Anthrax spores are living landmines.
Explosive charges hidden just underground of World War II battlefields are still taking life and limb from people mostly children who step on them. So are the millions of antipersonnel mines sown since, all over the world.
Spores of Bacillus anthracis are at least as ubiquitous, stubborn and treacherous. During World War II, the British military seeded anthrax spores on tiny Gruinard Island off the coast of Scotland. The organisms persisted and stayed viable for 36 years after conclusion of testing. Decontamination of Gruinard began in 1979 and lasted until 1987. The cleanup consumed 280 tons of formaldehyde and 2,000 tons of seawater.
“Anthrax spores’ whole thing,” observed microbiologist Philip Hanna, “is set up for long-term dormancy measured in centuries, and even beyond. Louis Pasteur put some of those spores down in 1868,” he recalled. “It was like pulling another Bacillus out of amber from 125 million years ago, and reviving it. People go to their Pasteur stocks and revive B. anthracis now.”
Hanna’s laboratory at the University of Michigan at Ann Arbor focuses on anthrax research.
“Those spores are spherical, 1.5 microns in diameter, rock hard and dehydrated,” he related. “They’re built in layers, like an onion. The inner layer, called the core, has the chromosome and ribosome and what would be the cell’s cytoplasm but there’s no water. Instead of water, there’s a lot of calcium and a chemical that together mimic the hydrogen bonding of water.
“Outside the core,” he continued, “there’s a big, thick layer called the cortex, separated from the core by a membrane. This would be the bacterial membrane but it’s paracrystalline, because of the extreme dehydration. The cortex is basically cell wall, and outside it there’s a layer of the protein called spore coat protein, which is really the unit of the spore. That’s all A. anthracis needs for endospore activity, resilience and longevity during its hibernation.”
Hanna is senior author of a paper in the March 2002 issue of the Journal of Bacteriology. Its title: “Amino acid- and purine ribonucleoside-induced germination of Bacillus anthracis DSterne endospores: gerS mediates responses to aromatic ring structures.”
Germination Is Anthrax Spore’s Grim Game
“What charms the spores into resurrecting themselves is the process of germination,” Hanna told BioWorld Today. “Although itself dehydrated, the spore will stay dormant, even in water. It’s not just moisture that it needs to come back to life. It requires different signals, which are small molecules we found to be combinations of amino acids the building blocks of proteins with nucleosides of RNA and DNA. We mixed those two and got a robust and rapid germination event. In fact, after months and years of dormancy, a spore under the influence of L-alanine germinated in 10 minutes.
“We reported that these dormant spores have evolved to recognize when they are where they want to be growing inside a mammalian host. They recognize signals of the body, and they need more than one signal to do this efficiently. Germination happens very quickly, and the multi-hits signaling ensures the anthrax bacterium that it won’t shed its armor until it’s pretty certain it’s in the right place for good growth.
“When that mammal is a human victim of anthrax infection,” Hanna pointed out, “we may wish for one thing to find ways of blocking this reactivation process. And the other thing, paradoxically, is that we may wish to enhance it, because one of the problems that we have in anthrax bioterrorism is that the spores stay in the lungs, non-germinated, and hang out, until patients complete an 80-, 90-, 100-day regimen of antibiotics. And then, when the spores do get to this right environment where the right signals are, they grow. And at that time the patient has done his treatment. So what you want to do is make the spores germinate in the lungs, where the antibiotics can kill them.
“Remember,” Hanna observed, “anthrax doesn’t cause pneumonia. You don’t get infection in the lung. The spores are taken up by professional phagocytes, macrophages and other immune system scavenger cells like those. They detach from the lung and go to the draining lymph glands or nodes. And that’s en route; it takes, two to four hours, and while that transport process is occurring is when the germination happens in the animal or the person.”
Homologous Germs: A Wild, Nasty Bunch
“We hypothesized that the genes that would resemble anthrax genes are its close microbial relatives spores of other Bacillus species. We found some of those from the anthrax genome sequencing project going on in TIGR [The Institute of Genetic Research]. When we removed those genes, we found that indeed there were defects in the germination process. Bacillus cereus, a food-poisoning germ, and B. thuringiensis, the gardener’s insecticidal friend, are the two closest relatives to anthrax. People are comparing their genomes now.
“Each relative, even though they’re very homologous and all endospore-forming responds to different germination signals, different sets of small molecules. Besides the other Bacillus group, there’s an anaerobe called Clostridium species. C. perfringens causes death by gas gangrene. C. botulinum makes the lethal botulinum toxin botulism. C. tetani enters puncture wounds to inflict tetanus lockjaw. A lot of them cause some pretty nasty diseases. And in some, the spores are the infectious particle.
“What we’re looking at now,” Hanna said, “is whether our knockout anthrax gene mutant, gerS, is deficient in germination, and whether what we found in our test tube can be maintained in a more infectious experimental model. We’ll also be looking at how the bacterium, once it does germinate inside a macrophage or phagocyte, gets out and into the blood. And how does it grow? Because it grows so fast and in such high numbers it must be very efficient at gaining the nutrients it needs.
“There’s no reason why anthrax is a serious human threat,” Hanna observed. “It’s been worked with safely from the beginning of microbiology. I’d say we should declare a truce with A. anthracis. So if things are being done to it with the intent of making it into a potential human threat, that’s scarier than the disease or the bacterium itself.” He concluded: “It’s people trying to weaponize anthrax that’s the threat.”