Like a three-chamber revolver, the anthrax spore loads three pathogenic toxins: The first, and deadliest, is LF - lethal factor. The second is edema factor (EF). The third, protective antigen (PA), transports the first two to do their damage, but by itself is harmless. Anthrax, Bacillus anthracis, bundles its triple-threat store of chemical ammunition into bacterial spores. These microscopic, hard-shell, spherical particles 1.5 microns in diameter lie low in latency within macrophages, the immune system's strategic action cells.
"Bacterial spores," microbiologist Jimmy Ballard at the University of Oklahoma in Norman recounted, "can hole up doggo under the soil, or inside mammalian living bodies or corpses, for days, weeks, months, years or forever, awaiting their opportunity to pounce and kill. It's not completely clear in the disease process," observed Ballard, "where macrophages make contact with the B. anthracis toxins. We do know," he continued, "that nearing infection, macrophages engulf the anthrax spores.
"Within those macrophages," Ballard explained, "the spores will germinate into a vegetative form of the microorganism. Then, that form will escape the macrophage and spread systemically around several points in the human body.
"It's the form of the organism," he explained, "that grows and replicates, divides, and is metabolically active. It multiplies in very high numbers in the bloodstream - as opposed to the spore form, which is quiescent and very environmentally resistant.
"There were 18 reported anthrax-attack cases in addressed envelopes containing spores, delivered by the U.S. postal system in the fall months of 2001," Ballard recalled. "Five of the 18 died from inhalational poisoning - the quickest, surest death-dealing version of the pathogen."
Ballard is senior author of an article in the Proceedings of the National Academy of Sciences (PNAS) released online Sept. 30, 2003. Its title: "Toxin-induced resistance in Bacillus anthracis lethal toxin-treated macrophages."
Taming LT, The Deadlier Toxin, If Possible
"Our findings are two," Ballard observed. "The first message is that macrophages can adapt to and respond to anthrax lethal toxin. That means it should be possible to train these immune cells under the right conditions to develop resistance to the toxin during the disease process. It also means," he added, " that as the microorganism starts to grow and produce lower amounts of LT, these macrophages may be able to adapt during the infection and resist higher doses of the toxin that may eventually occur as the disease progresses.
"The most central finding," he continued, "is that low nontoxic doses of the toxin will activate the macrophages to resist otherwise higher toxic doses. It's significant from a therapeutic standpoint that one might be able to use nontoxic doses of the toxin to preemptively trigger this response and then get resistance to high doses that would be toxic.
"Besides those two talking points related to anthrax toxin," Ballard went on "there's a third point related to toxins in general. Historically, we've thought of toxins as being able to modulate cell physiology, with no concern of how the macrophage cell may respond to that. This may cause some of us to rethink that process, because in this case the cell is clearly adapted to resist higher doses of the toxin.
"Our hypothesis is very simple," he told BioWorld Today. "The macrophages develop resistance to extremely high does of anthrax lethal toxin. This doesn't happen with the other two toxins, as we report in the PNAS paper. The macrophage is the cell that initially engulfs the spores, especially in the case of inhalational anthrax, which is what most people are concerned with right now. Phages are the first cells we know of that are aggressively attacked by spores in attempts to destroy them. Spores are not killed by the macrophages. In fact, the toxic organism then uses the macrophages to grow and divide, so it can escape from those cells and become systemic in the human host."
One Man's Cipro Is Another's Antibiotic Dud
"Some people may have macrophages that adapt and resist more effectively than others," Ballard pointed out, "which is why the Cipro antibiotic was not uniformly effective during that anthrax attack. Not everyone was genetically the same as regards positive or negative response to this bacterial pathogen.
"The edema toxin is not necessarily targeted to a specific pathway," Ballard explained. "It generates high levels of cyclic AMP in the cell. This may make it more difficult for the macrophage to respond or adapt, because there's not a specific pathway that's being modulated. But with lethal toxin, if it does hit one or more of these selective pathways, there may be an opportunity for the cell to find some way to get around a lot of these obstacles."
Ballard and his co-authors deploy a factor they call TIR - toxin-induced resistance. "We don't know what makes TIR happen," he allowed. "Perhaps because of the pathway that's disrupted inside these macrophages, if intoxication happens at a low enough rate, the cells can find their way around the problem they face. At very high doses, it may simply be a race between the amount of toxin that's present and the ability of the macrophage to overcome B. anthracis," he concluded.
"In the current study," Ballard's PNAS paper summed up, "we show that macrophages adaptively resist anthrax lethal toxin through TIR activation. This we triggered by pretreatment of macrophages with a low dose of LT for at least six hours, resulting in resistance to high doses of LT for 96 hours. Activation of TIR required functional toxin, because LT subunits, mutants and heat-inactivated toxin were unable to trigger resistance. Despite the sustained loss of full-length, mitogen-activated protein kinase kinase (MAPKK), TIR macrophages regained diphosphorylated extracellular response kinases - suggesting an adaptation to recovery of this signaling pathway. These results," the paper concluded, "provide a paradigm for toxin-cell interactions and suggest that macrophages are capable of adapting to, and tolerating, toxic doses."