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Jean-Pierre GORVEL
BrucellaPathogenesis
Septses 09 2010
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
B.melitensis B.abortus B.suis (except biovar 2) B.canis
Human infection due to B.cetaceae or B.pinnipediae reported at 2004
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
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
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 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)
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
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