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. The primary receptor for PA is colony morphogenesis protein 2 (CMG-2). EF is a potent calmodulin-dependent adenylyl cyclase that causes large increases in intracellular cAMP concentrations and when injected with PA into animals, induces localized edema or systemic vascular collapse. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs) and the N-terminus of the inflammasome sensor NLRP1/NLRP1b. The cleavage of the MEKs inactivates important cellular signaling pathways. The cleavage of NLRP1 leads to caspase-1 activation and the maturation and release of the pro-inflammatory cytokines IL-1βand IL-18. When injected in animals, LT also causes a unique vascular collapse through unknown mechanisms. These toxins are considered the primary virulence factors of B. anthracis, and immunization against PA provides full protection against challenge with anthrax spores. The toxins play roles in different stages of infection. In the early stages of infection, both toxins work together to impair the innate immune response. At later stages, the induction of localized and systemic vascular dysfunction results in host death. Until the publication of our recent work, it was postulated that the vascular collapse induced by LT and ET was likely due to their actions on endothelial cells, although in vivo experimental evidence for this hypothesis was lacking. This year (2014) we published the culmination of years of analyses performed using genetically modified mice developed in our laboratory to investigate the key tissue targets responsible for the lethal effects of the anthrax toxins. Our laboratory created panels of cell-type specific anthrax receptor (CMG2)-deficient mice, as well as tissue-specific CMG2-expressing mice and challenged them with anthrax toxins or spore to assess the role of each tissue in pathogenesis. We found that LT and ET induce vascular collapse and lethality through targeting very different cell types. In a surprising finding, targeting of endothelial cells by either toxin did not play a dominant role in lethality. Instead, targeting of cardiomyocytes and vascular smooth muscles cells (but not hepatocytes) was found to be required for LT-induced lethality. In contrast, ET manifested its effects through direct targeting of hepatocytes, and despite the known impact of cAMP on vasculature, the direct targeting of the primary cells of the vascular system was not required for this toxins effect. These findings show that in anthrax, host lethality is the result of damage to two different vital systems, thereby providing a better understanding on how anthrax disease pathogenesis progresses. The knowledge of the targeted organs will also aid in development of therapeutics and supportive medical interventions for this disease. In other studies we further expanded on our recent discovery of a novel LF substrate, NLRP1. NLRP1 is a NOD-like receptor (NLR) protein which is part of the inflammasome, a multiprotein complex that activates caspase-1 in response to cytoplasmic danger signals. A consequence of NLRP1 inflammasome activation by LT is macrophage death with concurrent IL-1β/IL-18 maturation and release. Until recently, the only known activator of the NLRP1 was LT. We have now discovered that Toxoplasma gondii can also activate this sensor. T. gondii is an intracellular parasite that infects a wide range of warm-blooded species, but rodents can be sensitive or resistant to this parasite. We noted that the Toxo1 locus conferring Toxoplasma resistance in rats mapped to a small region of rat chromosome 10 containing Nlrp1. In a study published in 2014 we performed experiments to show that the parasite also activated NLRP1. In rats the differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation were found to be correlated with the same eight amino acid NLRP1 sequence that determines LT resistance. Thus, rat strain susceptibility was inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP and transcriptome analyses we narrowed candidate loci for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes which included Nlrp1. Knockdown of NLRP1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of resistant macrophages. Our findings reveal T. gondii as a novel activator of the NLRP1 inflammasome and suggest that the inbred strain susceptibility to infection by this parasite is controlled through inflammasome activation. We further extended these Toxoplasma studies to test the effects of both NLRP1 and the related inflammasome sensor, NLRP3, in parasite-infected mice. We found that Toxoplasma activates the NLRP3 inflammasome in mouse macrophages, leading to a pyroptosis-independent activation of IL-1β, but not IL-18. Strikingly, mice infected with the parasite produced large quantities of IL-18 (but not IL-1β) in a manner dependent on NLRP3 and caspase-1/11. Mice deficient in NLRP3 or NLRP1 infected with the parasite also show that both sensors play a role in controlling resistance to the parasite through an IL-18-dependent pathway which limits parasite replication. These findings established T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in mice and revealed a role for these sensors in host resistance to toxoplasmosis. In studies with collaborators we found that the PA channel translocates LF not only into the cytosol of cells but also the lumen of endosomal intraluminal vesicles (ILVs). LF persists in these ILVs (in both in vitro cell cultures and toxin-injected animals) for up to a week, fully protected from degradation by host enzymes and thus capable of maintaining cleavage of its substrates. This persistence results in a prolonged action of the toxin at various organ sites. We found that ILV-localized LF can also be transmitted to daughter cells upon cell division or delivered outside cells as exosomes, which could then potentially deliver LF to other cells in a manner independent of PA. The implications of these findings are significant for anti-LF therapeutics, because the toxin can maintain its action against the host at sites inaccessible to neutralizing antibodies for longer periods than previously anticipated. Furthermore, these studies may provide an explanation for the high mortality associated with anthrax long after bacteria have been killed by antibiotics. Finally, in another collaborative study, we reported on Nlrp1a regulation in a mouse model where expression of this gene was previously linked to sterol regulatory binding protein 1a (Srebp-1a). Srebp-1a knockout mice used for all previous studies carried the Nlrp1 locus from 129/Ola ES cells used to create this knockout, because the proximity of Srebp-1a and Nlrp1 makes it difficult for backcrossing to replace the 129 locus. The 129/Ola mouse Nlrp1a gene has a promoter defect that does not allow expression of NLRP1a. Our collaborators created a Srebp-1a knockout mouse carrying the functional Nlrp1 locus from C57BL/6J, with normal expression of NLRP1a restored. This study emphasized the importance of genetic fidelity in analyses of mouse phenotypes.
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