Jean-Pierre GORVEL

BrucellaPathogenesis

Septses 09 2010

ISOLATION OF BRUCELLAE

ISOLATION OF BRUCELLAE 19th Century (1860) first description of disease by

Marston

ISOLATION OF BRUCELLAE 19th Century (1860) first description of disease by

Marston

B.melitensis 1887 (Bruce - Carruana-Secluna)

ISOLATION OF BRUCELLAE 19th Century (1860) first description of disease by

Marston

B.melitensis 1887 (Bruce - Carruana-Secluna)

B.abortus 1895 (Bang)

ISOLATION OF BRUCELLAE 19th Century (1860) first description of disease by

Marston

B.melitensis 1887 (Bruce - Carruana-Secluna)

B.abortus 1895 (Bang)

B.suis 1914 (Traun)

ISOLATION OF BRUCELLAE 19th Century (1860) first description of disease by

Marston

B.melitensis 1887 (Bruce - Carruana-Secluna)

B.abortus 1895 (Bang)

B.suis 1914 (Traun)

B.ovis 1953 (Buddle and Boyes)

ISOLATION OF BRUCELLAE 19th Century (1860) first description of disease by

Marston

B.melitensis 1887 (Bruce - Carruana-Secluna)

B.abortus 1895 (Bang)

B.suis 1914 (Traun)

B.ovis 1953 (Buddle and Boyes)

B.canis 1966 (Carmichael)

ISOLATION OF BRUCELLAE 19th Century (1860) first description of disease by

Marston

B.melitensis 1887 (Bruce - Carruana-Secluna)

B.abortus 1895 (Bang)

B.suis 1914 (Traun)

B.ovis 1953 (Buddle and Boyes)

B.canis 1966 (Carmichael)

B.neotomae 1957 (Stoenner and Zackman)

ISOLATION OF BRUCELLAE 19th Century (1860) first description of disease by

Marston

B.melitensis 1887 (Bruce - Carruana-Secluna)

B.abortus 1895 (Bang)

B.suis 1914 (Traun)

B.ovis 1953 (Buddle and Boyes)

B.canis 1966 (Carmichael)

B.neotomae 1957 (Stoenner and Zackman)

B.cetaceae and B.pinnipediae in marine mammals 1994

HUMAN PATHOGENS

HUMAN PATHOGENS

B.melitensis

HUMAN PATHOGENS

B.melitensis B.abortus

HUMAN PATHOGENS

B.melitensis B.abortus B.suis (except biovar 2)

HUMAN PATHOGENS

B.melitensis B.abortus B.suis (except biovar 2) B.canis

HUMAN PATHOGENS

B.melitensis B.abortus B.suis (except biovar 2) B.canis

HUMAN PATHOGENS

B.melitensis B.abortus B.suis (except biovar 2) B.canis

Human infection due to B.cetaceae or B.pinnipediae reported at 2004

BRUCELLOSIS DUE TO B.melitensis

B.melitensis GLOBAL STATUS

B.melitensis GLOBAL STATUSB.melitensis never reported

B.melitensis GLOBAL STATUSB.melitensis never reported B.melitensis eradicated

B.melitensis GLOBAL STATUSB.melitensis never reported B.melitensis eradicatedB.melitensis infected

B.melitensis GLOBAL STATUSB.melitensis never reported B.melitensis eradicatedB.melitensis infected B.melitensis reported in the past

Data not available

Occupational disease (vet, farmer, scientist and slaughterhouse worker).Human brucellosis

Intermittent fever, weakness, weight loss, epididymitis and orchitis, arthritis, spondylitis (disabling sequelae).

Consumption of [fresh] unpasteurized dairy products.

No significant human to human transmission. No vaccine in human, several inefficient vaccines in animals

Prolonged (4 weeks) combined antibiotherapy (relapses, low compliance).

Human brucellosis: symptoms & evolution

Pedro-Pons,A., P.Farreras, A.Foz, J.Surós, R.Surinyach, and R.Frouchtman. 1968. Enfermedades infecciosas. II.A. Enfermedades producidas por bacterias. Brucelosis., p. 338-374. Patología y Clínica Médicas, vol. VI. Salvat Ed. S.A., Barcelona-Madrid.

Orchitis

Lumbar spondylitissacroiliitis

Hepato &splenomegaly

Fatigue, chills, sweatingArthralgia & myalgiaConstipation

Incubation10-12 daysto months

A new fever episode (undulant fever)

Leukocytes 4200/ml

Brucella

Gram negative

Cocobacilliα2-Proteobacteria

Brucella

Gram negative

Cocobacilliα2-Proteobacteria

Facultative intracellular pathogen

Able to escape from innate immunity Able to multiply in cells

Chronic disease

Type IV SS

2 chromosomesno plasmids

Brucella: a silent parasiteNoisy parasites

Flagella

OM

Type IIIand IV SS

Fimbriae

Exopolysaccharide

ExotoxinsExoenzymes

Virulenceplasmids

Brucella: DOES NOT bear classical virulence factors

Type IV SS

2 chromosomesno plasmids

Brucella: a silent parasiteNoisy parasites

Flagella

OM

Type IIIand IV SS

Fimbriae

Exopolysaccharide

ExotoxinsExoenzymes

Virulenceplasmids

Brucella: DOES NOT bear classical virulence factors

To develop a tough outer membrane Not to release host-damaging agents

Not triggering systemic alarms

To become simple and to reduce PAMPs

Bald bacteria such as Brucella abortus

Brucella Virulence Factors

Guzman-Verri Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12375-80.

Celli J Exp Med. 2003 Aug 18;198(4):545-56.O’Callagnan, Mol Microbiol, 1999Delrue, Cell Micro, 2001Comerci, Cell Micro, 2001Rohan & Tsolis, I&I, 2007

Forestier J Immunol. 2000 Nov 1;165(9):5202-10.

Conde-Alvarez Cell Microbiol. 2006 Aug;8(8):1322-35.

Arellano-Reynoso Nat Immunol. 2005 Jun;6(6):618-25.

Freer Infect Immun. 1999 Nov;67(11):6181-6.Giambartolomei, JI 2004

From Intracellular Niches of Microbes. Brucella, Monika Kalde, Edgardo Moreno and Jean-Pierre Gorvel

Gorvel JP, Moreno E, Moriyón I.Nat Rev Microbiol. 2009 Mar;7(3):250

Gram-negative bacteria envelope

Phophates

Negatively charged sugars

Omps

Peptidoglycan

cytoplasm

LPSGram-negative Bacteria

OM

IMPhospholipidsPE, PC(Brucella)

+

Ornithine Lipid(Brucella)

Periplasm

Brucella LPS

•Brucella mutants in components of the Outer Membrane :

- Phosphatidylcholine (BApcs,BApmtA, BApcspmtA) Conde et al. Cell Microbiology, 2006. PC is an important for evading lysosomal killing.

- Ornithine Lipids (BAOlsB)

- LPS: Phosphatase (BAI1212,BAII1103,BAI1212II1103)

Manosyltransferase (BALpcC)

- Phosphatases and ornithine lipids (BA I1212II1103/OlsB)

• H2O2

• NO

• Myeloperoxidase and aldehydes

• Phospholipase A2

• Metaloproteinases

• Complement

Brucella is resistant to bactericidal substances of cells

Bactericidal activity (%)

Bactenecin 7

PMNs extract

Bactenecin 5Cap18

Cecropin ACecropin P1

Defensin NP-2Lactoferricin B

LactoferrinLysozyme

Magainin 1Magainin 2

MelittinEMP-2

Poly-L-lysinePolymyxin B

Poly-L-ornithineEDTA

Tris

0 25 50 75 100

Salmonella Brucella

In B. abortus, an intact LPS core is required for:

Resistance to the bactericidal action of polycationic peptides and normal serum.

Multiplication in dendritic cells and inhibition of maturation.

Inhibition of recognition by MD-2.

As a consequence, SAID: Shield Anti-Immune Detection

lpcC mutants may be promising vaccines

In B. abortus, an intact LPS core is required for:

Resistance to the bactericidal action of polycationic peptides and normal serum.

Multiplication in dendritic cells and inhibition of maturation.

Inhibition of recognition by MD-2.

As a consequence, SAID: Shield Anti-Immune Detection

Brucella: a stealth pathogen

• resistance to host cell bactericidal molecules/activities

• intracellular survival and replication

• ability to hide from and modulate the host immune response

Brucella replicates within host cells

C57BL/6 mice bone marrow-derived macrophages

time post infection (h)

CFU

/wel

l

0 10 20 30 40 5010 4

10 5

10 6

10 7

10 8

Brucella abortus 2308-GFP

Brucella-containing vacuole(BCV)

Biogenesis of the BCV in macrophages

5 min

1 h

4 h

8 h

12 h

Early BCVEEA+, Rab5+, TfR+

Intermediate BCVLAMP1+, Rab7—, M6P—

(Pizarro Cerda, Many!!!)

Replicative BCVLAMP1—, Cathepsin D—

Calreticulin+, Calnexin+, Se61ß+, PDI+

Require Rab2, GAPDH (Fugier PloS Pathogen 2010)

Sar1 (Celli, J Exp Med, 2003)

BCV interact and fuse with the ER during maturation

glucose-6-phosphatase detection Chantal de Chastellier

Ly12h p.i.

Lack of ER fusion with virB mutant-containing vacuoles leads to fusion with lysosomes

BMDM infected with the virB10— Brucella strain glucose-6-phosphatase detection

Chantal de Chastellier

Celli et al. 2003 J Exp Med

Brucella infection of dendritic cells

Salcedo et al. 2008 PLoS Pathog.

New cell model for studying Brucella virulence: murine bone marrow-derived dendritic cells

Brucella replicates in DCs

Log

CFU

wt B. abortus

Salmonella

virB-

Time post-infection (h)

1

2

3

4

5

6

7

8

0 20 40 60

0

20

40

60

80

100

2 12 24 48time post-infection (h)

% D

Cs w

ith

intr

acel

lula

r ba

cter

ia 1-5

5-10

>10

LPS wt-DSRed

30 h

Brucella replicates in ER-derived vacuoles

Ba

ER

ER

Ba

ER

Ba

calnexin

0

20

40

60

80

100

0 20 40 60Time after infection (h)

BCVs

pos

itiv

e La

mp1

(%)

wt

virB-

24 h

KDELMHCIIwt-GFP

Murine bone marrow-derived DCs(C57BL/6 mice)

Immature Mature

change in morphology

transient formation of DALIS (DC aggresome-like induced structures)

increased surface expression of MHC and co-stimulatory molecules

cytokine secretion

antigen presentation

Does Brucella affect maturation of DCs?

Brucella does not induce formation of DALIS

Dendritic cell Aggresome Like Structures (DALIS) : • transient and insoluble structures that appear upon TLR activation

• site of storage/ubiquitination for newly synthesized defective proteinsImmature (0h)

8-14h E. coli LPS

Ub-proteinsMHC-II

Mature (24-36h)

Lelouard et al., 2002

negative

Salmonella

B. abortus

DCs

wit

h D

ALIS

(%)

Time after infection (h)

0

20

40

60

80

100

4 12 24 36 48

Brucella does not induce maturation of DCs

24hD

Cs w

ith

DAL

IS (

%)

nega

tive wt

0

20

40

60

80

100

virB

-HK

Bru

cella

MHCIILAMP1

E. coli LPS

FK2MHCII

Salmonella Brucella

Salmonella Brucella

Brucella does not induce surface expression of co-stimulatory molecules

Brucella interferes with the immune functions of DCs

Brucella-infected DCs do not induce T cell proliferation (neither MHCI nor MHCII)

Identification of Btp1 (Brucella TIR-containing protein)

Tir2

Btp1

MHCII

wt btp1-

3

4

5

6

7

8

0 20 40 60

wtvirB-

btp1-Log

CFU

Time post-infection (h)

Btp1 contributes to blocking DC maturation

IL12

neg

HKB wt

Infe

cted

ce

lls w

ith

DAL

IS (

%)

DALIS (24h)

0

20

40

60

80

100

btp1

-

neg

HKB wtbt

p1-

IL12

(ng

/ml)

0

20

40

60

80

0

400

800

1200

neg

HKB wtbt

p1-

TFNα

(pg

/ml) TFNα

48 h24 h

Lack of Btp1 had no significant effect on surface expression of CD40 and CD86 and only a very minor effect on MHCII and CD80 when analysed by flow cytometry

TLR2

0100200300400500

neg

+PAM

Btp1 + PAM PipB2

+PAM

050

100150200250

neg

+CpG

Btp1 + CpG PipB2

+CpG

TLR9

Rela

tive

luci

fera

se a

ctiv

ity

Rela

tive

luci

fera

se a

ctiv

ity

Btp1 interferes with TLR2 signalling

•Btp1 acts on the myd88/TLR2 pathway

Btp1 mutant

0

20

40

60

80

wt

myd

88

TRIF

TLR2

TLR4

TLR7

TLR9

% D

Cs w

ith D

ALI

S

mice

control

wt B. abortus

Wild-type Brucella

0

20

40

60

80

wt

myd

88

TRIF

TLR2

TLR4

TLR7

TLR9

% D

Cs w

ith D

ALI

S

mice

Controlcgs- mutant

Newman et al., I&I, 2006Salcedo et al., PloSpathogen, 2008Cirl et al., Nat Med, 2008Radakrishnan et al., JBC, 2009 (Tirap)Sengupta et al., JI, 2010 (Mal)

outer membrane

inner membraneperiplasm

E. coli

Brucella

Periplasmic glucans

Cyclic glucan prevents Brucella fusion with lysosomes

cathepsin D

Arellano-Reynoso et al. 2005 Nat Immun

cgs—

cgs— + CßG

Arellano Reynoso, Nat Immunol, 2005

Molecular properties

Brucella CβG

I: CβG is not toxic for cellsalso shown in vivo in mice

MβCD

MβCD: Methyl-beta-cyclodextrin

II: CβG extracts cholesterolfrom membranes but withLess efficiency than MβCD

III: CβG modifies lipid raft Characteristics (flotation gradient)

Conclusions

• Both macrophages and dendritic cells are targeted by Brucella and

provide a cellular niche for its intracellular replication

• Brucella controls the maturation of murine DCs

• Btp1 is a new Brucella virulence protein that interferes with TLR2

signaling and contributes to down-modulation of DC maturation

•Cyclic glucan is potent activator of the immune response

Fever. edema. leukocytosis. trombocytopenia

Endotoxic shock

Monocyte

Platelet aggregation

PMN Chemotaxis

Chemokines

CCL1 CCL3 CCL4

CCL5CXCL1 CXCL8

CytokinesTNF-α

IL-1βIL-6

IL-1

IL-12INF-γTNF-α Phagocytosis of

apoptotic bodies

Mature DC

Apoptosis

DegranulationROS. killing

Mφ

Activationkilling

Mφ

Tissue damage

Endothelial damage Plasmin

Fibrinopeptides

Fibrinogensynthesis

Bradykinin

C’ activation

C3a. C5a.C5b67

C3a

C5a

Mast cell

ProstaglandinsLeukotrinesHistamine

IL-18

DC

DefensinsMIP -1αMIP-1βCXCL8TNF-αIL-1β

Apoptotic body

1 2

3

5

4

67

8

9

10

11

12

13

14

16

15

17

18