The Study Of Inhibitors Of Anthrax Toxin

Bacillus anthracis is the bacterium responsible for causing anthrax. Anthrax infections are difficult to treat because flu-like symptoms appear only after the bacteria have multiplied inside the human host and started to produce the tripartite toxin that eventually causes death. If antibiotics are applied at this stage, the infection can still be lethal because of the accumulation of the toxins. Logically, an effective therapeutic approach would include simultaneous blocking of bacterial growth by antibiotics and neutralization of anthrax toxin with antitoxinsAnthrax toxin is an AB-type toxin, with a single binding B-moiety, protective antigen (PA), and two catalytic A-moieties, lethal factor (LF) and edema factor (EF). LF and PA combine to form lethal toxin, and EF and PA combine to form edema toxin. These toxins are responsible for disabling host innate and adaptive immune responses, causing vascular leakage, and leading to the death of animals and cultured cells. To exert its toxin effect, anthrax toxin must enter inside the cell compartment. PA binds to the ubiquitously expressed cellular receptors, after its proteolytic activation by the furin-like proprotein convertases and the release of the N-terminal 20-kDa fragment, generates the mature PA protein (PA63). PA63 heptamerizes and binds to both LF and EF. After endocytosis of the resulting complexes, the engulfed molecules of LF and EF are liberated and exert their toxic action inside the cell compartment.In this article, a strategy of anthrax-toxin neutralization based on soluble anthrax toxin receptor decoy was evaluated and the development of a new anthrax-toxin inhibitor that competitively binds to PA63 heptamer and protects animals from toxin challenge was described.Evaluating the potential that the soluble anthrax toxin receptors have as candidate therapeutic agents for anthrax1. Expression and purification of recombinant soluble anthrax toxin receptor CMG2 (human capillary morphogenesis protein 2).Three different forms of soluble anthrax toxin receptor was expressed and purified by employing Ni chelating column and Superdex 75 column. The CMG2 VWA40-217 protein consists of aa residues 40-217 of CMG2489 fused to a His tag. The CMG2 VWAC175A protein consists of aa residues 39-218 of CMG2489 fused to a His tag with a point mutation (C175A). The CMG2EP protein consists of aa residues 34-317 of CMG2489 fused to a His tag. 2. Protective antigen binding activity of sCMG2ELISA tests showed that all three sCMG2 can bind to protective antigen ,but its bind toⅣcollagen was not detected.3. Protection against intoxication in vitro and in vivo provided by soluble capillary morphogenesis protein 2 (sCMG2).Recombinant soluble anthrax toxin receptor decoy were tested for their ability to inhibit intoxication in vitro by incubating J774A.1 cells with 100ng/ml PA and 100ng/ml LF. The results showed a concentration of 200ng/ml CMG2 VWA40-217, 100ng/ml CMG2 VWAC175A or 200ng/ml CMG2EP was sufficient to confer complete protection. The lethal toxin neutralization effect of the recombinant soluble anthrax toxin receptor in vivo was also tested by using male Fisher344 rats as toxin challenge model. The result showed that these recombinant protein might successfully neutralize the lethal effect of lethal toxin if enough recombinant protein was injected at the same time (sCMG2: PA molar ratio≥15:1) or immediately after challenge (sCMG2:PA molar ratio is 15:1). If these recombinant sCMG2 were injected 30min or 15min before the challenge, Fisher344 rats were failed to be protected, even did not exhibited delays in time to death (TTD), compared with the rats receiving LeTx alone .That means the stability of these proteins is very low in vivo.Conclusion: Although it can neutralize the lethal effect of anthrax lethal toxin in vitro and in vivo effectively, low stability of recombinant sCMG2 in vivo would be a barrier to use it as an anti-toxin.The development of a new anthrax-toxin inhibitor that competitively binds to PA63 heptamer and protects animals from toxin challenge1. Protection against intoxication in vitro provided by LFM,LFn,EFn and LFn27.LFM(a E678A mutant of LF),LFn (aa residues 1-263 of LF ),EFn (aa residues 1-263 of EF )and 27LFn (aa residues 27-263 of LF ) were expressed and purified by employing fast flow column , Ni chelating column and Superdex 75 column. Cell tests showed they have different ability to inhibit intoxication of anthrax lethal toxin in vitro. The BiaCore was used to measure the kinetics and stoichiometry of the interaction between these recombinant protein and PA63. We found that the ability of LFM,LFn,EFn and LFn27 to inhibit intoxication of anthrax lethal toxin is consistent with their affinity to PA63.2. Fusion protein LE/27LE construction and its anthrax-toxin neutralization activity in vitro.To develop an anthrax-toxin inhibitor with higher affinity to PA63, LFn and EFn /27LFn and EFn were fused to form a fusion protein LE/27LE. The BiaCore results showed the affinity of LE/27LE to PA63 is higher than LFn, EFn or 27LFn.Their ability to inhibit intoxication in vitro was tested by incubating J774A.1 cells with 100ng/ml PA and 100ng/ml LF. A concentration of 625ng/ml 27LE, 1200ng/ml LE was sufficient to confer complete protection.3. Protection against intoxication in vivo provided by 27LE.Male Fisher344 rats toxin challenge model showed if 27LE was injected at the same time(27LE:PA molar ratio≥15:1) or 30min before challenge(27LE molar ratio is 45:1) Fisher344 rats were fully protected. And rats received 27LE at a molar ratio of 45:1 1 hour before challenge exhibited statistically significant delays in time to death (TTD), compared with the rats receiving LeTx alone.Conclusion: The effectiveness of the 27LE in protecting rats against LeTx challenge indicates the potential that 27LE have as candidate therapeutic agents for anthrax.