Florent SEBBANE

Bruno POTBenoit FOLIGNE Catherine DANIEL

U801Bactéries Lactiques

&Immunité des Muqueuses

Sabine POIRETJoëlle DEWULF

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Michaël MARCEAU

Interactions Cellulaires & Moléculairesdes Bactéries Pathogènes avec l’Hôte

Genetics of Yersinia Virulence

Y. enterocolitica Y. pseudotuberculosis Y.pestis

Pathogenic Yersiniae Harbora Plasmid Involved in Bacterial Virulence,

Encoding a Type III Secretion System

YscNYscL

YscS

YscT

YscQ

YscD

YscU

YscR

YscJ

YscC

YscF

Innermembrane

Outermembrane

YopN, TyeA, LcrG , LcrV

YopTYopO

YopBDYopE

YopH

YopM

YopJ

Cytoplasm

Trends Microbiol.,2006

YscV

the lethal weaponYsc

D

E O

phosphatase

kinase

cytotoxin

macrophage

Yersinia

M

E

B

H

J

O

D

J

N

BYsc YscYscH

N

M

T

T

macrophage

Ysc

• YopH (protein tyrosine phosphatase) dephosphorylates focaladhesion proteins p130cas, Fak, Fyb, paxillin , and YopE, YopO (YpkA) & YopT inactivate GTPases

• YopJ abrogates pro-inflammatory cytokine biogenesis in macrophages through inhibition of NF- κκκκB and MAPK-kinase activation

• YopJ induces macrophage apoptosis through caspase activation and/or inhibition of NF- κκκκB activation

YscNYscL

YscS

YscT

YscQ

YscD

YscU

YscR

YscJ

YscC

YscF

Innermembrane

Outermembrane

YopN, TyeA, LcrG , LcrV

YopTYopO

YopBDYopE

YopH

YopM

YopJ

Cytoplasm

YscV

the lethal weaponYsc

PNAS, 2005

�LcrV injection in vivo inhibits TNF- αααα and IFN-γ γ γ γ upon infection with LcrV-deficient Y. pestis

�LcrV upregulates IL-10 secretion in a TLR2- and CD14 -dependent fashion, which in turn suppresses TNF- αααα secretion in vitro

�The N-terminal region of LcrV determinesTLR-2-mediate d IL-10 induction

Infect. Immun., 1995

J. Exp. Med., 2002

Lactococcus

LactobacillusLactic acid bacteria (some members have probiotic properties) are

non-pathogenic, non-invasive micro-organisms

used for mucosal delivery of therapeutic agents

�Sensitive to acid

�Sensitive to bile

�Not persistent (< 2 days) in the mouse GIT

�Resistant to acid

�Resistant to bile

�Persistent (7 days) in the mouse GIT

Lactococcus lactis MG1363 Lactobacillus plantarum NCIMB8826

36 kDa

64 kDa

98 kDa

L.la

ctis

-Lcr

V +

Yer

sini

a

Recombinant L. lactis releases high levels of LcrV

Signal peptideUsp45

Pusp45 lcrV pst

Lactococcus lactis MG1363

L.la

ctis

-Lcr

V -

anti-LcrV antibody

bacterial culture supernatant

pMEC237

cat

Gastroenterology, 2007

Recombinant LcrV is functional in vitro

50

100

150

200

250

300

350

0

IL- 10 assay

IL-1

0 (p

g/m

l)

LcrV + ControlLcrV -

L. lactis

mouse peritonealmacrophages

IL-10 levelin cell supernatant

L. lactis LcrV -

L. lactis LcrV +

PBS (control)

LPS

24 h

3 h

(no LPS)

…and also in vivo

D1 D2 D3 D4 D5

L. lactis LcrV -, 108 i.gL. lactis LcrV +, 108 i.gNaHCO3 (control), i.g 0

200

400

600

800

1000

1200

1400

1600

LcrV + ControlLcrV -

IL- 10 assay

L. lactisIL

-10

(pg)

/ co

lon

cytokine quantification (at D5)in 9-cm length of colon

intranasalimmunization

intragastricimmunization

1 21 42days

109

109

53

anti-

LcrV

IgG

tite

r bloodan

ti-Lc

rV Ig

A ti

ter BAL Gut

lavages

L. lactis

LcrV -LcrV +

L. lactis

LcrV -LcrV +

L. lactis

LcrV -LcrV +

10

100

10,000

1,000

0.1

0.2

0.3

0.4

0.5

0.6

anti-

LcrV

IgA

tite

r

0.1

0.2

0.3

0.4

0.5

0.6

blood BAL Gut lavages

10

100

10,000

1,000

anti-

LcrV

IgG

tite

r

anti-

LcrV

IgA

tite

r

0.1

0.2

0.3

0.4

0.5

0.6

anti-

LcrV

IgA

tite

r

0.1

0.2

0.3

0.4

0.5

0.6

Vaccine, 2009

The humoral reponse against LcrV

intranasal immunization

Cyt

okin

e (p

g/m

l)IL-2 INF-γγγγ

L. lactis

LcrV -LcrV +

L. lactis

LcrV -LcrV +

Vaccine, 2009

3,000

6,000

9,000

12,000

The cellular reponse against LcrV

300

200

100

L. lactis LcrV + protects mice against pseudotuberculosis

10 20Days post-challenge

the oral model

Mou

se s

urvi

val

%

d 3 d 6 d 16 d 30

Days (d) post-challenge

9

7

5

3

1

log

10Y

ersi

nia

/g o

forg

an

d 3 d 6 d 16 d 30

7

5

3

1

spleen

Mesenteric lymph nodes

LcrV +

LcrV -

10080

60

40200

(4x108 bacteria, i.g)

30

intranasal immunization

L. lactis LcrV + protects mice against pseudotuberculosis

10 20Days post-challenge

the systemic model

Mou

se s

urvi

val

%

d 3 d 5 d 15 d 21

Days (d) post-challenge

9

7

5

3

1

log

10Y

ersi

nia

/g o

forg

an

d 3 d 5 d 15 d 21

7

5

3

1

spleen

liver

LcrV +

LcrV -

10080

60

40200

(103 bacteria, i.v)

intranasal immunization