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.
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