Anthrax toxin protective antigen protein (PA) binds to receptors on the surface of mammalian cells and transports two other toxin proteins, lethal factor (LF) or edema factor (EF) to the cytosol. EF is a potent calmodulin-dependent adenylyl cyclase that causes large increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). Anthrax lethal toxin (LT, the combination of PA and LF) is considered the primary virulence factor of B. anthracis and immunization against either of its components provides full protection against challenge with anthrax spores. Injection of this toxin or anthrax edema toxin (ET, the combination of PA and EF) into animals replicates the vascular shock induced in anthrax. Macrophages from some inbred mice are uniquely sensitive to an inflammasome-mediated rapid pyroptosis death induced by LT. Historically, an inverse relationship was observed between macrophage sensitivity to LT and animal susceptibility to spore infection across species, although the basis was unknown. Our studies in 2011 focused on understanding the role of the protein controlling sensitivity to LT-induced macrophage lysis, Nlrp1b, in anthrax infections. Nlrp1b is the sensor component of the Nlrp1 inflammasome, a multiprotein complex that activates caspase-1 in response to cytoplasmic danger signals. A consequence of caspase-1 activation by LT is macrophage death with concurrent IL-1b/IL-18 maturation and release. We used inbred and congenic mice harboring macrophage-sensitizing Nlrp1b alleles (which allow activation of caspase-1 and IL-1 release in response to anthrax LT challenge) to show that Nlrp1b was required for effective control of bacterial dissemination during anthrax infection. Caspase-1 and IL-1 receptor knockout mice which harbor sensitive Nlrp1b alleles were developed in our laboratory and used to implicate IL-1 signaling in resistance to anthrax infection. Sensitization of mice with the IL-1 receptor antagonist anakinra further supported a role for IL-1 signaling in control of anthrax infection. Our studies also showed that neutrophils are required for control of anthrax infection. We found that resistance to infection correlates with toxin-mediated caspase-1/IL-1 dependent neutrophil recruitment, and is independent of germination state or infection route. These studies highlight the importance of the toxin-mediated inflammasome-dependent innate immune response in controlling anthrax infection. In other studies utilizing knockout mice, we discovered a novel target organ for anthrax LT along with an associated innate immune pathway that was required for protection against the toxins effects. MyD88-deficient mice were found to have increased susceptibility to anthrax infection and LT challenge. We found that LT targets the intestinal epithelium and induces ulcerations in MyD88 knockout mice both during infection as well as after challenges with purified toxin. The acute toxin-mediated damage to the intestinal lining leads to enteric bacterial leakage, peritonitis and septicemia in mice. Our findings demonstrate that MyD88 plays a protective role against LT-induced impairment of the intestinal barrier. In a collaboration with Peter Eichacker (CC), we continued our studies on the mechanisms of anthrax toxin-mediated vascular shock. Previous studies showed that ET induces rapid hypotension and tachycardia in rodent and canine models, while LT appeared to reduce left ventricular output in the same systems. To analyze the direct effects of the toxins on the heart, we utilized an isolated perfused rat heart model and monitored cardiac functions in response to circulating toxin. We found that ET increased heart rate, coronary flow and myocardial cAMP levels, and that adefovir, an inhibitor of EF enzymatic function, and anti-EF neutralizing antibodies could reverse the toxin effects in this system. Surprisingly, LT, previously believed to target left ventricular function, had no effect on cardiac functions at any tested dose in the isolated heart model. These findings show that ET rapidly and directly targets the rat myocardium to induce cAMP-mediated changes in cardiac function, while LT does not have similar effects over the same short periods of toxin exposure in the isolated heart model. In other collaborative studies with Robert Purcell (NIAID) and Peter Eichacker (CC), we continued in vivo analyses and characterization of W1-mAb, a potent humanized chimpanzee-derived monoclonal antibody against anthrax toxin. W1-mAb neutralizes anthrax toxins by binding to the receptor binding domain of PA and has the highest affinity reported to date for an anti-anthrax toxin antibody. We demonstrated the protective efficacy of W1-mAb in rat LT and ET infusion models, as well as against mouse spore infections. The antibody was effective even when administered at 24 h post spore infections (in mice), or 6-12 h after toxin infusion commenced in rats. Increased survival with antibody treatments correlated with improved hemodynamic functions in the animals. Our studies confirm the value of this therapeutic agent against anthrax, and provided further in vivo data in establishing its potency. Finally, we collaborated with the company CombinatoRx, which had used a high-throughput screen to identify compounds acting in a synergistic inhibitory manner against LT. We deciphered the steps in inflammasome activation targeted by these compounds. The organogold molecule Auranofin, used as an anti-inflammatory in treatment of arthritis, was found to be a potent inhibitor of Nlrp1 inflammasome activation and caspase-1 activity. It acted synergistically with inhibitors of voltage-gated potassium channels in inhibiting LT activity.
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