The FIIND domain of Nlrp1b promotes oligomerization and pro-caspase-1 activation in response to lethal toxin of Bacillus anthracis by Vineet Joag A thesis submitted in conformity with the requirements for the degree of Masters of Science Graduate Department of Laboratory Medicine and Pathobiology University of Toronto ©Copyright by Vineet Joag (2010) The FIIND domain of Nlrp1b promotes oligomerization and pro- caspase-1 activation in response to lethal toxin of Bacillus anthracis Vineet Joag Masters of Science Laboratory Medicine and Pathobiology University of Toronto 2010 Abstract Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like- receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self- associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity. ii Acknowledgements I would first like to thank my supervisor, Dr. Jeremy Mogridge. His guidance, willingness, and open-mindedness in discussing science led to my development as a Masters Student. Jeremy leads by example and the clarity with which he seeks to answer biological questions encourages me to understand the subject matter comprehensively. I am also grateful for his financial support during the past 3 years. In addition to Jeremy, I would like to extend my gratitude to past and present members of the Mogridge lab: Sarah, Edith, Mandy, Teresa, Bradley, Kristopher, Mia, Vincent and Kuo Chieh. I would especially like to thank Kuo Chieh with whom I have worked closely. I would also like to thank my family and friends for their continual support during my graduate studies. I would like to thank members of my supervisory committee Dr. Philip Sherman and Dr. Dana Philpott for their valuable time and input as well as Dr. Harry Elsholtz and other faculty members as mentors. iii Table of Contents Chapter 1: Introduction………………..……………………………………………………..…1 1.1 Anthrax…………..………………………………………………………….….....1 1.2 Poly-D-glutamic acid capsule…………………….…………………………….…3 1.3 Anthrax toxins……….………………………………………………………….....3 1.3.1 Toxin assembly and internalization………….……………………………3 1.3.2 Edema toxin……………………………………….………………………6 1.3.3 Lethal toxin……………………………………………..…………………8 1.3.3.1 Lethal factor……………………………….…………....................8 1.3.3.2 LeTx and impairment of the immune response……………………9 1.3.3.3 Lethal toxin and macrophage death……………………………..10 1.4 Nlrp1b……………………………………………………………………….…...14 1.4.1 Nod-like-receptors……………………………………………………….15 1.4.2 Caspase-1 inflammasomes…………………………………………….....18 1.4.3 Inflammasome assembly and pro-caspase-1 activation……………...…..20 1.4.4 Key cellular events involved in intoxication, Nrlp1b activation, and cell death……………………………………………………………………..23 1.5 Characterization of Nlrp1b………………………………………………………26 1.5.1 Rationale of project………………………………………………………28 1.5.2 Hypothesis………..………………………………………………………29 1.5.3 Summary of project………………………………………………………29 Chapter 2: Results………………………………………………………………………………30 2.1 N1100 is the minimal region of Nlrp1b that constitutively activates pro- iv caspase-1 and self-associates…………….………………………………………30 2.2 Residues 1100EIKLQIK1106 are critical for pro-caspase-1 activation potential of N1100 and for self-association…...………………………………………………..32 2.3 The 7A mutation does not impair binding of N1100 to pro-caspase-1-C284A…...37 2.4 Residues 1100EIKLQIK1106 of Nlrp1b are critical for Nlrp1b mediated pro-caspase- 1 activation in response to LeTx……………………………………..…………..37 Chapter 3: Discussion……………………………………………………….………………….40 3.1 The FIIND domain promotes self-association of Nlrp1b and activation of pro- caspase-1………………………………………………………………..………..40 3.2 Cleavage of Nlrp1b in the FIIND domain correlates with potential of Nlrp1b to promote pro-caspase-1 activation………………………………………………..43 3.3 Future Directions……………………………………………………………...…44 Chapter 4: Materials and Methods………………………………………………………........49 4.1 Cell lines…………………………………………………………………………49 4.2 Plasmid construction and site-directed mutagenesis……………………………..49 4.3 IL-1β and LDH release assays…………………………………………………...52 4.4 Detection of TAP-tagged proteins……………………………………………….53 4.5 Co-immunoprecipitation assay - self-association of Nlrp1b truncation mutants...53 4.6 Co-immunoprecipitation assay - association between pro-caspase-1-C284A and N1100, and N1100-7A………………………………………………………...…….54 4.7 Antibodies……………………………………………………………………..…54 References……………………………………………………………………………………….56 v List of Tables Table 1. Nomenclature for N-terminal truncation mutants of Nlrp1b and for alanine substitution mutants of Nlrp1b1100-1233………………………………………………………………………...29 List of Figures Figure 1. Anthrax toxin assembly and internalization…………………………………………….4 Figure 2. Domain organization of various Nod-like-receptors (NLRs), CARDINAL, ASC, and pro-caspase-1…………………………………………………………………………………….17 Figure 3. Assembly of NLRP3, NLRP1, and Nlrp1b inflammasomes……...…………………...22 Figure 4. N1100 is the minimal region of Nlrp1b that activates pro-caspase-1 and self-associates in the absence of LeTx……………………………………………………….……………………..31 Figure 5. Residues 1100EIKLQIK1106 are critical for pro-caspase-1 activation by N1100…………33 Figure 6. Substitution of residues 1100EIKLQIK1106 with alanine abolishes self-association of N1100 but does not affect its binding to procaspase-1.……...……………………………….…....36 Figure 7. Substitution of residues 1100EIKLQIK1106 with alanine in full-length Nlrp1b inhibits LeTx-induced pro-caspase-1 activation and cell death…………..………..…….……………….39 Figure 8. The A3/1 mutant differs from Nlrp1b allele 3 by 6 amino acids in the FIIND domain…………………………………………………………………………………………....46 vi List of Abbreviations ANTXR1 Anthrax toxin receptor 1 ANTXR2 Anthrax toxin receptor 2 ASC Apoptosis associated Speck-like-protein ATP Adenosine triphosphate BMDM Bone Marrow Derived Macrophages BSA Bovine Serum Albumin cAMP Cyclic adenosine monophosphate CARD Caspase Activation and Recruitment Domain Casp1 Caspase-1 Casp5 Caspase-5 CBP Calmodulin Binding Peptide CMG2 Capillary Morphogenesis Protein 2 CRE Cyclic AMP responsive element CREB Cyclic AMP responsive element binding protein DC Dendritic Cell DMSO Dimethylsulfoxide DRM Detergent resistant membrane EdTx Edema Toxin EDTA Ethylenediaminetetraacetic acid EF Edema Factor EGFP Enhanced Green Fluorescent Protein ERK Extracellular-Signal Regulated Kinase FIIND Function Unidentified Domain GST Glutathione S-transferase IFN Interferon IL Interleukin JNK c-Jun N-terminal kinase LD Lethal Dose LDH Lactate Dehydrogenase LeTx Lethal Toxin LF Lethal Factor LMP Lysosomal Membrane Permeabilization LPS Lipopolysaccaride LRR Leucine-rich-repeat Ltxs Lethal-toxin sensitive MAMP Microbe-Associated-Molecular-Pattern MAPK Mitogen Activated Protein Kinase MDP Murymyl Dipeptide MEK1 MAPK/ERK kinase 1 MHC Major Histocompatibility Complex MKK Mitogen Activated Protein Kinase Kinase NACHT NAIP CIITA HET-E TP1 NF B Nuclear Factor Kappa B vii NLR Nod-like receptor NLRP Nod-like receptor protein NOD Nucleotide-binding and oligomerization domain PA Protective Antigen PAGE Polyacrylamide gel electrophoresis PMA Phorbol 12-myristate-13-acetate PMN Polymorphonuclear leukocyte PKA Protein Kinase A PG Peptidoglycan PRR Pattern-recognition-receptor PYD Pyrin SDS Sodium dodecyl sulfate TEA Tetra-ethyl ammonium acetate TEM8 Tumor Endothelial Marker 8 TLR Toll-like receptor TNF Tumor Necrosis Factor UVB Ultraviolet B VEGF Vascular Endothelial Growth Factor viii Chapter 1 Introduction 1.1 Anthrax Study of the bacterium that causes anthrax has played an important role in microbiology. Robert Koch first identified the microbe which caused anthrax disease in 1875, and this was the first demonstration that established a causal relationship between microbes and disease. Based on this work, Koch developed a set of guidelines called the Koch’s Postulates which continues to inform the approach to microbiologic diagnosis even today. Following Koch’s discovery, Louis Pasteur successfully tested the first anti-anthrax vaccine in domesticated animals (Turnbull, 2002). The study of anthrax has led to many scientific advances in the field of microbiology and vaccinology. However, the development and misuse of anthrax as a biological weapon, exemplified by the accidental release of weapons-grade anthrax in Sverdlovsk, Russia and the terrorists attacks in September 2001 in the United States have caused serious concern in the international community. Bacillus anthracis, a rod shaped, Gram-positive, spore-forming bacterium is the causative agent of anthrax. As a spore the bacterium does not replicate, and can survive adverse environmental conditions (Gould,