New virulence strategy of P. aeruginosa clonal outliers
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1 New virulence strategy of P. aeruginosa clonal outliers Ina Attrée Bacterial Pathogenesis and Cellular Responses CNRS, CEA Grenoble University Grenoble-Alpes Grenoble, France
2 Pseudomonas aeruginosa virulence Gram-negative bacillus, motile 10% of nosocomial infection acute and chronique infections cystic fibrosis and COPD wounds + Ventilator-Associated Pneumonia (Intensive Care Units) + septicemia New targets for «anti-virulence» drugs?
3 Pseudomonas aeruginosa T3SS & toxins Accepted dogma : Pa virulence depends on its Type III Secretion System and Exotoxins PBS/Mock PAO1 (ExoS+) toxin injection by T3SS ExoU ExoS Host cell ExoT ExoY two types of T3SS strains 70% ExoS + (PAO1) 30 % ExoU + (PA14) T3SS target for «anti-virulence» drugs
4 Clinical P. aeruginosa outliers without T3SS Classical strains (T3SS+) PAO1 PA7-like strains (T3SS-) PA1689 T3SS locus (35kb) Type I deletion bglx pscu pscl PA14 Environmental strains Clinical isolates CF COPD urinary tract Hyper virulent in mice Haemorragic pneumonia Type II deletion
5 Exolysin is a pore-forming toxin expressed in clonal outliers Identification of the toxin, Exolysin, by comparative proteomics of bacterial secretoms Classical TPS system (T5SS) SP POTRA POTRA ExlB - Outer Membrane Protein (63kDa) SP RGD RGD RGD RGD RGD TPS HAEMA Fil HAEM ExlA - secreted protein (172 kda) Epithelial membrane disruption by ExlA + P. aeruginosa Basso et al., mbio, 2017 Elsen et al., Cell Host Micro, 2014
6 Draq7 Hoechst ExlA activity requires adhesion by Type IV pili Tn library (7000 mutants) in ExlA + isolate Screen : Live/Dead assay on infected A549 cells/ High-content imaging Observations : Need for live bacteria for cytotoxicity Purified rexolysin is inactive Transwell system : no cytotoxicity WT Tn_ mutant All pil mutants are affected in twiching All pil mutants do not adhere to cells All pil mutants secrete ExlA Cytotox >80% Cytotox <30%
7 Exolysin (ExlA) is a unique pore-forming toxin Different mechanism of obtaining optimal local toxin concentration Pore forming toxins: active as soluble Concentration-dependent membrane insertion Requirement of specific cellular receptor ExlA : Secreted toxin alone biologically non active Requires active secretion (live bacteria) Requires adhesion/proximity (pili) Basso et al., 2017, mbio
8 ExlA shapes P. aeruginosa virulence PAO1 (ExoS+) CLJ1 (ExlA+) Broncho-alveolar lavages Mock PAO1 PA7 CLJ1 (ExoS) (ExlA-) (ExlA+) Bouillot et al., 2016, Sci. Rep.
9 Effects of ExlA on adherens jonctions Lung Epith 90 kda NTF 120 kda FL ExlA Basal lamina Blood vessel E-cad VE-cad Endoth 30 kda CTF E-cad Host Cell 00:30 01:30 02:00 02:30 03:10 04:00 04:40 05:10 06:00 CLJ1 IHMA87 IHMA87 exla 00:30 01:30 02:00 02:30 03:10 04:00 04:40 05:10 06:00 00:30 06:00 00:30 06:00 PAO1F Reboud et al., PLOS Pathogens, A549 E-cadherin-GFP Propidium iodide (nuclei) Hypothesis ExlA ADAM kda FL E/VE-cad 90 kda NTF 30 kda CTF
10 ExlA activates ADAM10 and Cadherin cleavage through Ca 2+ influx GM6001 metalloprotease inhibitor PROTECTION IHMA87 GI254023X ADAM10 inhibitor cell-permeable calcium probe Fluo3-AM IHMA87 Ca 2+ entry PI entry KO ADAM10 cells are resistant to ExlA-induced Cad cleavage Two specific inhibitors protect cells from Cad cleavage Ca 2+ Maturation of ADAM10 IHMA87 exla Dissociation of Calmodulin-proADAM10 complex IHMA87 exla
11 Responses of macrophages to ExlA High-content live-imaging of bacterial cytotoxicity > several genotypes of host-cells versus different strains/bacteria > access to kinetics of infection Basso et al., Env Microbiol., 2017
12 Death pathways induced by bacterial infection Pyroptosis Apoptosis Apoptosis Necroptosis Necroptosis Pyroptosis pyros (greek; fire, heat) inflammasomes Caspase-1 activation IL-1β production
13 Bone Marrow Derived Macrophages (BMDMs) 5 different genotypes Femur Thomas Henry Pierre Wallet 5 different genotypes of macrophages (BMDMs) Wild-type Casp1 -/- Casp11 -/- ASC -/- NLRP3 -/- AIM2 -/-
14 Caspase-1 KO BMDMs are resistant to ExlA ExlA + Wild-type BMDMs Caspase-1 activation IL-1ß maturation Casp-1 KO NI LPS +Nig Pa ExlA + ExlA + P. aeruginosa induces pyroptosis of primary macrophages LPS+Nig ExlA+ ExlA-
15 Caspase-1 is activated through NLRP3-ASC inflammasome ASC and NLRP3 KO BMDMs are resistant to ExlA + infection P. aeruginosa expressing Exolysin activate inflammasome in macrophages ExlA induces pyroptosis of macrophages accompagnied by Caspase-1 cleavage and IL1- β release
16 ExlA+ P. aeruginosa ExlA-like toxins are distributed among Pseudomonas species P. fluorescens P. entomophila P. protegens P. putida ExlA-like sequences were analyzed by neighbor joining method using Phylo-widget.
17 Insights in Exolysin regulation 3
18 Take-home message two clades of P. aeruginosa clinical strains two different strategy of virulence Exolysin (ExlA) is a TPS pore-forming toxin Activity requires contact/pili (T4P) Is responsible for * adherens jonction cleavage * inflammasome NLRP3 activation * pyroptotic death of macrophages ExlA-like toxins are present in several Pseudomonas sp. Origins?
19 Pauline Basso Stéphanie Bouillot François Cretin Sylvie Elsen Eric Faudry Philippe Huber Antoine Maillard Tuan Dung Ngo Stéphane Pons Michel Ragno Émeline Reboud Mylène Robert-Genthon Erwin Santausa Julian Trouillon Thomas Henry Pierre Wallet Laëtitia Kurzawa (Confocal) Emmanuelle Soleilhac (HC) Yohann Couté (Proteomics) Stephen Lory
20 % Hemolysis Pore-forming activity of Exolysin-producing Pa Pore forming activity on red blood cells (RBCs) H 2 O EBM2 RBC Osmotic lysis (hemolysis A 560nm ) α-hemolysin (Staphylococcus aureus) P. aeruginosa ExoU (phospholipase) P. aeruginosa ExlA % Hemolysis Glucose Sucrose Raffinose Maltopentose PEG 1000 PEG 2000 Inhibition of hemolysis by sugars and PEGs Estimation of pore-size 0 α-hemolysin Pa ExoU+ * pore size estimation (1.6 nm) α-hemolysin (1.3 nm) Streptolysin O (30 nm) Pa ExlA+ Basso et al., mbio, 2017
21 Evidence for pore-formation in epithelial cells (A549) Incorporation of Yo-Pro (629 Da non-permeable nucleic acid dye)
22 Cytotoxicity (% of LDH release) camp and Vfr regulate ExlA expression Vfr binding site exlb exla Alice Berry and Sylvie Elsen, non published data
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