Pathophysiological Actions of Anthrax Virulence Determinants

National Institute Of Allergy And Infectious Diseases

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Abstract

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 that causes large increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves and inactivates several mitogen-activated protein kinase kinases (MEKs). In certain strains of rodents LF also cleaves and activates the inflammasome sensor NLRP1. Inflammasomes are intracellular complexes that play a role in innate immune sensing for defense against pathogens. The cleavage of NLRP1 in macrophages and dendritic cells leads to caspase-1 activation and a rapid cell death termed pyroptosis. Caspase-1 activation, which follows from activation of many other inflammasome sensors, including the NLRP3, NAIP/NLRC4 and AIM2 sensors, also leads to maturation and release of the pro-inflammatory cytokines IL-1 and IL-18. Interestingly, the only other known activator of NLRP1 is Toxoplasma gondii, but the mechanism for its activation is currently unknown. The inhibition of the MEK pathways and NLRP1 cleavage-mediated activation of the immune response have a wide range of consequences for the host. Continuing a long-term mouse gene mapping project, in 2017 we initiated use of the NIAID-supported Collaborative Cross recombinant inbred mouse collection. This mouse collection is a unique resource, a large panel of inbred strains developed from 8 unique parental founders which include three wild strains. The collection represents a wider genetic diversity than in any other inbred model, with segregating polymorphisms at every 100-200 bp. We are in the process of doing crosses and SNP analyses to identify the genetic basis for unique LT-induced phenotypes. In 2017 we also continued our studies into the role of NLRP1 inflammasome activation in the rapid, 1-hour death induced by LT in rats. While we previously mapped sensitivity to the toxin to the NLRP1 locus, the mechanism by which activation of an inflammasome sensor leads to rapid animal death remains unknown. We are in the process of assessing the contribution to lethality of the hematopoietic cells in which NLRP1 is primarily expressed. During 2017 we also continued our studies on the role of inflammasome activation in both murine and rat resistance to Toxoplasma gondii. Specifically, we investigated the contribution of activation of different inflammasome sensors, including NLRP1, to the induction of protective cytokine responses in rodents and the cellular sources of these cytokines. A collaborative study completed during 2017 identified molecular consequences of ET-mediated cAMP release, and showed disruption of endocytic recycling dependent on the small GTPase Rab11. This disruption impacts cellular junctions and contributes to edema induced by the toxin by preventing transport of crucial junction proteins to cell membranes. Using both Drosophila and mammalian cells we showed that over-activation of the cAMP effectors PKA and Epac/Rap1 interferes with Rab11-mediated trafficking at two distinct steps. We further described conserved roles of Epac and the small GTPase Arf6 in ET-mediated disruption of vesicular trafficking and showed that chemical inhibition of either pathway prevents ET-induced edema in mice. These studies further our understanding of ET-induced vascular leakage at the cellular level. In a continuation of other collaborative studies the cardiovascular effects of ET and LT on rat aortic rings were analyzed. While we had previously demonstrated that ET but not LT inhibits phenylephrine stimulated contraction of aortic rings prepared from healthy rats, in this period we examined arterial function in rats pretreated with toxin infusions. Only ET was found to reduce arterial pressure and contractile force. Adefovir, an ET inhibitor, reversed these effects and protected animals from toxin challenge. This study showed that in ET-treated rats, hypotension and lethality are associated with reduced arterial contractile function.