The protective antigen component of anthrax lethal toxin, produced in vitro, has a molecular mass of 83 kDa. Cell-culture studies by others have demonstrated that upon binding of the 83 kDa protective antigen to cell-surface receptors, the protein is cleaved by an unidentified cell-associated protease activity. The resultant 63 kDa protein then binds lethal factor to form lethal toxin, which has been proposed to be internalized by endocytosis. We found that, in the blood of infected animals, the protective antigen exists primarily as a 63 kDa protein and appears to be complexed with the lethal factor component of the toxin. Conversion of protective antigen from 83 to 63 kDa was catalysed by a calcium-dependent, heat-labile serum protease. Except for being complexed to protective antigen, there was no apparent alteration of lethal factor during the course of anthrax infection. The protective antigen-cleaving protease appeared to be ubiquitous among a wide range of animal species, including primates, horses, goats, sheep, dogs, cats and rodents.

Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1 → 4)-[3-O-acetyl]-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNH(2)-(1→.

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...

Gamma phage specifically lyses vegetative cells of Bacillus anthracis and serves as part of the basis for identification of isolates from agar cultures. We report our study to standardize gamma phage production and preparation and to validate the assay for routine use. Unstable phage preparations were largely reduced through propagation of phage on blood agar cultures of the avirulent B. anthracis strain CDC684 and were adequately stable for extended storage beyond 1 to 2 years at 4°C, provided that the preparation initially gave rise to clearly discernible plaques (macroplaques, 5 to 10 mm in diameter) on dilution at 1:8 or greater during potency testing with the Sterne strain or its equivalent. The primary intent of the assay was to test nonhemolytic, ground-glass-appearing bacterial B. anthracis-like colonies arising from culture of clinical or nonclinical samples on 5% sheep blood agar. Specifically, the assay was designed to show clear or primarily clear circular zones of lysis on bacterial lawns at the site of gamma phage inoculation after incubation at 35°C ؎ 2°C for 20 h. When tested with 51 B. anthracis strains and 49 similar non-B. anthracis Bacillus species, the analytical specificity was >95%, a value that is intentionally low because our study design included two rare nonsusceptible B. anthracis strains as well as a rare susceptible non-B. anthracis strain, B. cereus ATCC 4342. Repeatability, day-to-day precision, and analyst-to-analyst precision were superior. The assay was rugged to variations among phage lots, phage concentration, amounts of bacterial inoculum, and incubation times as short as 6 to 8 h. System suitability evaluation showed improved robustness when bacterial lawns were tested with highand low-density inoculum using the first and second quadrants of a serial four-quadrant streak on 5% sheep blood agar plates.Bacteriophages that are active on B. anthracis but not specific were first described in 1931 by Cowles (7). In 1951, McCloy isolated a bacteriophage called "phage W" from what she described as an atypical Bacillus cereus strain, designated strain W, ATCC 11950 (9). The phage was highly specific for 171 strains of B. anthracis and failed to react with 19 other Bacillus species, except for two unusual strains of B. cereus, strains NCTC 1651 and NCTC 6222 (ATCC 4342). In a subsequent study, McCloy (10) isolated two types of phage from aging cultures of Bacillus strain W, referred to as "phage ␣" (rare mutant) and "phage ␤" (dominant form), which specifically produced plaques on agar cultures of B. anthracis. The two phages differed only in that phage ␣ attacked strains of B. anthracis and Bacillus strain W. Phage ␤ attacked all B. anthracis strains tested by McCloy but not strain W; however, it was not able to attack smooth or encapsulated forms of B. anthracis. In 1955, Brown and Cherry (4) isolated a variant of the original phage W, designated gamma phage. This phage differed from phage ␣ and phage ␤ in that it lysed encapsulated smooth forms of B. anthracis, failed to lyse any ...

A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non–B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens.