Anthrax, made infamous by a rash of mail attacks after the events of September 2001, can kill if inhaled. Scientists have known that a trio of proteins act together to invade and destroy cells, but had not been able to decipher the form of the third critical component that multiplies the effects of the other two factors a hundred-fold. Last month, scientists announced they had deciphered the three-dimensional structure of the final protein, edema factor (EF).
By mapping the 3-D structure of the edema protein, researchers at the University of Chicago and the Boston Biomedical Research Institute gave scientists a much better chance of developing a treatment that halts the damaging effects of anthrax infection. The researchers published their results in a January 2002 issue of Nature.
Andrew Bohm led the research at the Boston laboratory, and collaborated with Wei-Jen Tang’s lab at the University of Chicago. Bohn worked with Chester Drum, an M.D./Ph.D. student from U. Chicago, to solve the crystal structures of. In parallel, Wei-Jen unraveled the biochemistry of EF.
Bacillus anthracis is a bacterium that occurs naturally in soil. Livestock and those who work with animals or animal products, such as herders or wool processors, are the most common victims of infection.
During times of drought or other environmental shock, anthrax forms durable spores and can remain inactive indefinitely. It is this spore-forming ability that makes anthrax such an attractive bioweapon. Since September, the Center for Disease Control has confirmed seven cases of anthrax skin infection and 11 cases of inhalation anthrax, five of which have resulted in death.
Although antibiotic treatments such as Cipro can stop an anthrax infection, by the time they are applied, fatal levels of the toxins produced by the bacteria may have already built up in the body.
Anthrax that enters the body through a cut in the skin will lead to a localized “cutaneous” infection. Black sores and swelling may occur. If ingested, the spores may cause vomiting and abdominal pain and bleeding. However, if the infection persist for a long enough time, the bacteria may enter the bloodstream to cause a “systemic” infection.
Anthrax in the bloodstream is attacked by phagocytes, cells that engulf and dissolve foreign invaders. However, anthrax fights back with two “virulence factors.” One factor produces a strong shell, preventing the phagocytes from breaking down the invader.
The other virulence factor enters the phagocytes themselves, preventing them from effectively fighting any other bacteria. Once in the bloodstream anthrax makes its way to the lungs, where the activities of its toxins come into play.
Anthrax toxin is actually three proteins. “Protective antigen,” PA, is made up of several subunits. When PA is dispatched to the target cell membrane, one of the cell’s enzymes cuts off the end subunits. The remaining units form a seven-sided ring that attaches to the membrane to form a pore, allowing the other two components access to the interior of the cell. “Lethal factor,” LF, interferes with normal cell signaling and other functions.
The final component, “edema factor” or EF, by interfering with the way ions and water flow across the cell into and from the cell. The cells release water into surrounding tissue. This fluid accumulation, or edema, in the lungs in cases of “systemic” anthrax, and causes the swelling in more localized cutaneous anthrax where only regions of the skin are affected.
The edema factor of anthrax severely promotes the production of a protein in the cell called cAMP. This protein plays a major role in the burning of fats and carbohydrates stored in the cell.
Edema factor is a type of molecule called an adenylyl cyclase. Normal mammal cells contain about 50 other adenylyl cyclases that perform similar functions in regulating cell signaling, but EF operates at a rate 100-1,000 times that of the mammalian cyclases, hence the deadly effect of anthrax infection.
If EF were present inside the anthrax bacterium, it would disrupt cellular processes just as surely as it does in humans. It has been known for years that EF only becomes active in the presence of a small protein called calmodulin, which is found only in the cells of the organisms anthrax infects.
In addition to EF’s effects on cAMP, EF also inactivates a cellular protein called “MAP kinase kinase.” The inactivation of this kinase severs the chain of molecular events that allows signals to be sent through the cell, and may cause death within 24 hours.
Although the other two anthrax toxins still kill even when EF is not present, they work about 100 times slower than with it. Therefore, EF is being targeted as a way to stop the deadly edema cause by anthrax colonization.
By understanding the exact structure of EF, scientists are able to create drugs that target specific parts of the protein itself, or specific steps in the chain of events that leads to symptoms of anthrax infection, rather than going through time-consuming trials of a wide range of non-specific pharmaceuticals.
“One thing to note is that as human diseases go, anthrax is not a very serious health threat,” said Bohm. “It has been estimated that there are 20,000-100,000 cases of human anthrax per year worldwide. Most of these are cutaneous anthrax and few result in death. There are EF-like toxins produced by other pathogenic bacteria (ie. Bordetella pertussis, which causes whooping cough). If anti-EF drugs are developed, there is a very good chance that these drugs will also work in combating these other much more common pathogenic organisms.”
The Boston lab researchers used X-ray crystallography to determine the three-dimensional structure of EF. Using a technique called fluorescence resonance energy transfer (FRET), they were able to understand further how EF binds to calmodulin. Finally, the lab made different mutated versions of the protein to see what portions of it were active in what process.
Bohn believes his research will accelerate the search for effective anthrax treatments. “Knowing the structure will help speed up the development of anti-EF drugs. Without a crystal structure you can screen lots of compounds, but you are stumbling around in the dark without ever getting to see what is actually going.”
“Drug design is a tricky business,” said Bohm. “Given the many potential pitfalls (the drug might not get into the correct cells, the drug might cause severe side effects, the drug may be broken down by the body before it does any good, etc.), it would be foolish to concentrate exclusively on LF when EF-based drugs can be developed in parallel.”
In October of last year, two groups of researchers from The Burnham Institute in La Jolla, California and the University of Wisconsin described in back-to-back papers in Nature how anthrax destroys cells.
Members of the research teams were able to identify the location on animal cells where anthrax enters. A molecule they named anthrax toxin receptor (ATR) attaches to a region on the PA protein produced by anthrax, allowing the protein to penetrate the cell membrane.
When ATR was put into solution with anthrax toxins and animal cells, the cells remained unharmed. The soluble ATR attached to the toxins before they could reach the cell, blocking the protein from making contact with the cell.
The researchers next identified the region of ATR where the toxin attached.
Using this information they then produced a shortened, free-floating version of the receptor that contained the toxin-binding domain. When they mixed that receptor fragment with rodent cells and anthrax toxin in a test tube, the cells were completely protected from destruction.
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