From bugs and men to treatments and vaccines Philippe Sansonetti & collaborators Chaire de Microbiologie et Maladies Infectieuses, Collège de France Unité de Pathogénie Microbienne Moléculaire, & Unité INSERM 786 Institut Pasteur 3 èmes rencontres internationales de la recherche June 10, 2011
Symbionts Innate immunity Physiological inflammation Surveillance/Tolerance Recognition network: TLRs,NLRs, Rig1, MDA5… Danger signals: (uric acid, ATP, cytochrome C,etc..) Pathogens Innate immunity Pathological inflammation Microbe & tissue destruction Amplification loop: TREM, HMGB1, Gal3, Severe sepsis Septic shock Regulation Loss of control Adaptive immunity Pathogens recognition, capture, completion of eradication process, protection Sansonetti, 2004, Nature Rev. Immunol. Sansonetti, 2006, Nat. Immunol. Sansonetti & Di Santo, 2007, Immunity Sansonetti & Medzhitov, 2009, Cell Sansonetti, 2010, Mucosal Immunology Man is a primate-microbes hybrid Concept of HOLOGENOME / SUPERORGANISM - Rupture of homeostasis = IBD - Dysbiosis = obesity, diabetes, metabolic syndrome Turnbaugh & Gordon. 2009. J.Physiol. Human colon: 10 11 bacteria / g feces 500-1000 species,10x human cells 150x human genes Quin, 2010, Nature
Bacterial life at mucosal surfaces « Seating on a volcano » O2 NO ROS Anti-microbial peptides Other: lysozyme, proteases, lectins, phospholipases Transmigrating phagocytes Epithelium Mucus Cationic antimicrobial peptide hBD3 Survive Subvert Arbibe et al., Nature Immunol., 2007 Sperandio et al., J. Exp. Med., 2008 Marteyn et al., Nature, 2010
Pro-inflammatory cascade TLR NLR Pro-inflammatory genes PMN Activated DC & M Th1 - Th17 / Treg OCTN2 PepT1 QSM MDP TLR NLR Regulatory cascade ? Regulatory genes Mucus Regulatory cytokines, chemokines Immature DC & M Treg / Th1 – Th17 PATHOGENS Mucinases Adhesins / Invasins Type III / IV secretory systems Hemolysins Massive engagement of TLRs, NLRs Eradication of commensal microbiota (niche occupancy) + Dampening innate / inflammatory responses SYMBIONTS Absence (limitation) of virulence factors PAMPs less agonist ? Sequestration, weak activity of TLRs Life in biofilms on mucus surface Controled diffusion and sampling of PAMPs and prokaryotic signalisation molecules, including regulatory molecules (i.e. PSA of B. fragilis (Mazmanian & al.) Antimicrobial molecules Pro-inflammatory cytokines, chemokines PMN DC Physiological inflammation Toleranceb Pathological inflammation PATHOBIONTS SFB H. hepaticus Effectors ?
Intestinal lumen « Milieu intérieur »/ organs : PAMPs, other bacterial factors/effectors metabolites CNS (maturation, behaviour, inflammation) Vessels (maturation, inflammation) Adipose tissue (insulin resistance, obesity, diabetes) Liver (sugar & lipid metabolism) Lymphoid organs (maturation, mucosal & systemic physiological inflammation) Microbiota as an extra organ PAMPs, as hormones + mediators produced by the epithelium under « pressure » of the microbiota - Crossing of organ barriers (endothelium, BBB, etc…) - Receptors / specialized cells : Host mediators chemokines cytokines hormones neuromediators Intestinal epithelial barrier The host-microbiota symbiosis affects development, metabolism, immunity, what else ?
Signature Tagged Mutagenesis in Lactobacillus casei Hélène Licandro, Jean-Françios Cavin, Thierry Pédron Sequencing of mutations Assembling in pools of high interest “ Cell surface” “ Regulation” “ Random” Gavage Quantification of mutants Transposase production pVI110 transposition X72 Development of an efficient tool to generate random mutants of L. casei with a two-step procedure. Adaptation for STM and generation of a 7000 random tagged-mutants library. All mutations currently sequenced (Sanger Center) to optimize the use of STM by assembling tagged mutants in pools of predicted high physiological interest. Tagged mutants pools tested in a mouse model to determine genes involved in colonization and understanding of the role played by these genes. In vitro/in vivo assays to identify effector functions on cell cycle, cell differentiation, metabolism, anti-inflammation, etc…
Colon Follicle-associated epithelium Small intestine Mucus Proliferative compartment (PC) Differentiating compartment (DC) Desquamating cells Cell cycle arrest Stem cells (SC) compartment Stem cells compartment Cell cycle arrest PC DC Paneth cell Crypt-Based Columnar (Lgr5+) SC +4 Radiation-resistant SC Crypt-Based Columnar (Lgr5+) SC Paneth cells ? M cells Dendritic cells Macrophages T lymphocytes B Lymphocytes IEC Paneth cell Goblet cell Enterochromaffin cell The 4 gut epithelial lineages Proliferative progenitors Absorbtive secretory cells Lieberkühn’s crypt A crypt-specific core microbiota in gut homeostasis, restitution… and cancer ? Crypt stem cells (Lgr5+) Lgr5-GFP Crypt-specific core microbiota FISH
Epithelial cells mucus Basolateral macropinocytosis (TTSS) Vacuole lysis (TTSS) Escape to autophagy Motility, cell to cell spread (TTSS) IcsA ? ? M cell M B Lympho M pyroptosis TTSS/IpaB <ul><li>Pyroptosis = proinflammatory apoptosis </li></ul><ul><li>Activation of caspase-1, Release of IL-1 and IL-18 </li></ul>DC «facilitated translocation» Follicle-associated epithelium PNN Nod1 PGN NF- B JNK TTSS Pro-inflammatory genes IL-8, other cytokines chemokines <ul><li>- Development of inflammation </li></ul><ul><li>Rupture of epithelial barrier </li></ul><ul><li>- Facilitation of invasion </li></ul><ul><li>Stimulation of epithelial bactericidal capacities </li></ul>Defensins and other bactericidal molecules CCL-20 Rupture, invasion and inflammatory destruction of the intestinal epithelium by Shigella : the key steps of TTSS function, a gold mine to identify new effectors and pathways of innate immune responses B Lympho T Lympho
VirB ipaA, ipaB, ipaC, ipaD, ipgB1, ipgD , icsB, ospC2/3/4, ospD1, ospD2 ospD3, ospE1/2, ospG , ipaH1/2, ipaH4, ipaH7, ipaH9.8 MxiE Expression / regulation / function of type III effectors before secretion after TTSS activation (target cell recognition) INVASION Modulation of INNATE RESPONSES IpaB, IpaC, IpaA, IpgB1,VirA, IpgD IpgD : phosphatidyl-inositol phosphatase, hydrolyses P in 4 in Pi(4,5)P2 (Niebuhr et al, 2002, Pendaries et al, 2006 EMBO J.). Anti-inflammatory +++ (Puhar et al., in preparation). OspG : kinase,binds/blocks ubiquitin transfer protein E2, protects I-kB from degradation. Anti-inflammatory +++ (Kim et al., 2005, PNAS). OspF : dephosphorylation of Erk1/2, epigenetic regulation of pro-inflammatory genes - i.e. IL-8. Regulates transmigration of PMNs through epithelium (Arbibe et al., 2007,Nat.Immunol.). Phosphothreonine lyase (Li et al., 2007, Science). IpaHs : (5 + 5 chromosomal copies): New family of Ubiquitin ligases (E3) (Rohde et al., 2007, Cell Host & Microbes ) IpaH9.8 targets NEMO (Ashida et al., 2010, Nat.Cell Biol.) OTHER PHENOTYPES IcsB: inhibion or autophagy (Ogawa et al., 2005, Science) VirA: inhibition of microtubules, facilitates actin-based motility (Yoshida et al., 2006, Science) ospB ospF ospC1 virA
cellules épithéliales mucus macropinocytose baso-latérale (TTSS) lyse vacuole (TTSS) motilité/passage cellule-cellule (TTSS) IcsA M cell M Lympho B M pyroptosis TTSS/IpaB <ul><li>- activation of caspase-1 </li></ul><ul><li>pyroptosis = pro-inflammatory apoptosis </li></ul><ul><li>release of IL-1 et IL-18 </li></ul>DC «facilitated translocation» follicle-associated epithelium Nod1 PGN NF- B JNK TTSS Osp(s) TTSS IL-8 CCL-20 PNN Suppression of humoral defense mechanisms Suppression of cellular defense mechanisms AMPs Arbibe et al. Nat. Immunol., 2007 Sperandio et al., J. Exp. Med., 2008 Puhar et al., in preparation ATP ATP Suppression of danger signaling PR
Tran Van Nhieu et al., Nat. Cell Biol. 2003 Puhar et al., in preparation ATP = danger signal Inflammasome activation Differentiation of naive T cells to « inflammatory » Th17 cells HEMICHANNEL (Connexins) xd xd xd xd xd xd xd xd xd IpgD Pi(4,5)P2 Pi(5)P IpgD IpgD neg. phenotype wt phenotype Pi(5)P IpgD impairs danger signaling
IpgD impairs T cell polarization PIP 2 binding causes ERM conformation change between inactive and active / phosphorylated forms ERM proteins Ezrin, Radixin, Moesin: crucial role in cell polarization during T lymphocyte migration Lee JH et al. 2003 IpgD-mediated PIP 2 cleavage with subsequent reduction of PIP 2 pool at the plasma membrane. What about pERM ? IpgD injection/expression causes hydrolysis of Pi(4,5)P2 and dephosphorylation of Ezrin IpgD-mediated hydrolysis of PI(4,5)P2 causes activated T cell depolarization and loss of oriented movement in presence of chemokine CXCL12 Konradt et al., Cell Host Microbe, 2011
CD4 + T cell dynamics at 4h mm/min Velocity Uninfected T3SS - WT Uninfected T3SS- WT Salgado-Pabon et al., in preparation Straightness Confinement Uninfected T3SS - WT *** *** *** % Arrest Arrest Uninfected T3SS - WT *** ***
Perspectives To continue to decipher the pathways of innate and adaptive protection against pathogens by analysing how bacterial « anti-immunity » effectors subvert the molecular and cellular mechanisms of immune defenses (i.e. target identification) To identify the molecular effectors and mechanisms by which symbionts regulate host local and systemic innate and adaptive immune responses, and other key areas reflecting the duration, intimacy and depth of the host-microbe symbiosis (i.e.: metabolism, tissue repair, cancer…) To rationally develop novel therapeutics and vaccines based on understanding microbe-host cross talks