A Reamerging Fear
Anthrax became a household word when letters intentionally laced with a white powder were mailed to senators and journalists last year. U.S. government officials now realize a large-scale anthrax attack could become a reality and scientists race to find new ways to treat the disease. What makes anthrax such an ideal biological weapon? How do the bacterial spores invade the host? And why is anthrax toxin so deadly?
Despite its recent infamy, in current times anthrax is actually a relatively rare disease–especially in humans. Anthrax is the disease caused by the rod-shaped bacterium, Bacillus anthracis. Spores of B. anthracis occur naturally in the soil where they can lay dormant for decades. Waiting for a grazing animal–such as a cow, horse, or sheep–to accidentally ingest it. Once inside the animal, the spore germinates, switches to its virulent form, and produces the anthrax toxin, which eventually kills the host. Historically, people only became infected after working with infected livestock products or eating contaminated meat.
Anthrax is a disease that has plagued man and his livestock for centuries, causing it to be one of the most well-studied and understood diseases. The name anthrax is derived from the Greek word for coal, anthracis, because of one of the symptoms of infection is the formation of black ulcers on the skin. Over one hundred years ago B. anthracis became the first bacterium to be shown to cause disease when Robert Koch injected live B. anthracis culture into laboratory animals. Shortly after being injected with the bacterium, the animals showed symptoms of anthrax infection and later died.
Using anthrax as a biological weapon is not a new concept. During the second half of the 20th century several governments investigated using the anthrax bacterium as a biological weapon, including the United States and the former Soviet Union. Of more recent concern is the belief that several well-organized extremist groups are developing anthrax to wage large-scale biological warfare. This is no surprise to scientists who work on the microbe. B. anthracis spores are virtually indestructible, remaining viable after being exposed to extreme temperatures and ultra-violet radiation. The spores are also light, odorless, and colorless, and can easily infect a large population quickly and without being detected. In short, anthrax makes the perfect deadly biological weapon.
There are three ways to acquire anthrax. The most common and least serious is called cutaneous (or skin) anthrax and occurs when B. anthracis spores make contact with abrased skin. This form of the disease results in the formation of black ulcers on the skin and is usually treatable with antibiotics. The skin form of anthrax can turn deadly if the microbes invade the blood stream and the infection becomes systemic. Anthrax can also be acquired by ingesting improperly cooked meat from an infected animal. This type of infection is extremely rare, especially in North America and Europe, but results in a gastrointestinal infection that is often lethal even after antibiotic treatment. The third and most deadly type of infection is inhalation anthrax. Infection occurs when 10,000 to 50,000 spores are inhaled into the lungs. Once in the lungs, the spores germinate, form active bacteria, and migrate to the lymph nodes where the bacteria get inside macrophages–the host’s immune system cells that normally seek and destroy bacteria. Later, the bacteria multiply and convert into a virulent form that produces the anthrax toxin. Anthrax toxin causes internal bleeding and destroys the body’s immune system, eventually causing septic shock and killing the host.
It is possible to be exposed to the B. anthracis microbe and never develop an anthrax infection. Last year, several government workers tested positive for exposure to the microbe but never acquired the disease. Unfortunately anthrax is difficult to diagnose during the initial stages of the flu-like infection when antibiotics such as Cipro are most effective in treating the disease. Because current therapies are not potent enough to be effective at later stages of infection is important to understand the anthrax toxin on the molecular level. Knowing how anthrax toxin works to evade and destroy the host’s immune system cells has enabled researchers to develop novel, more potent therapeutics.
Infection on the Molecular Level
After the anthracis spores germinate inside the host, becoming active bacteria, they switch from their avirulent (non-toxic) to virulent (toxic) form. The virulent form of the bacteria expresses new proteins that were previously not expressed, resulting in the production of a polypeptide coat and the anthrax toxin. These virulence factors help the bacteria to evade the host’s immune system and eventually destroy it.
The polypeptide coat protects the microbes from being ingested by the host’s bactericidal macrophages. This coat is composed entirely of poly-D-glutamate and serves to hide the bacteria’s proteins, which the host’s immune system normally recognizes and attacks. In this way the bacteria can exist without being detected or destroyed by macrophages. The B. anthracis cells look rough in their avirulent state but adopt a smooth appearance once they surround themselves with this protective polypeptide coat.
Death from virulent B. anthracis is due to the production of a toxin that shuts down the host’s immune system and causes cell death. This deadly toxin is called anthrax toxin. The anthrax toxin has three components, all of which are fairly large, soluble proteins. Two of the components, edema factor (EF) and lethal factor (LF), cause either swelling of the cells (edema) or cell lysis (death) within the host’s cells. The third component, protective antigen (PA), binds to the host cell receptor and facilitates delivery of EF and LF into the cell. Separately these components are non-toxic, but PA combined with either EF or LF forms either edema toxin or lethal toxin, respectively.
The mechanism of anthrax toxin entry into the cell has been thoroughly studied. In 2001, it was discovered that the protective antigen (PA) binds a receptor called the anthrax toxin receptor (ATR) present on the mammalian cell membrane. Once bound to the ATR, a host protease protease cleaves off the N-terminus of PA, inducing a switch to its activated form. The activated PA binds to six other activated PAs, forming a heptamer on the surface of the mammalian cell. This heptameric complex then binds either EF or LF. The mammalian cell draws in the PA/EF (or LF) complex through endocytosis, a process where the cell ingests receptor-bound molecules by pinching off internalized parts of the membrane and forming an endosome inside the cell. The environment inside the endosome becomes more acidic, causing the PA molecules to change shape. This conformational change in the PA molecules forms a pore in the endosomal membrane and either EF or LF gets injected into the cytosol of the cell. Once in the cytosol, the EF and LF components disrupt the cell by affecting cell signaling.
EF, which is referred to as edema toxin once inside the cell, is an adenylate cyclase, an enzyme, which catalyzes the conversion of ATP to cyclic AMP (cAMP). Because cAMP is an important regulatory molecule in the cell, production of more cAMP by edema toxin disrupts normal cell function. One of these functions of cAMP in the cell is to maintain proper osmotic pressure by regulating the flow of small ions and water in and out of the cell. An increase in the amount of cAMP causes the cell to swell. Additionally, since edema toxin uses ATP to form cAMP, the cell becomes depleted of ATP. Macrophages need the energy-rich ATP in order to engulf and destroy bacteria. As a result, infected macrophages become bloated and useless.
The lethal factor, or lethal toxin, disrupts normal cell function by another route. Lethal toxin is a zinc protease that targets members of the mitogen-activated protein kinase kinase (MAPKK) family. This leads to inhibition of several cell signaling pathways and results in an increased amount of cytokines, protein that act as cell mediators. While the exact role these cytokines play in causing cell lysis remains unknown, it is believed that high cytokine levels cause an increase in harmful oxidative molecules. If the concentration of these harmful molecules gets too high within the cell, the macrophage ruptures and dies.
Treating Anthrax: How Cipro Works
Cipro, or ciprofloxacin“ (Bayer Pharmaceutical), is a powerful broad-spectrum antibiotic used to treat many diverse bacterial infections, including anthrax. One of the ways bacteria and mammalian cells differ is that bacteria store their DNA in a circular plasmid form. This plasmid is too large for the bacterial cell in its uncoiled form so bacteria enlist an enzyme called DNA gyrase to twist the DNA into a more compact, supercoiled form. Gyrase cuts one strand of the double stranded DNA, winds the DNA around itself and pastes the DNA back together to form supercoiled DNA. Cipro blocks the reannealing stage of this process by sitting in the active site of the DNA gyrase, leaving the double-stranded DNA broken. With their genomic DNA damaged, the bacteria cannot replicate. Therefore Cipro is only really effective in stopping the bacteria from multiplying, but does little to kill the bacteria or to stop the production of the anthrax toxin.
New Approaches–Antitoxin Therapeutics
Since it is unfeasible to vaccinate the entire population against anthrax and antibiotics are often ineffective in treating a severe anthrax infection, new “antitoxin” therapies are currently being investigated. Scientists are developing new therapies to treat anthrax by blocking the anthrax toxin from ever entering the mammalian cell.
One antitoxin approach is to block the PA component from binding ATR on the cell. Soluble anthrax toxin receptor (sATR) is being investigated as a decoy to keep PA away from its intended target on the surface of the cell. Perhaps a more powerful blocking agent has been designed by scientists at the University of Texas at Austin who use genetically engineered antibodies that bind PA fifty times more tightly than ATR.
A second antitoxin approach is to block the assembly of the PA heptamer. A group at Harvard University has developed a peptide that tightly binds the PA monomer, rendering it unable to form a heptamer with other PA monomers. Additionally, there has been some investigation into the design of small molecules or peptides to sit on the surface of the PA heptamer so that the EF and LF are blocked from binding the complex and therefore cannot enter the cell.
The prevalence of anthrax in the news and support from government-funded agencies are fostering the development of novel antitoxin therapeutics at an ever-increasing rate. While we are years away from seeing any of these therapeutics available for use by the public, we are getting closer to finding a cure. In the event of a large-scale bioterrorist attack, the key to effectively treating anthrax may be to use an antibiotic, such as Cipro, in combination with a novel antitoxin therapeutic. This “drug cocktail” would simultaneously kill the bacteria and render their deadly toxins impotent.
Copyright 2002, John Wiley & Sons Publishers, Inc.