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Principal Investigator: Stephen Leppla
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2019
Award: $971,268
Funding agency: National Institute of Allergy and Infectious Diseases
Anthrax toxin protective antigen protein (PA) binds to receptors on the surface of mammalian cells, is cleaved by cellular proteases, forms an oligomer, and transports two other toxin proteins, lethal factor (LF) or edema factor (EF) to the cytosol. EF is a potent calmodulin-dependent adenylyl cyclase. LF is a metalloprotease that cleaves and inactivates several mitogen-activated protein kinase kinases (MEKs). In rodents LF also cleaves and activates inflammasome sensor NLRP1. The inflammasomes are intracellular complexes that play a role in innate immune response. The cleavage of NLRP1 in macrophages and dendritic cells leads to caspase-1 activation, a rapid cell death termed pyroptosis, maturation and release of the pro-inflammatory cytokines IL-1 and IL-18 and recruitment of innate immune cells.
The inactivation of the MEK pathways by the toxin, alongside the ability to redirect the toxins to specific cell types through modification of the cleavage site on the receptor binding PA moiety allows the use of these toxins as anti-tumor therapeutics. For example, PA can be modified to be cleaved by specific matrix metalloproteinases (MMPs) which are overexpressed in certain cancers.
In FY2019 in a collaboration with UK colleagues, we utilized radiolabeling of modified toxin proteins to image MMP activity in tumor tissues. We first characterized PA mutants in which the furin cleavage site in PA was modified to an MMP-specific sequence. DOTA-GA maleimide was conjugated to LF to allow its labeling by two different methods and functionality of the toxin for cellular translocation was verified post-labeling. After development of efficient 111-Indium-labeling for the purpose of imaging MMP activity in tumors, the labeled LF toxin was used in conjunction with mutant PA proteins which require MMP for activation to assess MMP activity in cancer cell lines. MMP activity of a panel of cancer cells could be correlated to levels of activation of mutant PA. Furthermore, SPECT/CT imaging in vivo allowed tracking of labeled PA and tumor visualization in animals. Only modified PA proteins and not wild type toxin delivered labeled LF molecules to MMP-expressing tumors, showing the specificity of the tumor-targeting by mutant PA molecules. We believe labeled LF and modified PA molecules may be promising imaging agents for proteolytic activity in cancer.
In other collaborative studies during this reporting period we utilized the unique characteristics of the anthrax toxins for delivery of cargo to neuronal cells. We created a novel fusion of the N-terminal region of LF to nuclear receptor-related 1 (Nurr1), a protein which is important in maintenance of dopaminergic neurons. Delivery and subsequent cytoplasmic release of Nurr1 was associated with increased levels of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, and with a concentration-dependent protection of cells against a neurotoxin, 6-hyroxydopamine. These protective effects suggest Nurr1 delivery as a potential new treatment option for Parkinsons disease.
We continued our work on the multicomponent Bacillus cereus pore-forming toxin HBL, associated with food poisoning induced by this bacterium. We had previously demonstrated stepwise sequential assembly of the tripartite toxin on cells. In these new studies the ability of the HBL toxin to activate the NLRP3 inflammasome to induce pyroptosis and secretion of inflammatory cytokines was investigated. We found the NLRP3 inflammasome played an important role in mediating lethality by B. cereus in mice, as knockout mice were resistant to infection at doses where wild type mice succumbed. Furthermore, pharmacological inhibition of the NLRP3 inflammasome was able to protect against bacterium-induced lethality, suggesting one of the roles the HBL toxin plays is in induction of inflammasome-mediated pathology.
Finally, during this year, in a continuation of collaborative studies on anthrax toxin-induced effects on pulmonary vascular permeability in the isolated rat lung model, we found that lethal toxin (LT) increases pulmonary arterial pressure and pulmonary permeability, while edema toxin (ET) decreases pulmonary artery pressure and does not alter permeability in isolated lungs. Both toxins may induce organ dysfunction through these opposing effects during lethal infection. These findings suggest potentially different therapeutic approaches may be needed at different stages of infection depending on levels of each toxin.
Terms: <3'5'-cyclic ester of AMP><3,5 cyclic AMP synthetase><ATGN><ATP-protein phosphotransferase><Adenosine Cyclic 3',5'-Monophosphate><Adenosine Cyclic Monophosphate><Adenosine, cyclic 3',5'-(hydrogen phosphate)><Adenyl Cyclase><Adenylate Cyclase><Adenylyl Cyclase><Animal Model><Animal Models and Related Studies><Animals><Anthrax><Anthrax disease><Antibiotic Agents><Antibiotic Drugs><Antibiotics><Antigens><Apoptosis-Related Cysteine Protease Caspase 1><B anthracis><B anthracis LeTx><B anthracis LeTx toxin><B cereus><B. anthracis><B. cereus><Bacillus anthracis><Bacillus anthracis LF protein><Bacillus anthracis lef protein><Bacillus anthracis lethal toxin><Bacillus cereus><Bacteria><Binding Proteins><CASP-1><CASP1><CASP1 gene><CAT scan><CSAID-Binding Protein 1><CSAID-Binding Protein 2><CSBP2><CT X Ray><CT imaging><CT scan><Calcium-Dependent Activator Protein><Calcium-Dependent Regulator><Calmodulin><Cancer cell line><Cancers><Caspase-1><Caspase-1 Gene><Cell Body><Cell Communication and Signaling><Cell Death><Cell Protection><Cell Signaling><Cell model><Cells><Cellular biology><Cellular model><Cellular translocation><Characteristics><Cleaved cell><Collaborations><Common Rat Strains><Complex><Computed Tomography><Cyclic AMP><Cytokine-Suppressive Antiinflammatory Drug-Binding Protein 1><Cytokine-Suppressive Antiinflammatory Drug-Binding protein 2><Cytoprotection><Cytosol><DA Neuron><Dendritic Cells><Development><Disease><Disorder><Dopamine><Dopamine neuron><Dose><Dropsy><Dysfunction><EMI scan><Edema><Enzyme Gene><Enzymes><Esteroproteases><Food Poisoning><Functional disorder><Goals><Hydrops><Hydroxytyramine><ICE Protease><IFN-gamma-Inducing Factor><IGIF><IL-1><IL-1 Gamma><IL-1 beta Convertase><IL-1 beta-Converting Enzyme><IL-18><IL-1BC><IL-1b Converting Enzyme><IL-1g><IL1><IL18 Protein><IL1B-Convertase><IL1BC><IL1BCE><IL1F4><Image><Imagery><Immune><Immunes><In-111><In111 isotope><Indium-111><Infection><Inflammasome><Inflammatory><Innate Immune Response><Interferon-gamma-Inducing Factor><Interleukin 1-B Converting Enzyme><Interleukin 1-Beta Convertase><Interleukin 18 (Interferon-Gamma-Inducing Factor)><Interleukin 18 Proprotein><Interleukin I><Interleukin-1><Interleukin-1 Beta Converting Enzyme><Interleukin-1 Converting Enzyme><Interleukin-1 Gamma><Interleukin-18><Interleukin-18 Precursor><Intracellular Communication and Signaling><KO mice><Kinase Family Gene><Kinases><Knock-out Mice><Knockout Mice><Label><LeTx><Ligand Binding Protein><Ligand Binding Protein Gene><Lung><Lung Respiratory System><Lymphocyte-Stimulating Hormone><MAP Kinase Kinases><MAPK Kinases><MAPK14><MAPK14 Mitogen-Activated Protein Kinase><MAPK14 gene><MAPKKs><MGC12320><MMPs><Macrophage Cell Factor><Maintenance><Maleimides><Malignant Cell><Malignant Neoplasms><Malignant Tumor><Mammalian Cell><Matrix Metalloproteinases><Mediating><Metallopeptidases><Metalloproteases><Metalloproteinases><Methods><Mice><Mice Mammals><Miscellaneous Antibiotic><Mitogen-Activated Protein Kinase 14><Mitogen-Activated Protein Kinase Kinases><Modeling><Modification><Murine><Mus><Mxi2><N-terminal><NH2-terminal><Nerve Cells><Nerve Unit><Neural Cell><Neurocyte><Neurons><Neurotoxins><Nuclear Receptors><Null Mouse><Organ><Paralysis Agitans><Parkinson><Parkinson Disease><Parkinson's><Parkinson's disease><Parkinsons disease><Pathogenesis><Pathology><Pathway interactions><Peptidases><Peptide Hydrolases><Permeability><Pharmacology><Phosphodiesterase Activating Factor><Phosphodiesterase Protein Activator><Phosphotransferase Gene><Phosphotransferases><Physiopathology><Play><Primary Parkinsonism><Protease Gene><Proteases><Protein Binding><Protein Kinase><Proteinases><Proteins><Proteolytic Enzymes><Pulmonary Artery><Pulmonary artery structure><Radiolabeled><Rat><Rats Mammals><Rattus><Receptor Protein><Reporting><Resistance><Rodent><Rodentia><Rodents Mammals><Role><SAPK2A><SPECT><SPECT imaging><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Single-Photon Emission-Computed Radionuclide Tomography><Site><Stress-Activated Protein Kinase 2A><Surface><T Helper Factor><Therapeutic><Tomodensitometry><Toxin><Transphosphorylases><Tumor Tissue><Tyrosine 3-Monooxygenase><Tyrosine Hydroxylase><Veiled Cells><Virulence><Visualization><Wild Type Mouse><Work><X-Ray CAT Scan><X-Ray Computed Tomography><X-Ray Computerized Tomography><Xray Computed Tomography><adenosine 3'5' monophosphate><adenylcyclase><anthracis><anthrax lethal factor><anthrax lethal toxin><anthrax toxin><anthrax toxin LF><biological signal transduction><bound protein><cAMP><cancer cell><catscan><cell biology><cell type><cleaved><computed axial tomography><computerized axial tomography><computerized tomography><cytokine><developmental><dopaminergic neuron><edema factor><glycogen synthase a kinase><hydroxyalkyl protein kinase><imaging><imaging agent><imaging in vivo><immunogen><in vivo imaging><insight><lethal factor><lethal toxin><lymphocyte activating factor><macrophage><malignancy><model of animal><model organism><mutant><necrocytosis><neoplasm/cancer><neuronal><neurotoxicant><novel><overexpress><overexpression><p38><p38 MAPK Gene><p38 Mitogen Activated Protein Kinase><p38-Alpha><p38Alpha><pathophysiology><pathway><phosphorylase b kinase kinase><pressure><protective effect><pulmonary><pulmonary vascular permeability><receptor><receptor binding><receptor bound><recruit><resistant><sensor><single photon emission computed tomography><social role><tool><tumor><tumor specificity>