results correspond to counts of 1600 infected cells in 3 to 10 distinct experiments per condition.

 


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  		Fig. 9. Rho GTPases are involved in N. meningitidis internalization into endothelial host cells. HBMECs were either left untreated (control) or pretreated for 18 hours with 1 ng/ml of C. difficile toxin B (ToxB) or for 2 hours with 30 µM Y27632 (Y27632). Cells were infected with the ROU strain of N. meningitidis for 4 hours in the presence of the inhibitors, and the number of adherent bacteria (A) and internalized bacteria (B) were determined as in Fig. 3. These experiments, carried out in triplicates, were performed four times independently and results from one representative experiment are shown.

 




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