Antibiotic development is the first priority in responding to terrorist use of anthrax. But structural studies offer new leads in the hunt for more effective anti-toxin treatments.
In 1990, during the Gulf War, and then again in 1998, the decision was made to vaccinate US military personnel against the possible use of anthrax as a biological weapon. Never before had vaccinations been carried out as a response to anything but the natural occurrence of a disease. Now, spores of the causative bacterium, Bacillus anthracis, are being used to kill civilians and create panic: the question of how to deal with anthrax has shot straight to the top of the medical agenda.
There are three ways to tackle the disease: vaccination to prevent bacterial infection in the first place; antibiotics, to attack infection if it occurs; and anti-toxin treatments for the bacterium's toxic effects. Papers by Bradley et al.1 and Pannifer et al.2 on pages 225 and 229 of this issue, which follow their on-line publication on 23 October, will help in developing the last approach. The possible emergence of antibiotic- and vaccine-resistant organisms3 adds further impetus to the search for new approaches to preventing or treating anthrax.
The existence of toxins of B. anthracis was postulated by Robert Koch in the nineteenth century, when he discovered the cause of anthrax. Since their subsequent discovery almost 50 years ago, the toxins (known as 'lethal' and 'oedema'), along with the capsule that surrounds the bacterium, have been recognized as the main components that enable anthrax to cause harm. However, the manifestations of anthrax infection are not solely due to the effects of the toxins, as is the case with diphtheria, tetanus or botulism.
Rather, in anthrax the bacterium invades and grows to high concentrations in the host; the toxins act mainly by damaging defensive cells called phagocytes, causing the immune system to malfunction. Late in the infection, toxins may be present in large amounts in the blood and contribute directly to the death of the infected organism. Some studies, in non-human primates, suggest that lethal toxin by itself is not even particularly potent, requiring milligram quantities to cause death. But the results of other work4, in mice, imply that further non-toxin components contribute to virulence that have yet to be identified. So antibiotics constitute the mainstay of treatment, although anti-toxins have long been considered5 an essential 'adjunctive' therapy, and remain so.
The toxins are composed of three proteins: a cell-receptor binding protein, known as protective antigen; and two enzymes, lethal factor and oedema factor. The papers by Bradley et al.1 and Pannifer et al.2 respectively report on the identity of the cellular receptor for protective antigen, and the crystal structure of lethal factor. This valuable information about the toxins will allow the identification of vulnerable targets for anti-toxin therapy.
Lethal factor is a zinc protease, a type of enzyme that contains zinc and cleaves other proteins. Oedema factor belongs to a class known as adenylate cyclases. When combined with lethal factor, protective antigen constitutes lethal toxin; with oedema factor it makes oedema toxin. From cell-culture studies it seems that anthrax operates as follows. First, protective antigen is cleaved, and so activated, by a protease on the surface of the cell under attack. It then forms heptamers — aggregations of seven — and subsequently binds one or more molecules of lethal or oedema factor, or both. The complex passes into the cell through the receptor for protective antigen and on into an acidic compartment inside the cell. There the heptamer inserts into the compartment's membrane, releasing lethal and oedema factors into the cell body where they exert their toxic effects. The precise molecular targets remain unknown.
Several features of these events remain unclear, such as the ratios of molecules in the complex between protective antigen and lethal and oedema factors, and whether both factors are included. A protease in the blood stream can also activate protective antigen, and complexes with lethal factor occur in the blood of infected animals. So the relative importance of the two proteases in toxin action in vivo is unknown.
The structure of lethal factor2 will help in the identification of drugs that interfere with its binding, and maybe that of oedema factor, to protective antigen; indeed, such a peptide inhibitor has been described6. Investigating inhibitors of the activities of the two factors themselves is another route, and will be aided by knowledge of the crystal structures2. Therapies might include soluble toxin receptors and other drugs to prevent protective antigen from binding to its receptor. Non-toxic mutants of protective antigen have been shown to neutralize toxin7,8, and inhibitors of the protease(s) that activates it might have the same effect. Here, detailed knowledge of toxin kinetics during infection will be required, and the timing of drug delivery is critical. Another tactic may develop from understanding how a recently discovered motor protein confers resistance to lethal toxin in some phagocytes9.
Further adjunctive treatments might include antibodies to the toxin, spore and bacillus. Other bacteria that cause sepsis exert pathological effects by triggering an inflammatory response from molecules known as cytokines; treatment with activated protein C has been reported10 to be effective in such cases. The possible role of cytokines in anthrax warrants further evaluation, as do other ways of preventing the physiological consequences of the toxins. The extensive experience of testing adjunctive therapies for sepsis will be invaluable in guiding this work. In the current circumstances, however, priority should go to evaluating antibacterial drugs, initially antibiotics that have already been licensed for use.
Two final points are that information from sequencing of the B. anthracis genome, currently underway, may prove invaluable in tackling anthrax. And it is extraordinarily difficult to test anti-anthrax therapies in humans, so large clinical trials with non-human primates may well be needed.
In the early days of microbiology, 125 years ago, anthrax was significant mainly as an economically damaging disease of domesticated animals. The world's scientific community addressed that problem and developed effective countermeasures. It is now necessary once again to focus on anthrax, along with other pathogenic microorganisms, this time as agents of biological terrorism and threats to civilization. During the Second World War, the Office of Scientific Research and Development in the United States, and similar agencies in other countries, coordinated the application of science to warfare11. The same level of organization and commitment is needed today.
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