Author: De Michele Jennifer
Institution: Natural Resources
Date: September 2005
On November 9, 2001, the FBI released a linguistic and behavioral assessment of the person responsible for mailing anthrax-laced letters on September 18, and October 9, 2001. At national and global levels, United States Government agencies have so far been unsuccessful at locating the alleged anthrax mail-sender. Investigations remain without decisive conclusions, and are instead filled with speculations and frustration.
At quite a different level and scale - one that involves agar gels, petri dishes, microscopes, and fluorescent tags - numerous scientists have made substantial progress within the last few decades toward unlocking the mysteries, strategies, and fatal mechanisms of the anthrax-causing bacteria, Bacillus anthracis.
In light of recent bioterrorism events, microbiologists and immunologists affiliated with institutions such as the Michigan's Van Andal Research Institute, University of Wisconsin Medical School, and Harvard Medical School have come forward to share with a concerned American public their key findings on how anthrax toxin functions to destroy cells, and how certain genes and their associated proteins can protect against anthrax-induced death.
In the not-too-distant future, the efforts and findings of these researchers have the potential to add yet another weapon to the arsenal used to combat B. anthracis, a large, Gram-positive, spore-forming, non-motile bacteria (1-1.5 mm x 3-10 mm.). Combined with antibiotics and vaccinations, clinicians may soon be able to offer anti-toxins to exposed individuals that will inhibit B. anthracis from poisoning and killing its human host. "As we struggle with rather non-specific therapies and rather imperfect vaccines for anthrax, we are going to be looking towards fundamental science as the means to get us where we really need to be in this rather significant crisis, " said Anthony S. Fauci, M.D., director of the National Institute of Allergy and Infectious Diseases (NIAID).
B. anthracis already possesses an extensive and equivocal autobiography. Believed to have existed since the Egyptian era, and having played a crucial role in establishing both the origins of microbiology and immunology in the early 20th century, this rod-shaped bacterium is now aiding the present advancement of these scientific fields.
Although several types of anthrax exist, researchers' efforts have been most concerned with understanding the most fatal form - inhalation anthrax. Inhalation anthrax results when B. anthracis spores, minuscule (1-2 mm), bead-like, and resistant, are breathed in and transported to the alveolar spaces of a host's lungs. According to scientific investigator Nicholas Duesbery, Ph.D, at the Van Adel Research Institute in Michigan, once B. anthracis's spores find themselves inside the host's alveolar spaces, macrophages (a set of cells responsible for aiding in the protection of the human host against invading organisms) engulf these spores, and transport them to the mediastinal and peribronchial lymph nodes located between the lungs and the chest. During transport, B. anthracis finds itself bathed in an environment rich in amino acids, nucleotides, and glucose, and as a result begins to germinate and transform into rod-shaped bacteria.
Wearing an antiphagocytic poly-D-glutamic acid capsule, B. anthracis, is protected from phagocytotic-cell engulfment and immediate death, and in this vegetative form is able to release its toxin unimpeded. This toxin, composed of three components (edema factor (EF), lethal factor (LF) and protective antigen (PA)) is what eventually causes the lysies (or rupture) of macrophage cells, the release of cytokines, and eventual host death by systemic shock, Duesbery said.
Until recently, anthrax research at University of Wisconsin Medical School's McArdle Laboratory for Cancer Research and at Harvard Medical School received little attention. Biological warfare and bioterrorism were only hypothetical threats, and an anthrax attack seemed highly unlikely. Prior to anthrax's use as a biological weapon in September and October, research focused on scientific questions that had little direct application towards combating biological warfare. Rather, researchers examined the unique transport mechanisms of anthrax's toxin, or its ability to disrupt cell-to-cell communication in hopes of advancing vaccination and cancer research.
However, since anthrax's use as a bioterrorism agent, the research that John A. Young, the Howard M. Temin Professor of Cancer Research at the McArdle Laboratory, and his graduate student Kenneth Bradley have conducted has proved vital in both understanding the detailed mechanisms of how B. anthracis's toxin actually gets into cells and ways that toxin absorption can be inhibited. Specifically, Young, Bradley and their collaborators at Harvard Medical School have identified a single cell receptor, which they have named anthrax-toxin receptor (ATR). When combined with seven other receptors, ATR serves as a mission control station for B. anthracis to guide and shuttle its two virulent toxins, EF and LF, from the bloodstream into its host's macrophage cells.
The identification of ATR began two-and-a-half years ago when Bradley was mutating cells to find those that were resistant to infections caused by retroviruses - the family of viruses that includes the Human Immuno-deficiency Virus (HIV). His initial graduate work focused on identifying unknown cellular proteins that aid in virus production.
Upon genetically mutating cell after cell, Bradley, Young, and Jeremy Mogridge, a post-doctoral fellow of John Collier's laboratory at Harvard Medical School, eventually realized that certain cells remained virus-free because they no longer contained the genes to produce the cell receptors to which viruses could attach. Failing to attach to cells, viruses remained bobbing in a river of blood plasma, not unlike glass bottles lost at sea, inactive and unable to infect their hosts.
Recognizing that this method could be used to isolate other unknown cell receptors, Bradley's curiosity led him to ask what the nature of the receptor was that permitted anthrax's toxins to wreak havoc inside cells. He knew from earlier research conducted in the 1990s that anthrax's protective antigen (PA) bound to receptors expressed on macrophage surfaces, yet its exact identity remained a secret.
For two months, often working from 9 a.m. to 7 p.m. weekdays and half days on weekends, Bradley searched for a mutant cell out of millions that were resistant to anthrax's poison. According to Bradley, the process of randomly subjecting mutant genes to anthrax toxin to determine if cells lacked the appropriate receptor was like searching for a needle in a haystack. Yet once Bradley found a viable mutant cell, this cell and its million clones served as his blank canvases.
Upon these 'blank canvases'- tissue cultures of cells without ATR on their cell membranes - Bradley would spend the next two years determining the gene responsible for re-expressing ATR, the cell-receptor that serves as the PA docking site. He would use fluorescence-associated cell sorting (FACS), a cDNA library purchased from Clonetech (a biotechnology firm located in San Diego), polymerase chain reaction (PCR), gel electrophoresis, and enormous amounts of determination and patience to find the gene that caused mutant cells to re-expresses ATR. "It is sometimes frustrating that research is so slow. Often, I would like it to be faster and be applicable immediately. Unfortunately, scientific research is not as fast as that," Bradley said.
As a painter applies paint to his or her canvas to create a picture, Bradley applied fluorescently-tagged PA to his canvases of mutant cells so that he could see which cells re-expressed ATR. However, even before applying PA to his mutant tissue cultures, Bradley had to thaw and extract portions of Clonetech's cDNA library from one of two test tubes.
Each test tube contained millions of non-toxic E-coli bacteria. Of these, each bacterium possessed one cDNA in its circular plasmid. To ensure a stable integration of the cDNA into injected mutant cells, Bradley had to remove cDNA from the E-coli, and transfer them into retroviruses. These retroviruses acted as delivery vehicles, transporting cDNA into mutant cells. The number of retroviruses that were transferred into each cell would later be identifiable by recognizing certain marker genes, Bradley said.
After these retrovirus vehicles transferred their cDNA to Bradley's blank canvas cells, he was able to begin "painting." Over and over again, Bradley applied fluorescently tagged PA molecules to the various tissue cultures containing disparate cDNA. In each trial, he was looking for those cells that became coated with his fluorescently-tagged PA. To determine fluorescence, Bradley took advantage of the powerful FACS technique, which permitted him to examine one cell at a time. If a cell fluoresced, Bradley knew he had a cell that had regained receptor expression.
Yet, finding a cell that regained receptor expression is not the same as identifying the exact cDNA sequence that encodes for ATR. The fluorescent cell could have as many as 10 cDNAs. To determine the exact cDNA that causes ATR expression, Bradley had to use two more techniques: polymerase chain reaction (PCR) and gel electrophoresis.
According to Bradley there were times when things were not going right, and he just wanted to bang his head against the wall. It was Young's incessant enthusiasm and positive attitude toward scientific endeavors that continuously instilled in him the desire to continue tackling his research. Young's " try-it-one-more-time" attitude was one of the driving forces that eventually lead to Bradley's success in determining the nature of the ATR receptor. Yet at this point in his investigation, Bradley still had many more hours, weeks, and months ahead of him at the McArdle Lab.
After Bradley isolated his fluorescent cell, allowed it to replicate, and removed its DNA, he was able to use PCR to amplify each DNA segment. Upon amplification of the DNA, Bradley was able to narrow down the 2 million possible cDNAs that could have caused ATR expression to seven. Yet seven was not one, and Bradley's investigation continued in search of the specific gene that caused ATR expression.
His next step was to separate the seven retroviruses, which contained the possible cDNA that caused ATR expression. Here, he used a technique known as agarose gel electrophoresis to take advantage of the fact that each nucleic acid molecule within cDNA carries a negative charge. On running an electrical current through the gel, separation of the seven possible cDNA occurs by weight. As each cDNA molecule moves uniformly away from a negative electrode toward a positive electrode, the lighter cDNA segments move further down the gel while the heavier cDNA molecules remain closer to the negative electrode. After running the gel electrophoresis apparatus, Bradley determined the nucleotide sequences of all seven cDNAs, made purified copies of each band, and reapplied them back onto his canvases of mutant cells.
Bradley's reapplication of the seven possible cDNA into the mutant cells failed six times. At this point in his research, his dissertation committee told him to give up. Yet, due to his determination, and in part inspired by Young's "try-it-one-more-time" attitude, Bradley tried one last time to identify the gene that caused ATR expression. Fortunately for Bradley, and now the rest of the American public, his seventh attempt proved successful. Bradley had found the single gene, coined ATR, that restored receptor production.
As is often true in science, one scientific discovery leads to the next. This finding and the bioterrorism events spurred the need for rapid advancement of anthrax research, leading Young, Bradley, and their collaborators at Harvard Medical School's John Collier Laboratory to genetically engineer the portion of ATR that protrudes outside the cell membrane. According to Young, injection of a genetically-engineered, free-floating soluble portion of the ATR receptor into tissue cultures grown on plastic petri dishes soaks up PA like a sponge before it gets a chance to attach itself to macrophage cells. If PA is prevented from attachment, the virulent factors of anthrax (EF and LF) have no way into the cell, says Young.
These findings will eventually lead pharmaceutical companies to develop more efficient ways to stop Bacillus anthracis's deadly attack. "Our short-term goals are to study the mechanism of toxin uptake through ATR and to make enough of the toxin-blocking form of the receptor so that it can be tested in animals systems," says Young. "A more long-term application would be for pharmaceutical companies to use the receptor along with anthrax toxin to screen the millions of compounds they've already synthesized to identify toxin inhibitors."
For Bradley, these recent findings and bioterrorist events have caused a shift in focus of the McArdle Lab. "It is interesting. One moment you are not being paid attention to and then the next you are thrown into a media frenzy. Nobody used to care about what we were researching, but now that our research has the potential for immediate application, there is a new motivation and driving force behind our current research." says Bradley. "For the last month, everything has just been one big adrenaline rush."
Many of the scientific questions that the McArdle Lab is now asking differ from those prior to September and October's anthrax exposures. Determining what experiments need to be conducted is now facilitated by the urgent need to understand anthrax in its entirety. "Before, millions of interesting scientific questions existed that could still get you up in the morning," says Bradley, "but now the questions that need to be answered about anthrax are geared to the more practical side."
As Dr. Fauci, director of NIAID said, "You can't rush the science, but when the science points you in the right direction, then you can start rushing. Now that we have these very important findings, it is incumbent upon us to do as much as we can to translate [this research] into something for the public health. Current events are now showing the importance of scientific diligence."
While United States officials' investigations continue their struggle to discover the sender of the anthrax letters, researchers are succeeding in piecing together the detailed mechanisms of how B. anthracis causes death in its hosts. Much is still left unknown, yet if the researchers affiliated with labs such Young's and Collier's demonstrate the same amount of effort that has been put forth so far, within the next year or two, anthrax-induced death, given correct diagnosis, has the potential of becoming a non-fatal disease.
In the not too distant future, anthrax's infamous autobiographical history may be brought to closure, yet the use of this bacterium to further vaccination and cancer research will live on. As Bradley says, "We hope to be able to understand the normal function of ATR, which may shed new insights into cancer as well as anthrax toxin action."