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), although the cleavage of these substrates has not yet been linked to the physiological effects of this toxin. Anthrax lethal toxin (LT, the combination of PA and LF) is considered the primary virulence factor of B. anthracis, 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 induces a unique vascular leakage in animal models in a manner similar to that seen in anthrax disease. Rats are uniquely susceptible to anthrax lethal toxin, and can die in as little as 37 minutes when challenged with saturating doses of toxin. Macrophages from some inbred rat are uniquely sensitive to an inflammasome-mediated rapid lysis when treated with anthrax LT. Our previous studies mapped sensitivity of both rats and their macrophages to 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/caspase-1 activation by LT is macrophage death with concurrent IL-1b/IL-18 maturation and release. Nlrp1 is one of many studied NLR sensors, each of which are activated by unique danger signals through unknown mechanisms that allow specificity to some, and a broad range of activation signals for others. The response of these sensors to intracellular danger signals is critical to the innate immune response. Until our recent studies, published in 2012, the biochemical mechanism of activation was unknown for any inflammasome NLR sensor. We discovered that anthrax LF cleaves rat Nlrp1 in certain rat strains, and that this cleavage event is required for toxin-induced inflammasome activation, IL-1 release, and macrophage rapid death. We identified the cleavage site in rat Nlrp1 using purified protein constructs and mass spectrometry, and established cells lines expressing uncleavable mutant Nlrp1 proteins which are resistant to anthrax toxin. Our studies represent two major steps in the anthrax and inflammasome fields: First, the identification of a physiologically relevant new substrate for anthrax LT, and second, the discovery of a novel method of activation for an NLR protein. In collaborative studies with the laboratory of Dr. Russell Vance, of the University of California at Berkeley, the rapid 30-minute death of mice in which the Nlrc4/Naip5 inflammasome was activated by bacterial flagellin delivered to cells as an LF fusion protein was studied. In a major discovery in the innate immunity field, the activation of caspase-1 in mice was linked to an eicosanoid storm resulting in rapid release of lipid mediators in a COX-1-dependent manner. This study is the first in which eicosanoid responses were found to be downstream consequences of inflammasome and caspase-1 activation. In a separate set of studies using the other anthrax toxin, ET, we analyzed the effects of this toxin in vivo, in a mouse model of intoxication. We discovered a new effect of this toxins actions in vivo, in which the toxin alters circulatory protein and small molecule pharmacokinetics, allowing for prolonged circulation of virulence factors. In these studies, we found that in mouse models of toxemia and infection, anthrax PA and EF levels were significantly higher following ET treatment. We showed that ET manifested its effects on general protein clearance as it impaired clearance of other proteins (ovalbumin and green fluorescence protein) injected into mice, as well as small molecules such as biotin. Clearance was impacted in a manner independent of general fluid loss. In these studies, we also demonstrated that ET treatment rapidly leads to phosphorylation and activation of the aquaporin-2 water channel in collecting ducts of kidneys, which are responsible for fluid homeostasis. Finally, in a collaborative study on anthrax receptor transcripts, we surveyed a wide range of tissues and identified two new splice variants. We analyzed expression of these and the previously known anthrax toxin receptor splice variants in different tissues and tested their functionality and relative efficiencies.

Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2012
Total Cost
$676,462
Indirect Cost
City
State
Country
Zip Code
Al-Dimassi, Saleh; Salloum, Gilbert; Saykali, Bechara et al. (2016) Targeting the MAP kinase pathway in astrocytoma cells using a recombinant anthrax lethal toxin as a way to inhibit cell motility and invasion. Int J Oncol 48:1913-20
Li, Yan; Cui, Xizhong; Xu, Wanying et al. (2016) Nitric oxide production contributes to Bacillus anthracis edema toxin-associated arterial hypotension and lethality: ex vivo and in vivo studies in the rat. Am J Physiol Heart Circ Physiol 311:H781-93
Greaney, Allison J; Maier, Nolan K; Leppla, Stephen H et al. (2015) Sulforaphane inhibits multiple inflammasomes through an Nrf2-independent mechanism. J Leukoc Biol :
Greaney, Allison J; Leppla, Stephen H; Moayeri, Mahtab (2015) Bacterial Exotoxins and the Inflammasome. Front Immunol 6:570
Maier, Nolan K; Leppla, Stephen H; Moayeri, Mahtab (2015) The cyclopentenone prostaglandin 15d-PGJ2 inhibits the NLRP1 and NLRP3 inflammasomes. J Immunol 194:2776-85
Moayeri, Mahtab; Leysath, Clinton E; Tremblay, Jacqueline M et al. (2015) A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model. J Biol Chem 290:6584-95
Remy, Kenneth E; Cui, Xizhong; Li, Yan et al. (2015) Raxibacumab augments hemodynamic support and improves outcomes during shock with B. anthracis edema toxin alone or together with lethal toxin in canines. Intensive Care Med Exp 3:9
Gorfu, Gezahegn; Cirelli, Kimberly M; Melo, Mariane B et al. (2014) Dual role for inflammasome sensors NLRP1 and NLRP3 in murine resistance to Toxoplasma gondii. MBio 5:
Cirelli, Kimberly M; Gorfu, Gezahegn; Hassan, Musa A et al. (2014) Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii. PLoS Pathog 10:e1003927
Liu, Shihui; Moayeri, Mahtab; Leppla, Stephen H (2014) Anthrax lethal and edema toxins in anthrax pathogenesis. Trends Microbiol 22:317-25

Showing the most recent 10 out of 36 publications