Lorne Protein report: beating anthrax

Molecular geneticist John Collier has devised a potentially perfect counter-measure to the bioterrorist's current weapon of choice -- the deadly anthrax bacterium.

Collier, professor of microbiology and genetics at Harvard Medical School, told the 29th annual Lorne Conference on Protein Structure and Function yesterday that the microbe could be detoxified simply by dosing its victims with a subtly modified version of the powerful cell-killing toxin with which it overwhelm its victims.

Bacillus anthracis has made headlines on three occasions in recent years: Saddam Hussein plotted to use aircraft to spray it on his enemies, Shoko Asahara pumped it into the air from a factory in Tokyo, and an anonymous US scientist mailed anthrax spores to the US Senate, and others, killing several postal workers who handled the letters.

Collier told the conference, at Lorne's Erskine on the Beach resort, that anthrax was still easily controlled with antibiotics. But in a bioterrorism attack, people will be unaware they have been infected, and by the time they develop symptoms, antibiotics are useless -- a single millilitre of their serum may swarm more than a billion toxin-secreting anthrax cells.

Scientists have known since the 1950s that the anthrax toxin has three components, called oedema factor (OF) protective antigen (PA) and lethal factor (LF), carried on two plasmids called Px01 and Px02.

As the Japanese Aum sect's botched bioterrorism attempt confirmed, if either plasmid is missing, the bacterium is non-pathogenic. Individually, the three proteins are harmless, suggesting they team up to work their lethal mischief. When the victim inhales the spores, they are ingested by macrophages, which would normally signal the immune system to mount an antibody response against the microbe.

By determining the structure of the three proteins of anthrax toxin, and showing how they fit together, Collier's team, and others, have now shown the microbe deactivates the immune response by short-circuiting the macrophages' internal signalling processes.

The process begins with the PA protein binding to a receptor on the macrophage's outer surface. It is cleaved, yielding an oligomer; the oligomers then cluster in groups of six to form a pre-pore: a circular cluster of molecules to which both EF and LF bind strongly.

The outer membrane of the macrophage then invaginates, and the complexed proteins are internalised in a pocket-like endosome.

Proteins flood into the endomes, activating the pore, and the attached EF or LF enzymes are then 'fired' through the endomsome wall into the cell's interior, where they wreak havoc with the cell's MAP kinase signalling systems.

But by studying natural mutants of the anthrax bacterium, Collier's team has found that if a mutant monomer of the pore-forming PA protein is incorporated in the heptameric protein that will form the pore, it no longer works -- the EF and LF toxins are no longer released into the cell.

That suggests that a mutant monomer could be used as a peptide drug to disrupt assembly of the pore. Better still, the mutant monomer retains the immunogenicity f the natural form of the molecule, so it will double as a vaccine, conferring protection if the individual -- say, a soldier -- encounters anthrax again.

'A nice decoy'

Prof Robert Liddington, director of the Burnham Institute in La Jolla, California, described his team's research into two other, potentially life-saving therapies for anthrax.

The microbe's PA monomers bind to two different types of receptors on the macrophage's surface, as a prelude to forming an active pore inside the cell.

Liddington told the conference a soluble form of the receptor would make "a nice decoy", to which the pore-forming protein would bind, preventing it from attaching to the normal, membrane-bound receptors on macrophages.

The LF enzyme is another potential drug target is the LF enzyme, which cleaves one specific bond common to the MAP kinase molecules that link the cell's external signalling pathways to genes in its nucleus.

Liddington said the LF enzyme contains a pronounced groove, into which the 'tail' of the MAP kinase molecule fits neatly -- and is then cleaved.

His team is trying to devise a 'mimic' drug, resembling the cleaved tail, which would jam itself into the groove, blocking the LF enzyme's ability to disrupt internal signalling.

The team has already developed several promising molecules, but Liddington says they will need to be made much more potent to be useful as a frontline therapy for anthrax.

According to Collier, countries that might be the target of anthrax attacks would need to stockpile any new blocker drug, in a rapidly deliverable form, to ensure it was readily available to save lives in an emergency.

He said that another issue was whether people would be prepared to take a drug that had only been proven effective in cell-culture systems -- because nobody would risk being experimentally infected with anthrax, it was impossible to test it in humans.

Australian Biotechnology News reporters will be on the ground at the Lorne conferences, presenting daily updates.