Bacillus anthracisToxins Inhibit Human Neutrophil NADPH Oxidase Activity

Abstract

Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, spore-forming bacterium. B. anthracis virulence is ascribed mainly to a secreted tripartite AB-type toxin composed of three proteins designated protective Ag (PA), lethal factor, and edema factor. PA assembles with the enzymatic portions of the toxin, the metalloprotease lethal factor, and/or the adenylate cyclase edema factor, to generate lethal toxin (LTx) and edema toxin (ETx), respectively. These toxins enter cells through the interaction of PA with specific cell surface receptors. The anthrax toxins act to suppress innate immune responses and, given the importance of human neutrophils in innate immunity, they are likely relevant targets of the anthrax toxin. We have investigated in detail the effects of B. anthracis toxin on superoxide production by primary human neutrophils. Both LTx and ETx exhibit distinct inhibitory effects on fMLP (and C5a) receptor-mediated superoxide production, but have no effect on PMA nonreceptor-dependent superoxide production. These inhibitory effects cannot be accounted for by induction of neutrophil death, or by changes in stimulatory receptor levels. Analysis of NADPH oxidase regulation using whole cell and cell-free systems suggests that the toxins do not exert direct effects on NADPH oxidase components, but rather act via their respective effects, inhibition of MAPK signaling (LTx), and elevation of intracellular cAMP (ETx), to inhibit upstream signaling components mediating NADPH oxidase assembly and/or activation. Our results demonstrate that anthrax toxins effectively suppress human neutrophil-mediated innate immunity by inhibiting their ability to generate superoxide for bacterial killing.

Anthrax Lethal Toxin Increases Superoxide Production in Murine Neutrophils via Differential Effects on MAPK Signaling Pathways

“…Although neutrophils provide host protection during bacterial infections through the killing and clearance of bacterial pathogens, they have also been implicated as mediators of host damage during systemic inflammatory responses (20,21). The role of anthrax LT in modulating human neutrophil function has been somewhat controversial; both proinflammatory and immunosuppressive effects have been reported (22)(23)(24)(25)(26)(27)(28). These apparent contradictions could be partially explained by differences in the mode and/or timing of stimulation.…”

“…In selected experiments, the effect of anthrax LT on neutrophil superoxide production was also confirmed using a ferricocytochrome C reduction assay (28), modified for our purposes as described below. Purified bone-marrow derived neutrophils from C57BL/6 mice were cultured for 24 h or 48 h in the presence or absence of anthrax LT. Neutrophils (2 ϫ 10 6 ) were then resuspended in 100 l PB buffer (1 mM CaCl2 and 20 mM glucose in PBS).…”

Treatment of Experimental Anthrax with Recombinant Capsule Depolymerase

“…The capsule is essential for virulence (3,10,18,21), and its antiphagocytic property is a primary mechanism of immune cell evasion utilized by B. anthracis (21,22,26). The anthrax lethal toxin (PA plus LF) and edema toxin (PA plus EF) are known to inhibit some neutrophil functions (1,8,12,21,34,43), but in contrast to a deleterious effect of lethal toxin on macrophage survival from some animals (13,14), neither affects human neutrophil viability (8). Methods to counter the antihost properties of the capsule and toxins represent current areas of anthrax research and may lead to new vaccines and treatments.…”

Gastrointestinal anthrax in coastal south India: a critical alert on a fatal masquerader

“…The protective antigen binds to the cell surface and allows oedema toxin and lethal toxin to enter the cell and inhibits the mitogenactivated protein kinase pathway. This leads to the inhibition of upstream signalling components that mediate NADPH oxidase assembly and thus suppresses human neutrophil-mediated innate immunity (Crawford et al, 2006). A recent outbreak of injectional anthrax among intravenous drug abusers was reported in Scotland (Booth et al, 2014).…”