Purified Bacillus anthracis Lethal Toxin Complex Formed in Vitro and during Infection Exhibits Functional and Biological Activity

Abstract

Anthrax protective antigen (PA, 83 kDa), a pore-forming protein, upon protease activation to 63 kDa (PA 63 ), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell. The relatively small size of the heptameric PA 63 pore (ϳ12 Å) raises questions as to how large molecules such as LF and EF can move through the pore. In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA 63 . In this study, we found that purified (PA 63 ) 7 -LF complex exhibited biological and functional activities similar to the free LF. Purified LF complexed with PA 63 heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogenactivated protein kinase kinase substrates and kill susceptible macrophage cells. Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA 63 pore. Complexes of (PA 63 ) 7 -LF found in the plasma of infected animals showed functional activity. Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF. Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.Lethal and edema toxins play key pathogenesis roles as virulence factors produced by Bacillus anthracis, the etiologic agent of anthrax. The toxins show commonality in that both of their enzymatic components, lethal factor (LF, 1 90 kDa) and edema factor (EF, 88 kDa), require protective antigen (PA) for translocation into host cells. LF, a Zn ϩ2 -dependent metalloprotease, cleaves several members of the mitogen-activated protein kinase kinase (MAPKK) family (1-4), and in complex with PA, is responsible for the lethal action of anthrax toxin. EF is a calcium-and calmodulin-dependent adenylate cyclase that elevates intracellular levels of cyclic AMP, causing deregulation of cellular physiology and resulting in edema (5).The proposed in vitro model for the binding, assembly, translocation, and subsequent killing of the target cells by anthrax lethal toxin involves a series of steps (6). PA binds to the ubiquitous cellular receptors tumor endothelial marker 8 (TEM8) (7) and capillary morphogenesis protein 2 (CMG2) (8). Proteolytic cleavage of PA by cell surface proteases such as furin (9, 10) generates a 63-kDa fragment that oligomerizes and forms a ring-shaped heptameric prepore. The LF and EF proteins bind to the prepore, and the whole complex then undergoes receptor-mediated endocytosis. The acidic environment in the endosomes causes PA to undergo a conformational change that promotes membrane insertion and formation of a transmembrane channel. In a recent study, this process of insertion was shown to occur in the early endosomes, whereas the delivery of the enzymatic moieties required the invo...

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

“…For example, anthrax toxin harvested from the blood of infected animals can be isolated in a complex of PA 63 oligomer, polyglutamic acid-based capsule material, and LF or EF. 24,25,54 This harvested complex has adverse effects when exposed to macrophages in vitro and when injected into animals. 55 Interestingly, LF is enzymatically active in a complex with the PA 63 channel 25 and the complex reconstitutes into planar bilayer membranes.…”

“…24,25,54 This harvested complex has adverse effects when exposed to macrophages in vitro and when injected into animals. 55 Interestingly, LF is enzymatically active in a complex with the PA 63 channel 25 and the complex reconstitutes into planar bilayer membranes. 24 The latter result led to the hypothesis that the anthrax toxin complex (LF or EF bound to the channel) might enter the cytoplasm and cause cell intoxication.…”

“…[21][22][23] Previous electrophysiological studies demonstrated that full-length LF and EF (≈760 amino acids each) [24][25][26][27] fragments (LF N and EF N , ≈254 amino acids each) [28][29][30][31][32] bind strongly but reversibly to the channel and reduce the pore conductance. Reversal of the LF N -induced channel current blockade is kinetically dependent on the bulk pH and transmembrane pH gradients.…”

Detection and Quantification of Anthrax Lethal Factor in Serum by Mass Spectrometry

“…10 PA83 may also be activated to PA63 by serum protease activity, and the LTx complex is present in blood from B. anthracis infected animals. 11,29 Since the two proteins are so closely associated in vivo, it was important to test whether the presence of PA83 and PA63 interferes with detecting and measuring LF in this assay.…”

A recombinant 63-kDa form of Bacillus anthracis protective antigen produced in the yeast Saccharomyces cerevisiae provides protection in rabbit and primate inhalational challenge models of anthrax infection

“…Our rationale for expressing the 63-kDa form of the antigen was: (a) interest in extracellular production which was thought to be more achievable with the mature protein; (b) concern that intact PA83 would be difficult to isolate due to endogenous yeast protease activity; (c) literature reports suggesting that protective epitopes were present on PA63 [1,[22][23][24]; and (d) the potential that PA63 might contain additional protective epitopes generated by conformational changes resulting from protease cleavage of the 83-kDa protein. Indeed both PA63 monomer and PA63 heptamer/LF complexes have been detected in the blood of infected animals, whereas PA83 has not [66,67]. S. cerevisiae was chosen based on in-house experience with this expression system for vaccine manufacturing, and because of the excellent safety profile of currently licensed and experimental yeast-derived vaccines such as Recombivax TM [68,69], and Gardasil TM [38].…”