Although antibiotics can kill the bacteria, anthrax produces toxins that can cause death even after the bacteria have been eradicated. Since some forms of anthrax infection produce few symptoms until the disease is advanced, physicians need better drugs to counteract these toxins.
"Knowing the structure of edema factor and how it works allows us to design drugs that can block its effects," said Wei-Jen Tang, Ph.D., associate professor in the Ben May Cancer Research Laboratories at the University of Chicago and director of the study.
Anthrax produces three toxins that work together. One, called protective antigen, allows the other two to enter target cells. The second, lethal factor, destroys cells of the immune system. When immune cells die they release inflammatory agents that can cause septic shock, leading to death. The structure for protective antigen was published in February, 1997, and that of lethal factor in November, 2001.
The structure of the third toxin, called edema factor because it causes fluid accumulation, provides the last piece of this pathogenic puzzle. Tang, his graduate student Chester Drum, and Andrew Bohm of BBRI, spent three years deciphering the structure of this deadly molecule and determining how it damages infected cells.
Edema factor can cause death on its own by releasing fluid into the lungs or other infected areas. It makes lethal factor 10 to 100 times more potent and can disrupt immune function.
Tang, who studies how cells sense their environment and communicate with other cells, became interested in edema factor several years ago as a way to understand a basic cellular process -- how an enzyme called adenylyl cyclase helps regulate cell-to-cell signaling. In response to certain chemical signals, such as adrenaline, adenylyl cyclase converts a common cellular chemical to cyclic AMP, which, in turn, modulates multiple processes such as heart rate, blood sugar level, and learning and memory.
Although the presence of cyclic AMP is normal, too much can be lethal, causing affected cells to become hyperactive, as if constantly drenched with adrenaline. Cells that produce too much cAMP devour their energy stores and soon lose the ability to regulate their environment. They release water, causing edema to surrounding tissues, and die.
Edema factor is harmless until it comes in contact, inside an infected cell, with a protein called calmodulin. The researchers found that when calmodulin binds to edema factor, it changes the toxin's shape, creating a pocket that functions just like adenylyl cyclase -- but is 1000-fold more active.
"This pathogen has evolved a very clever tactic," said Tang. Edema factor alone is benign because one key section of the structure is incomplete. But when it connects with calmodulin, it changes shape and becomes a relentless version of adenylyl cyclase."
Fortunately, the 3D structure of edema factor appears to provide an ideal drug target. The active site, which mimics adenylyl cyclase, is a deep, narrow pocket that should be comparatively easy to block with a small molecule. It is also very different from mammalian adenylyl cyclase, so there is little risk that the drug would interfere with the normal activity of this important enzyme.
At least two other disease-causing bacteria rely on a similar process: Bordetella pertussis, which causes whooping cough; and Pseudomonas aeruginosa, which infects patients with cystic fibrosis and those with impaired immunity.
Besides it's value in developing better anti-anthrax drugs, the finding is an important one for basic cellular biology. Calcium is not only used for bone formation but also plays a vital role in the communication between cells. Calmodulin is a ubiquitous calcium sensor that interacts with more than 50 protein targets.
"People have studied calmodulin when it is bound to a small piece of various protein targets before," said co-author Andrew Bohm, Ph.D., a crystallography expert at the Boston Biomedical Research Institute, "but the structure of calmodulin with edema factor, a complete protein target, is significantly different from these earlier findings. A lot of the previous studies will have to be reconsidered."
This discovery "is an example of the unexpected benefits of basic science," said Paula Flicker, Ph.D., a program director at the National Institute of General Medical Sciences, which partially supported the work. (The American Heart Association also contributed.)
"Three years ago," said Tang, "when we started this project, Bacillus anthracis was an obscure agricultural pathogen with interesting biological properties. Now anthrax is front and center in every clinician's mind, and within months of the first bioterrorism case we have the structures for all three toxins. We hope this work will quickly lead to new therapies."
The work resulted from a collaboration between Wei-Jen Tang's Chicago laboratory -- including Chester Drum (who also worked on this project in the Bohm lab), Shui-zhong Yan, Yuequan Shen, Dan Lu, and Sandriyana Soelaiman -- Andrew Bohm's Boston laboratory, including Joel Bard; and Zenon Grabarek's lab, also at Boston Biomedical.
Journal
Nature