Purification of Factor I and Recognition of a Third Factor of the Anthrax Toxin

Abstract

SUMMARYFactor I of the anthrax toxin was isolated and showed one major component in the ultracentrifuge and on paper electrophoresis; it contained less than 5.5% of extraneous antigens detectable by serological precipitation in gels. The final preparation contained all the usual amino acids (N = 10*1%) and some carbohydrate (6 yo, calculated as glucose) and phosphorus (0.7 yo). The most striking aspects of its analysis were a high ash (10-13y0) and a light absorption a t 260 mp. The high ash was not due to one element but to a highly variable metal content (mainly Ca, Mg, Ni, Cu) indicating a powerful and indiscriminate chelating action of factor I. This chelating action might have been due to the chemical entity which absorbed light a t 260 mp and which was not RNA or DNA.The final preparation of factor I was not toxic when injected alone but when mixed with purified factor I1 it evoked oedema in the skin of a rabbit and killed mice. However, the concentration of this mixture which killed mice formed a much larger skin reaction in rabbits than a comparable dose (based on mouse LD50) of either crude toxin or a mixture of crude factors I and 11. An investigation of this fact led to the demonstration and partial purification of a third factor (111) of the anthrax toxin which: (1) was different serologically from factors I and 11; (2) was present in anthrax toxin produced in vivo; (3) was non-toxic when injected alone; (4) was lethal for mice when mixed with factor I1 but not with factor I ; ( 5 ) increased the lethality of mixtures of factors I and I1 for mice and decreased their capacity to produce oedema in the skin of rabbits. A mixture of factors I, I1 and I11 showed synergic action in toxicity tests in mice; the mixture killed guinea pigs which showed signs of oligaemic secondary shock (as did guinea pigs killed by anthrax infection).

Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.

“…These acute effects might be expected to mask any cAMP-mediated responses. In the only studies directly implicating EF as a virulence factor, mice were found to be killed by lower doses ofthe lethal toxin (PA and LF) when EF was administered simultaneously (4).…”

“…Experiments involving sequential injections of PA and LF led to the hypothesis that the factor common to both activities, PA, binds to tissue receptors and permits subsequent action of either LF or EF (6). This model was supported by the demonstration that LF blocks the action of EF (4).…”

“…The crude toxin subsequently was produced in broth cultures and resolved into three components, which American workers (2, 3) designated edema factor (EF), protective antigen (PA), and lethal factor (LF). British workers call these materials factors I, II, and III, respectively (4,5). The individual toxin components have no known biological effects when administered alone, but EF injected with PA into the skin of rabbits or guinea pigs causes edema, and PA injected with LF into rats causes death in as little as 60 min.…”

Alanine-scanning Mutations in Domain 4 of Anthrax Toxin Protective Antigen Reveal Residues Important for Binding to the Cellular Receptor and to a Neutralizing Monoclonal Antibody

“…Each is composed of a common binding component, protective antigen (PA), 1 together with an enzymatic component, edema factor (EF), in the case of edema toxin and lethal factor (LF) in the case of lethal toxin (1)(2)(3). The current model for toxin entry into the cell illustrates the centrality of PA for toxin action.…”

70th Anniversary Collection for the Microbiology Society: Microbiology

“…Harry Smith published notable contributions to the study of bacterial pathogenesis in the journal, with studies on Bacillus anthracis, Brucella, and Neisseria gonorrhoeae (Smith, 1990). These included several papers on the purification and characterization of the tripartite anthrax toxin and the use of in vivo grown bacilli to facilitate toxin purification (Smith & Stanley, 1962;Stanley & Smith, 1961, 1963Stanley et al, 1960). Smith was already convinced in 1947 that 'to learn more about bacterial pathogenicity we should examine organisms grown in vivo' (Smith, 1990) -a concept that has truly come to fruition with modern tools in cellular and molecular biology.…”