Purification of Factors I and II of the Anthrax Toxin Produced in vivo

Abstract

SUMMARY:Highly labile factors (I and 11) of anthrax toxin were purified from the toxic plasma of guinea-pigs just dead from anthrax by fractionation methods which involved the xninimuxn of inaetivation. The final preparations of factors I and 11, which still contained constituents of guinea-pig plasma, were toxic when injected together but innocuous when injected separately.The specific, lethal and oedema-producing toxin of Bm*Zlw ar&hraciS found in the plasma of guinea-pigs dying of anthrax (Smith, Keppie & Stanley, 1955) has two components which form a synergic mixture (Smith et al. Further purification of these factors was hampered by their extreme lability. The present paper describes fractionations whereby they were considerably purified without appreciable loss of toxicity. A brief report of some of this work has appeared elsewhere (Stanley & Smith, 1958; Sargeant & Smith, 1958). METHODSAnthrax toxin produced in infected guinea-pigs was obtained as described by Smith et aZ. (1055). It was imperative that the plasma of guinea-pigs should be collected within a few minutes of death from anthrax. When delay occurred here, or in the subsequent removal of bacteria, the tendency for factor I to lose activity and to aggregate and become insoluble during subsequent fractionation was more pronounced.This was described by Peterson & Sober (1956). It was made in the laboratory from I.C.I. solka floc S.W. 40.B or bought from Eastman Kodak Co.Assay of factors I and I I of arzthrax toxin. Serial descending dilutions (0.1 ml.) of the test sample (factor I or factor 11) were injected intradermally into the shaven side of a rabbit, immediately after mixing each dilution with a fixed amount (0.1 ml.) of the complementary factor (either crude factor I1 or crude factor I; for preparation see below) a t a concentration which just failed to produce a skin lesion when 0.2 ml. was injected alone. The oedematous lesions formed by these mixtures of factors I and I1 were observed 16 hr. after injection and the null point, i.e. the first dilution of the test sample not giving a positive reaction, was noted as a measure of factor I or factor 11. The assay would detect twofold differences in activity.

The Serological Relationship between Purified Preparations of Factors I and II of the Anthrax Toxin Produced In vivo and In vitro

“…( 8 ) The purified preparation of factor I produced in Viwo (Stanley et al 1960) contained, in addition to serological component A (associated with factor I activity), some component B despite the fact that it had no factor I1 activity. Since our antiserum to this preparation neutralized factor I1 activity, it was possible that component B was here present in a non-toxic but antigenic form.…”

“…This solution was added to crude factor I1 (Stanley et al 1960) in slight excess of the amount required to suppress serological component B (11 ml. crude factor I1 required 8 ml.…”

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

“…In gel diffusion plates against 'spore' antiserum (H533) it (10,ug.) formed two lines which were also formed by crude anthrax toxin produced in vivo and by an impure preparation of factor I from this source (see Stanley et al 1960); these lines were different from those formed by the final preparations of factor I (see above) and of purified factor I1 (see Sargeant et al 1960). Against 'antigen' (H25) antiserum the preparation of factor I11 formed a line which very easily dispersed and dissolved in antigen excess, indicating a low content of factor I11 antibody in this antiserum.…”

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.…”