Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization - PubMed (original) (raw)

doi: 10.1038/nm.3563. Epub 2014 Jun 1.

Nandi Simpson 2, Olivier Join-Lambert 3, Christian Federici 4, Marie-Pierre Laran-Chich 4, Nawal Maïssa 4, Haniaa Bouzinba-Ségard 4, Philippe C Morand 5, Fabrice Chretien 6, Saïd Taouji 7, Eric Chevet 7, Sébastien Janel 8, Frank Lafont 8, Mathieu Coureuil 9, Audrey Segura 9, Florence Niedergang 4, Stefano Marullo 4, Pierre-Olivier Couraud 4, Xavier Nassif 3, Sandrine Bourdoulous 4

Affiliations

Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization

Sandra C Bernard et al. Nat Med. 2014 Jul.

Abstract

Neisseria meningitidis is a cause of meningitis epidemics worldwide and of rapidly progressing fatal septic shock. A crucial step in the pathogenesis of invasive meningococcal infections is the adhesion of bloodborne meningococci to both peripheral and brain endothelia, leading to major vascular dysfunction. Initial adhesion of pathogenic strains to endothelial cells relies on meningococcal type IV pili, but the endothelial receptor for bacterial adhesion remains unknown. Here, we report that the immunoglobulin superfamily member CD147 (also called extracellular matrix metalloproteinase inducer (EMMPRIN) or Basigin) is a critical host receptor for the meningococcal pilus components PilE and PilV. Interfering with this interaction potently inhibited the primary attachment of meningococci to human endothelial cells in vitro and prevented colonization of vessels in human brain tissue explants ex vivo and in humanized mice in vivo. These findings establish the molecular events by which meningococci target human endothelia, and they open new perspectives for treatment and prevention of meningococcus-induced vascular dysfunctions.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. CD147 accumulates at sites of meningococcal adhesion independently of β2-AR activation.

(a) CD147 staining in noninfected and infected polarized monolayer of hCMEC/D3 cells. CD147 (green) and meningococcal colonies (red) in reconstructed images of 2-h infection; image reconstructions along xz and yz axes are shown. Scale bars, 10 μm. (b) Magnification of an adherent diplococcus 10 min after infection. Scale bar, 5 μm. (c) hCMEC/D3 cells expressing exogenous YFP-tagged β2-AR (β2-AR–YFP) left untreated (NT) or preincubated for 1 h with 10 μM isoproterenol (ISO) to induce endocytosis of the β2-AR before 2-h infection with wild-type meningococci. Arrows indicate the location of adherent meningococcus colonies. Scale bars, 10 μm. Images are representative of 3 independent experiments. (d) Percentage of the bacterial colonies associated with ezrin, CD44 or CD147 recruitment to sites of meningococcal adhesion in untreated cells or after cell pretreatment with 10 μM isoproterenol to promote β2-AR endocytosis. Mean ± s.e.m.; n = 3 experiments; ***P < 0.001, two-way analysis of variance (ANOVA).

Figure 2

Figure 2. CD147 depletion, soluble CD147 and anti-CD147 antibodies inhibit N. meningitidis adhesion to human endothelial cells.

(a) Left, adhesion of Nm2C4.3 to CD147-depleted (siRNA CD147) or control (siRNA CTL) hCMEC/D3 cells, quantified following a 30-min infection. Right, FACS analysis of CD147 expression in hCMEC/D3 cells, with isotype control as shaded area and mean fluorescence intensities in parentheses. Mean ± s.e.m., n = 4; **P < 0.05, two-tailed Student's _t_-test. (b) Left, adhesion of Nm2C4.3 to control hBMEC cells (siRNA CTL + empty vector), CD147-depleted (siRNA CD147 + empty vector) or CD147-complemented HBMECs (siRNA CD147 + plasmid encoding CD147), quantified following a 30-min infection. Right, FACS analysis of CD147 expression in HBMEC cells, with isotype control as shaded area and mean fluorescence intensities in parentheses. **P = 0.0025, two-tailed Student's _t_-test. (c) Left, adhesion of Nm2C4.3 to control (empty plasmid) or CD147-overexpressing (CD147 plasmid) hBMEC cells, quantified following a 30-min infection. Right, FACS analysis of CD147 expression in HBMEC cells, with isotype control as shaded area and mean fluorescence intensities in parentheses. Mean ± s.e.m., n = 3; **P = 0.0094, two-tailed Student's _t_-test. (d) Adhesion of Nm2C4.3 to CD147-depleted or control hCMEC/D3 cells, quantified following a 10-min infection under shear stress (0.04 dynes/cm2). **P < 0.01, two-tailed Student's _t_-test. (e) Adhesion of meningococcus strains belonging to different sequence types and capsular serotypes to CD147-depleted or control hCMEC/D3 cells, quantified following a 30-min infection. Mean ± s.e.m., n = 4; *P = 0.025, **P = 0.001, ***P = 0.0008, two-tailed Student's _t_-test. (f) Adhesion of Nm2C4.3 pilin variant strains expressing PilESA or PilESB to CD147-depleted or control hCMEC/D3 cells, quantified following a 30-min infection. Mean ± s.e.m., n = 6; ***P < 0.001, two-way ANOVA. (g) Adhesion of Nm2C4.3 to HBMECs in the absence (negative) or in the presence of purified CD147-Fc or ICAM-1–Fc (both at 5 μg ml−1), quantified following a 30-min infection. **P < 0.01, one-way ANOVA. (h) Adhesion of Nm2C4.3 to HBMECs and hCMEC/D3 cells preincubated with 10 μg ml−1 of antibodies targeting ICAM-1 (11C81), the N-terminal Ig domain of CD147 (MEM-M6/1) or the C-terminal Ig domain of CD147 (MEM-M6/6), quantified following a 10-min infection under shear stress (0.04 dynes/cm2). Abs, antibodies. Mean ± s.e.m., n = 3; **P < 0.01, ***P < 0.001, one-way ANOVA.

Figure 3

Figure 3. The major pilin PilE and the minor pilin PilV interact directly with CD147.

(a) Adhesion of wild-type (WT) and nonpiliated (Δ_pilE_, as control) meningococci to immobilized CD147-Fc, or to ICAM-1–Fc or no immobilized protein as controls, quantified following a 1-h infection. Mean ± s.e.m., n = 4; ***P < 0.001, one-way ANOVA. (b) Adhesion of Nm2C4.3 to hCMEC/D3 cells in the absence (Nontreated, NT) or in the presence of soluble MBP-pilins (PilESA, PilESB, PilV, PilX or ComP) or MBP as a control, quantified following a 30-min infection. Mean ± s.e.m., n = 4; **P < 0.01, ***P < 0.001, one-way ANOVA. (c) Left, CD147-Fc coprecipitation with staphylococci conjugated with the indicated MBP-pilins, or MBP alone as a control. Right, quantification of CD147 immunoprecipitation. Mean ± s.e.m., n = 4; **P < 0.01, ***P < 0.001, one-way ANOVA. (d) AlphaScreen analysis of the interaction between CD147-Fc (500 nM) or vehicle (−) as a control, and increasing MBP-PilESB concentrations. Mean ± s.e.m., n = 2; ***P < 0.001, two-way ANOVA. (e) AlphaScreen analysis of the interaction between CD147-Fc (500 nM) and MBP, MBP-PilX or MBP-PilV at various concentrations. Mean ± s.e.m., n = 2 experiments; ***P < 0.001, two-way ANOVA. (f) AlphaScreen analysis of the interaction between CD147-Fc (100 nM) and MBP-PilESB or MBP-PilV (500 nM) in the presence of increasing concentration of the anti-CD147 antibody (MEM-M6/6) or anti–ICAM-1 (11C81, as control). mAb, monoclonal antibody. (g) AFM analysis of the retraction forces between cantilever tips functionalized with MBP, MBP-pilins (PilX, PilV, PilESA and PilESB) or cyclophilin (Cyclo, positive control) and CD147-Fc or activated leukocyte-cell adhesion molecule-1 (ALCAM-1)-Fc (control). Bar plots indicate the percentage of force curves with interaction events. Mean ± s.e.m.; n = 7. ***P < 0.001, one-way ANOVA.

Figure 4

Figure 4. The major pilin PilE and the minor pilin PilV are essential bacterial components for vascular colonization.

(a) Adhesion of Nm2C4.3 wild-type strain or Δ_pilE_ and Δ_pilV_ mutant derivatives to hCMEC/D3 cells and HBMECs. Mean ± s.e.m., n = 4; ***P < 0.001, one-way ANOVA. (b,c) Colonization of human vessels in grafted human skin by Nm2C4.3 wild-type strain or the Δ_pilE_ or the Δ_pilV_ mutant derivatives. (b) Mean ± s.e.m. of the vascular colonization index. Analysis of 10 sections per graft, n = 6 grafts per condition. ***P < 0.001, one-way ANOVA. (c) Representative fluorescence microscopy showing the different bacterial derivatives (red) associated with human vessels (basal lamina collagen IV, green) in the skin. Scale bars, 100 μm. Images are representative of 6 different human skin grafts.

Figure 5

Figure 5. In situ interaction of N. meningitidis with human brain vessels requires both PilE and PilV expression.

(a) Immunohistochemical analysis of bacteria (brown) in a whole human brain section infected with Nm2C4.3 wild-type strain. Arrows indicate bacterial aggregates within brain vessels in the cortical region. Scale bar, 1 mm. (b) Detailed immunohistochemical analysis of the bacterial staining (blue) in the cortical region of serial human brain sections from the same donor infected with Nm2C4.3 wild-type strain or the Δ_pilE_ or the Δ_pilV_ mutant derivatives. Scale bar, 100 μm. (c) Detailed immunofluorescence analysis of bacteria (red), actin (green) and nucleus (blue) in the meningeal region (Virchow-Robin space) of serial human brain sections from the same donor infected with Nm2C4.3 wild-type strain or the Δ_pilE_ or the Δ_pilV_ mutant derivatives. Scale bars, 100 μm. (d) Quantification of adherent meningococci per 100-μm2 section, expressed as percentage of adherent wild-type bacteria. Mean ± s.e.m., analysis of 3 brain sections per brain from 3 different donors; ***P < 0.001, one-way ANOVA.

Figure 6

Figure 6. In situ infection of fresh human brain sections by N. meningitidis requires pilus interaction with CD147.

(a) Immunofluorescence analysis of CD147 (green), meningococcus colonies (red) and nucleus (blue) in the Virchow-Robin space and cortical region of serial brain sections from the same donor infected with Nm2C4.3 wild-type strain for 1 h. Scale bars, 20 μm. Images are representative of 6 different human skin grafts. (b) Immunohistochemical (top) and immunofluorescence (bottom) analysis of serial sections of cortical regions of the same donor, treated with 10 μg ml−1 anti–ICAM-1 (11C81) or anti-CD147 (MEM-M6/1 and MEM-M6/6) antibodies before infection with Nm2C4.3 wild-type meningococci. Scale bars, 100 μm (top) and 20 μm (bottom). Images are representative of 3 different human skin grafts. (c) Mean ± s.e.m. of total fluorescence per 100 μm2 relative to fluorescence in absence of antibody, n = 4 brain sections per donor from 3 different donors; *P < 0.05, one-way ANOVA.

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