Similar to other A-B binary toxins, the putative mechanism of anthrax toxin-induced toxinemia suggests that the B-protein, protective antigen (PA), 2 binds to a cell surface receptor (2, 3) and is subsequently cleaved at the N terminus into two fragments, PA 20 and PA 63 (4, 5). Following cleavage, PA 63 oligomerizes into a heptameric prepore on the cell membrane (6). The A-proteins, lethal factor (LF) and edema factor (EF), competitively bind to the heptameric PA 63 , forming the (PA 63 ) 7 -LF/EF complex which is endocytosed (7). Upon acidification of the endosome, the heptameric PA 63 is thought to undergo a conformational change, insert into the membrane, and form a functional transmembrane pore. In this model, LF and EF are subsequently translocated across the endosomal membrane into the cytosol (8). Determining the mechanism of PA 63 -mediated translocation of EF/LF into the cytosol remains an interesting challenge. Here we present proof-of principle for a potential biosensor in anthrax diagnostics and therapeutics. EXPERIMENTAL PROCEDURESProteins-Purified PA 63 , LF, and EF proteins (List Biological Laboratories Inc.) from Bacillus anthracis were used for these studies.Antibody-Hybridoma PA 63 1G3-1-1 was prepared by fusing spleen cells from female BALB/c mice injected with PA 63 with SP2/O-Ag 14 myeloma cells and subcloning positive hybridomas twice by limiting dilution (9, 10). Ascites was produced in BALB/c female mice and was clarified by centrifugation. This ascites monoclonal antibody, 1G3-1-1, prevents binding of LF to PA 63 through steric hindrance (9).Electrophysiology-Channel recordings were carried out with planar lipid bilayer membranes as previously described (11). Briefly, solventfree diphytanoyl phosphatidylcholine membranes were formed on a 50 -100-m diameter hole in a thin Teflon partition. The partition separated two identical Teflon chambers that each contained ϳ2 ml of aqueous solution (0.1 M KCl, 5 mM MES, pH 6.6). Voltage was applied across the membrane via Ag-AgCl electrodes. The current was amplified using a patch-clamp instrument (Axon Instruments 200B), recorded with an analog to digital converter (Axon Instruments DigiData 1322), and analyzed off-line. A negative transmembrane potential drove anions from the cis to the trans chamber. Details of this particular method were summarized elsewhere (12).Channels were formed by adding small aliquots (ϳ100 ng) of the purified PA 63 to the aqueous electrolyte solution bathing one side of the membrane (herein called cis). The formation of individual channels was indicated by the stepwise increases in ionic current monitored at ϩ50 mV applied potential. During recording of the ionic current in the steady-state experiments, the membrane potential was held constant at either ϩ50 or ϩ100 mV. To determine the instantaneous current-voltage (I-V) relationships, we generated current by applying brief (0.5 s) voltage pulses (typically ϩ200 to Ϫ200 mV in 10-mV steps) across the membrane and averaged the first 100 ms of the signal to obtain the inst...

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...

Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA(63)). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus alpha-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pore's PEG molecular mass cutoff (approximately 1400 g/mol). The results suggest that the limiting diameter of the PA(63) pore is<2 nm, which is consistent with an all-atom model of the PA(63) channel and previous experiments using large ions.

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm. [http://dx