Pathophysiological Actions of Anthrax Virulence Determinants

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Bacillus anthracis toxins (anthrax toxin) are made of three polypeptides. Protective antigen (PA) binds cellular receptors to transport two enzymes, lethal factor (LF) and 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) and a completely unrelated substrate, the rodent inflammasome sensor, NLRP1. During the current FY2021 review period, we discovered a third family of LF substrates, the regulatory subunits of phosphoinositide-3 kinase (PI3K). The p85alpha (PI3KR1) and p85beta (PI3KR2) proteins are cleaved by LF in a proline-rich linker region between their N-terminal SH3 (Src homology) and BH (Bcr homology) domains. LF cleavage in this region disrupts the N-terminal domains responsible for homo-dimerization of the regulatory subunits and their association with downstream signaling partners. LF cleavage modifies PI3K signaling in a cell-type dependent manner. We found cleavage was robust in mouse tissues. Importantly, a knock-in mouse in which p85 was mutated within the LF cleavage site to abrogate toxin-mediated proteolysis had greater resistance to toxin challenge. In a complementary approach, we also identified a mutant toxin with a single amino acid change, LF W271A, which was altered in the ability to cleave p85 and was nontoxic to mice. This mutant was lethal to mice in combination with PI3K pathway inhibitors. Our studies show that anthrax LF has evolved to affect two important signaling pathways in mammalian hosts. Discovery of the PI3K pathway as a target of anthrax lethal toxin is a major step forward in understanding anthrax disease. In another study during Fy2021, we developed a unique tool to tackle a limitation in the study of anthrax pathogenesis. Measuring toxin activity in different cell types and organs through the course of infection, in relation to inoculation dose, genetic background, or therapeutic treatments has presented a challenge to the field. The distribution of functional PA receptors is also not known in different organs. In a collaboration with colleagues at NIDCR, we developed a system for imaging and quantification of anthrax toxin delivery to the cytosol of cells in live mice. A chimeric protein using the N-terminal domain of LF fused to Cre recombinase was created with a nuclear localization signal. The ability of this protein to be translocated into cells was measured through Cre-dependent conversion of fluorescence in Cre-activity reporter mice. We can now visual anthrax toxin-mediated delivery of Cre at single cell resolution by confocal microscopy. This assay will be an important tool in the study of anthrax pathogenesis and in identifying off-target events for anthrax toxin-based cancer therapeutics. We continued our years-long collaboration with the Eichacker lab (NIH Clinical Center) in using an isolated perfused rat lung model to study the physiological effects of EF. New studies during FY2021 demonstrated that this adenylate cyclase toxin has an inhibitory effect on hypoxic pulmonary vasoconstriction (HPV). Treatments with PA neutralizing antibody or the EF inhibitor adefovir were found to reverse this effect. These findings suggest that inhibition of HPV by EF may aggravate hypoxia during anthrax pulmonary infection. In another collaboration with this group, a review was published of our multi-year efforts to identify beneficial effects of various FDA-approved neutralizing anti-PA monoclonal antibodies or immune globulin sera in antibiotic-treated B. anthracis infected animals (rabbits, non-human primates and canines). Finally, in a third collaborative effort, this time with researchers at Harvard University, we studied the effects of EF on nociceptive sensory neurons, where the receptor for PA is enriched. EF was found to modulate pain in multiple mouse models of inflammatory and chronic neuropathic pain. These studies suggest EF or modified anthrax toxins may be used as a mechanism for delivering cargo to neurons, and potentially, as a platform for treatment of pain.