Neisseria meningitidis, les secrets de la subversion de l’endothélium

vasculaire cérébral

Xavier Nassif�

INSERM U1002�

Faculté de Médecine Paris Descartes�

Paris, France�

Meningitis

Purpura

Human +

N. meningitidis

Human +

M.tuberculosis

Commensalism Pathogenesis Pathogenesis Latency

No disease Dissemination

Disease Dissemination

Disease No dissemination

No disease No dissemination

Environment (Influenza virus)

Host factors + Deficiency in late complement components + Deficiency in maltose binding protein + Lack of immunity (young children)

Bacterial factors : capsule (+++), Iron adhesines/pili, Opa.. filamentous phage

Cerebrospinal fluid MENINGITIS

Blood SEPTICEMIA

PURPURA FULMINANS

Commensalism Human- nasopharynx + N. meningitidis

Neisseria meningitidis, a paradigm of extra cellular bacterial pathogen�

Meningococcal infections, a public health burden�

-  1 to 1. 5 per 100 000 habitants,�-  Two peaks before 2 years and at 15-25 years of age�

-  Fulminant septicemia (30% death) even when treated �-  Meningitis (3% death)�- Can be responsible for small epidemics in developed countries�and large epidemics in Africa (the meningitis belt)�

-  Two major serogroups in Europe : B (60%) and C (40%). �-  A vaccine against C is available�-  No vaccine against B serogroup�

2 8 α�

NeuNAc 2 8 α�

NeuNAc NeuNAc O

OS

N

2 8 α�

NeuNAc 2 8 α�

NeuNAc NeuNAc O

P O O

OCH2

CH

CH2

O C R

O C R

O

OCOOH H2N

binding cell attachment n-CAM

Serogroup B

Structural relation of the capsular serogroup B of N.meningitidis to the carbohydrate terminal of the neonatal neural cell adhesion

molecule (n-CAM); NeuNac = N-acetylneuraminic acid.

THE AFRICAN MENINGITIS BELT

How N.meningitidis once in the bloodstream can cross the blood brain

barrier ?�

Subpial vein Pia mater

Dura mater

Arachnoid

Glia limitans

Subpial space

Brain post-capillary venules and venules (Virchow-Robin Perivascular space)

Ventricle

Choroid plexus

Subarachnoid space

Gray matter

White matter

Sulcus

Brain capillary (gliovascular unit)

Skull

CSF Neuropyle

CSF

Blood-Brain barriers Meningeal Blood-CSF barriers Ventricular Blood-CSF barriers

Brain vein

Secretory epithelial cells

A- Choroid plexus

Stroma

Fenestred capillary

B- Meningeal veins Subarachnoid space

Glia limitans

Blood-CSF barriers

Subpial space

Perivascular macrophage

Pia mater

Subpial vessel

Blood-brain barriers

A- Brain capillaries B- Brain post-capillary venules and venules

5 µm 10-50 µm

Astrocytic end-foot

Endothelial cell

Pericyte

Perivascular macrophage

Microglial cell

Tight junction belt

Adherent junction Virchow-Robin Perivascular space NS tight junction

Brain capillary inter-endothelial junctional complexes

Claudin

Occludin

JAM

VE-cadherin

Tight junctions

Adherent junctions

ZO-1 ZO-2,3 F-actin

Catenin

Vinculin

Luminal face

Abluminal face

Brain�

Heart�

Kidney�

117 colonies

24 in the cortex 93 in the white matter

White matter�Cortex�

Meninges�

Meningeal spaces

Subarachnoidal space

Pia mater

Arachnoid

Glia limitans

Subpial vein

Brain vein Subpial

space

Capillaries (gliovascular unit)

Lumen

pericyte

Astrocytic foot

Endothelial cell

Microglial cell

Junctional complex

Post-capillary venule, venules and veins

pericyte

Astrocytic foot

Endothelial cell

Virchow-Robin perivascular spaces

Lumen

Perivascular macrophage

117 colonies

24 in the cortex 93 in the white matter

White matter�Cortex�

Meninges�

IMMORTALIZATION OF HUMAN BRAIN ENDOTHELIAL CELLS

Primary culture

hCMEC/D3 cell line

The D3 cell line express junctional proteins and makes tight junctions

Brain�

Heart�

Kidney�

Type IV pili (Tfp)

PilD

PilF PilT

E

Assembly Retraction

Outer membrane

Inner membrane

PilC PilQ

PilW

Assembly PilM, N, O, P

Stabilization

PilC, G, H, I, J, K, W

Maturation PilC, I, J, K, W

PilE� Minor pilins� (PilX, PilV, ComP)�

Prepilin�Minor pilins�

assembly�

re�traction�(PilT)�

emergence � on the�(PilQ)�

Functional Tfp (PilE, PilX, PilV, ComP)�

(PilD, F, M, N, O, P)� (PilC, G, H, I, J, K, W)�

cell surface�

(Craig et al, Molecular Cell 2006)

Signaling to endothelial cells by Neisseria meningitidis

ErbB2

CD44 ICAM-1

? CD46

ezrin

F-actin

Rho Cdc42

Actin polymerisation

cortactin src

?

Lambotin et al. J.Cell Science, 2005, 18:3806�

Doulet et al., J.cell.Biol., 2006,173:627�

Actin Ezrin Bacteria Overlay

Pili signaling lead to the formation of a « cortical plaque ”�

Host cell surface reorganization is responsible for mechanical resistance of Nm colonies growing onto the apical surface

Mikaty et al, Plos Pathogens, 2009�

Summary

1.  Pili are responsible for the interaction of Nm with endothelial cells and signaling

2.  How pilus mediated adhesion is responsible for the crossing of the BBB ?�

Nm recruit adherens and tight junction proteins

Nm VE-Cadherin Actin Merge

10 µm

p120-catenin beta-catenin

ZO-1 ZO-2 claudin-5

10 µm

Formation of junctions (epithelial cells)

The Par3/Par6/aPKC complex is needed for the recruitment of junctionnal proteins

and then segregation of adherens and tight junctions

Nm recruits the polarity complex Par3/Par6/PKCζ in a Cdc42 dependent manner

Actin

Cdc42

Control siRNA

Cdc42 siRNA

Tagged VE-cadherin is recruited from cell-cell junctions TEA1/31-bound VE-cadherin – BASO-LATERAL TEA1/31-bound VE-cadherin - APICAL

*

*

MERGE TEA1/31-bound VE-cadherin – BASO-LATERAL Dapi

1

6 4

2

5 3

z

y x

View 1 VE-cadherin

View 1

x

y z

3 1

4 5

? ErbB2

Translocation of AJ proteins

- Cell-cell junction

leakage ?

VE-cadherin

Er

?

p120

VE-cadherin

Arp2/3 Cortactin

p120

p120

Actin polymerization (honeycomb)

Unknown receptor

Membrane protrusion

The polarity complex is required to open the paracellular route

1

1

2 2

Z

Z

Z

3

4

3

4 Z

VE-cadherin

PKC

η P

SPK

C ζ

PS

Number and surface of gaps observed

in hcmec/D3 monolayer

0

4

8

12

16

20

24

1.7-3.4 3.4-7.6 7.6-15 15-30 > 30 µm2

Nm

Nm PKCζPS

Nm ΔpilE

Lucifer Yellow Permeability Bacterial diffusion through hCMEC/D3 monolayer

1,8

0

0,2

0,4

0,6

0,8

1

1,2

Nm ΔpilE Nm +PKCζ PS

Fold increase

Nm PKCζ PS

1

1,2

1,4

1,6

3

* *

Nm Nm PKCη PS

ΔpilE Control D-mannitol

* Fold

increase

Type IV pilus-mediated signaling induces the opening of the paracellular route�

Nm recruit the polarity complex and open the intercellular junctions

Cortical plaque

p120

Acin polymerisation

ZO-1

VE-cadherin

VE-cadherin

p120

Claudin-5

ZO-1

eeeCdc42 c42c4c4

GPCRs GPCRs + agonist

Neisseria meningitidis activate the ββ2-adrenergic receptor (β2AR)

β2-adrenoceptor/βarrestins are sufficient to induce formation of the cortical plaque

Ezr

in s

tain

ing

Recruitment of proteins in CEACAM-1/β2AR/βarrs transfected cells

Nm

β2-adrenoceptors/barrestins pathway open junctions

Nm transmigration 5h after infection

Nm recruit the polarity complex and open the intercellular junctions

Cortical plaque

p120

Acin polymerisation

ZO-1

VE-cadherin

VE-cadherin

p120

Claudin-5

ZO-1

eeeCdc42 c42c4c4

Beta2 adrenoreceptor�

Conclusions�

1.  N.meningitidis franchit la BHE grâce à ses pili qui permettent adhésion �et signalling par deux récepteurs différents�

2. Le passage de la BHE est due à l’ouverture des jonctions intercellulaires�suite à un recrutement au siège de l’interaction bactérie-cellule des protéines�

de jonction intercellulaire�

3. Le récepteur membranaire induisant la signalisation cellulaire est le�récepteur beta2 adrenergique�

Applications�

1.  Utilisation des composants du pilus interagissant avec le récepteur �pour ouvrir la BHE�

2. Utilisation de ces mêmes épitopes pour une application vaccinale contre �N.meningitis de sérogroupe B�

INSERM U1002/ Necker-Enfants Malades

E. Bille E. Carbonnelle

M.Coureuil M.Drab

G. Duménil E. Frapy S.Hélaine

A.Jamet O.Joinlambert

H.Lecuyer P. Martin G.Mikaty H.Omer V.Pelicic

Institut Cochin, INSERM U561

Paris

S. Bourdoulous P-O. Couraud

F.Miller S.Marullo M.Scott

Institut du fer à moulin,

INSERM U839 Paris

R-M. Mège