Brucella Pathogenesis

Transcription

1 Brucella Pathogenesis Septses Jean-Pierre GORVEL

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3 ISOLATION OF BRUCELLAE

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

5 ISOLATION OF BRUCELLAE 19 th Century (1860) first description of disease by Marston B.melitensis 1887 (Bruce - Carruana-Secluna)

6 ISOLATION OF BRUCELLAE 19 th Century (1860) first description of disease by Marston B.melitensis 1887 (Bruce - Carruana-Secluna) B.abortus 1895 (Bang)

7 ISOLATION OF BRUCELLAE 19 th Century (1860) first description of disease by Marston B.melitensis 1887 (Bruce - Carruana-Secluna) B.abortus 1895 (Bang) B.suis 1914 (Traun)

8 ISOLATION OF BRUCELLAE 19 th 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)

9 ISOLATION OF BRUCELLAE 19 th 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)

10 ISOLATION OF BRUCELLAE 19 th 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)

11 ISOLATION OF BRUCELLAE 19 th 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

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13 HUMAN PATHOGENS

14 HUMAN PATHOGENS B.melitensis

15 HUMAN PATHOGENS B.melitensis B.abortus

16 HUMAN PATHOGENS B.melitensis B.abortus B.suis (except biovar 2)

17 HUMAN PATHOGENS B.melitensis B.abortus B.suis (except biovar 2) B.canis

18 HUMAN PATHOGENS B.melitensis B.abortus B.suis (except biovar 2) B.canis

19 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

20 BRUCELLOSIS DUE TO B.melitensis

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22 B.melitensis GLOBAL STATUS

23 B.melitensis GLOBAL STATUS B.melitensis never reported

24 B.melitensis GLOBAL STATUS B.melitensis never reported B.melitensis eradicated

25 B.melitensis GLOBAL STATUS B.melitensis never reported B.melitensis eradicated B.melitensis infected

26 B.melitensis GLOBAL STATUS B.melitensis never reported B.melitensis eradicated B.melitensis infected B.melitensis reported in the past Data not available

27 Human brucellosis Occupational disease (vet, farmer, scientist and slaughterhouse worker). Consumption of [fresh] unpasteurized dairy products. Intermittent fever, weakness, weight loss, epididymitis and orchitis, arthritis, spondylitis (disabling sequelae). Prolonged (4 weeks) combined antibiotherapy (relapses, low compliance). No significant human to human transmission. No vaccine in human, several inefficient vaccines in animals

28 Human brucellosis: symptoms & evolution Incubation days to months Fatigue, chills, sweating Arthralgia & myalgia Constipation Hepato & splenomegaly Lumbar spondylitis sacroiliitis Orchitis A new fever episode (undulant fever) Leukocytes 4200/ml Pedro-Pons,A., P.Farreras, A.Foz, J.Surós, R.Surinyach, and R.Frouchtman Enfermedades infecciosas. II.A. Enfermedades producidas por bacterias. Brucelosis., p Patología y Clínica Médicas, vol. VI. Salvat Ed. S.A., Barcelona-Madrid.

29 Brucella Gram negative Cocobacilli α2-proteobacteria

30 Brucella Gram negative Cocobacilli α2-proteobacteria Facultative intracellular pathogen Able to escape from innate immunity Able to multiply in cells Chronic disease

31 Brucella: DOES NOT bear classical virulence factors Flagella Noisy parasites Brucella: a silent parasite Type IV SS Type III and IV SS Fimbriae 2 chromosomes no plasmids Virulence plasmids OM Exopolysaccharide Exoenzymes Exotoxins

32 Brucella: DOES NOT bear classical virulence factors Flagella Noisy parasites Brucella: a silent parasite Type IV SS Type III and IV SS Fimbriae 2 chromosomes no plasmids Virulence plasmids OM Exopolysaccharide To become simple and to reduce PAMPs To develop a tough outer membrane Not to release host-damaging agents Exoenzymes Exotoxins Not triggering systemic alarms

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35 Bald bacteria such as Brucella abortus

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37 Brucella Virulence Factors Forestier J Immunol Nov 1;165(9): Celli J Exp Med Aug 18;198(4): O Callagnan, Mol Microbiol, 1999 Delrue, Cell Micro, 2001 Comerci, Cell Micro, 2001 Rohan & Tsolis, I&I, 2007 Guzman-Verri Proc Natl Acad Sci U S A Sep 17;99 (19): Freer Infect Immun Nov;67(11): Giambartolomei, JI 2004 Arellano-Reynoso Nat Immunol Jun;6(6): Conde-Alvarez Cell Microbiol Aug;8(8): From Intracellular Niches of Microbes. Brucella, Monika Kalde, Edgardo Moreno and Jean-Pierre Gorvel Gorvel JP, Moreno E, Moriyón I. Nat Rev Microbiol Mar;7(3):250

38 Gram-negative bacteria envelope Gram-negative Bacteria Ornithine Lipid (Brucella) LPS OM Periplasm IM + cytoplasm Omps Negatively charged sugars Phophates Peptidoglycan Phospholipids PE, PC(Brucella) Brucella LPS

39 Brucella mutants in components of the Outer Membrane : - Phosphatidylcholine (BApcs,BApmtA, BApcspmtA) Conde et al. Cell Microbiology, 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)

40 Brucella is resistant to bactericidal substances of cells Bactenecin 7 Bactenecin 5 Cap18 Cecropin A Cecropin P1 Defensin NP-2 Lactoferricin B Lactoferrin Lysozyme Magainin 1 Magainin 2 Melittin EMP-2 Poly-L-lysine Polymyxin B Poly-L-ornithine EDTA Tris PMNs extract Bactericidal activity (%) H 2 O 2 NO Myeloperoxidase and aldehydes Phospholipase A2 Metaloproteinases Complement Salmonella Brucella

41 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

42 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

43 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

44 Brucella replicates within host cells 10 8 C57BL/6 mice bone marrow-derived macrophages 10 7 CFU/well time post infection (h) Brucella abortus 2308-GFP Brucella-containing vacuole (BCV)

45 Biogenesis of the BCV in macrophages 5 min 1 h 4 h 8 h 12 h Early BCV EEA +, Rab5 +, TfR + Intermediate BCV LAMP1 +, Rab7, M6P (Pizarro Cerda, Many!!!) Replicative BCV LAMP1, Cathepsin D Calreticulin +, Calnexin +, Se61ß +, PDI + Require Rab2, GAPDH (Fugier PloS Pathogen 2010) Sar1 (Celli, J Exp Med, 2003)

46 BCV interact and fuse with the ER during maturation glucose-6-phosphatase detection Chantal de Chastellier

47 Lack of ER fusion with virb mutant-containing vacuoles leads to fusion with lysosomes Ly 12h p.i. BMDM infected with the virb10 Brucella strain glucose-6-phosphatase detection Chantal de Chastellier Celli et al J Exp Med

48 Brucella infection of dendritic cells New cell model for studying Brucella virulence: murine bone marrow-derived dendritic cells Salcedo et al PLoS Pathog.

49 Brucella replicates in DCs Log CFU wt B. abortus 5 4 Salmonella 3 2 virb Time post-infection (h) % DCs with intracellular bacteria time post-infection (h) >10 30 h LPS wt-dsred

50 Brucella replicates in ER-derived vacuoles BCVs positive Lamp1 (%) virb - wt Time after infection (h) KDEL MHCII wt-gfp 24 h Murine bone marrow-derived DCs (C57BL/6 mice) ER Ba Ba Ba ER ER calnexin

51 Does Brucella affect maturation of DCs? 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

52 Brucella does not induce formation of DALIS Ub-proteins MHC-II Dendritic cell Aggresome Like Structures (DALIS) : transient and insoluble structures that appear upon TLR activation Immature (0h) site of storage/ubiquitination for newly synthesized defective proteins 8-14h E. coli LPS Mature (24-36h) DCs with DALIS (%) Time after infection (h) negative Salmonella B. abortus Lelouard et al., 2002

53 Brucella does not induce maturation of DCs 24h DCs with DALIS (%) negative wt virb - HK Brucella FK2 MHCII Salmonella Brucella MHCII LAMP1 E. coli LPS Salmonella Brucella

54 Brucella does not induce surface expression of co-stimulatory molecules

55 Brucella interferes with the immune functions of DCs Brucella-infected DCs do not induce T cell proliferation (neither MHCI nor MHCII)

56 Identification of Btp1 (Brucella TIR-containing protein) Btp1 8 wt btp1-7 Tir2 6 Log CFU 5 4 wt virb - btp Time post-infection (h) MHCII

57 Btp1 contributes to blocking DC maturation Infected cells with DALIS (%) neg HKB 0 DALIS (24h) wt btp1 - TFNα (pg/ml) IL12 (ng/ml) neg neg IL12 HKB wt TFNα HKB wt btp1 - btp1-24 h 48 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

58 Btp1 interferes with TLR2 signalling Relative luciferase activity neg +PAM Btp1 + PAM PipB2 + PAM TLR2 Relative luciferase activity neg +CpG Btp1 + CpG PipB2 + CpG TLR9

59 Btp1 acts on the myd88/tlr2 pathway Wild-type Brucella Btp1 mutant % DCs with DALIS Control cgs- mutant % DCs with DALIS wt B. abortus control wt myd88 TRIF TLR2 TLR4 TLR7 TLR9 mice wt myd88 TRIF TLR2 TLR4 TLR7 TLR9 mice Newman et al., I&I, 2006 Salcedo et al., PloSpathogen, 2008 Cirl et al., Nat Med, 2008 Radakrishnan et al., JBC, 2009 (Tirap) Sengupta et al., JI, 2010 (Mal)

60 Periplasmic glucans outer membrane periplasm inner membrane Brucella E. coli

61 Cyclic glucan prevents Brucella fusion with lysosomes cgs cathepsin D cgs + CßG Arellano-Reynoso et al Nat Immun

62 Molecular properties MβCD Brucella CβG I: CβG is not toxic for also shown in vivo in II: CβG extracts cholesterol from membranes but with Less efficiency than MβCD III: CβG modifies lipid raf Characteristics (flotation gra MβCD: Methyl-beta-cyclodextrin Arellano Reynoso, Nat Immunol, 2005

63 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

64 Platelet aggregation 11 Monocyte PMN Prostaglandins Leukotrines Histamine Chemotaxis Mast cell 13 C3a. C5a. C5b67 C3a C5a C activation Mφ Bradykinin Endothelial damage Tissue damage 6 Plasmin 7 8 Fibrinopeptides 4 9 Fibrinogen synthesis Degranulation ROS. killing Defensins MIP -1α MIP-1β CXCL8 TNF-α IL-1β Fever. edema. leukocytosis. trombocytopenia Endotoxic shock 18 Chemokines CXCL8 CXCL1 CCL5 CCL4 CCL3 CCL1 Cytokines TNF-α IL-1 IL-1β IL-12 IL Mφ Activation killing INF-γ TNF-α Apoptosis IL-18 Apoptotic body 16 3 Phagocytosis of apoptotic bodies DC Mature DC