By Rob Mitchum
Brucella abortus is a particularly pesky pathogen. Frequently infecting cattle in many countries around the world, the bacterium causes the most common zoonotic infection, usually passing from animal to humans through ingestion of unpasteurized dairy products. While the infection, known as brucellosis or undulant fever, is rarely deadly, it can cause assorted flu-like symptoms including high fever, muscle pain, weight loss, and in severe cases, meningitis and encephalitis. These debilitating effects made it appealing to both the United States and Soviet Union for the development of biological weapons during the Cold War. Although these “brucella bombs” have long since been destroyed, the bacterium remains a concern around the world.
“It’s a substantial human health problem on a global level,” said Sean Crosson, assistant professor of biochemistry and molecular biology at the University of Chicago Biological Sciences. “The World Health Organization predicts that there are a half million cases annually, and it may be higher than that. It’s hard to know, because it’s more of a problem in the developing world where it’s difficult to quickly diagnose.”
Crosson’s laboratory is one of the few in the United States studying the bacterium and its surprisingly evasive behavior. The infection is very difficult to treat, requiring two different antibiotics to be taken for nearly two months — not an easy task in countries without easy access to pharmacies and the proper storage of the heat-sensitive drugs. Even when this rigorous treatment is followed under ideal conditions, the relapse rate for the disease is still high. When the Crosson lab searched through the genome of the bacterium, they found a potential reason for this resilience: a genetic poison and antidote pair usually thought of as a bacterial self-destruct mechanism.
“After treatment for brucellosis, there’s still a 15 percent chance of relapse,” said Brook Heaton, a Committee on Microbiology graduate student in Crosson’s lab. “This relapse is something that is obviously problematic, but also interesting when thinking of toxin-antitoxin systems, which are thought to play a role in persistence of infection.”
Toxin-antitoxin systems have been discovered in a variety of bacterial species, and studied primarily for their role in bacterial quality control. The tiny toxin and antitoxin genes were initially found on plasmids, small bits of bacterial DNA separate from the chromosomes. The two genes are typically transcribed together, producing both the toxin and antitoxin proteins simultaneously. Under normal conditions, the two proteins would stick together, nullifying the toxic activity like a sword in a sheath. But in this case, the antitoxin “sheath” degrades faster than the toxic “sword.” So if gene transcription stops for some reason or the cell divides and the daughter cell does not properly inherit a copy of the plasmid, then the self-destruct process begins.
But over the past decade, scientists started to find toxin-antitoxin genes on the chromosomes themselves, throwing the quality control theory into doubt.
“What exactly these things are doing is mysterious,” Crosson said. “They’ve been implicated in bacterial stress response, they get activated by certain stressors, and if they’re missing toxin-antitoxin genes then bacterial cells may not survive as well in certain environments. But the real biological function of toxin-antitoxin genes is still unclear.”
In a paper recently published by The Journal of Biological Chemistry, Heaton and her colleagues examined one toxin-antitoxin system from Brucella abortus with a wide variety of scientific techniques, from genetic manipulation and cell culture experiments carried out at the Ricketts Regional Biocontainment Laboratory at Argonne to biophysical analyses of protein structure. Functionally, when the toxin (named BrnT) was unleashed without its antitoxin (named BrnA), protein synthesis screeched to a halt and the bacterial cells stopped growing within an hour. But reports of the bacterial massacre were greatly exaggerated — subsequent activation of the antitoxin, even hours later, resurrected the cells to a normal, healthy state.
“On the surface, when you express the toxin, it looks almost like all the cells are dying,” Crosson said. “99.999% of the cells appear to die. But they’re not actually dead.”
The results suggested that this so-called toxin was given a bad rap. Instead of a self-destruct mechanism, the toxin appears to have a “bacteriostatic” effect, locking the cells in a dormant state but not killing them off. Further experiments demonstrated that the toxin is activated by a variety of stressful conditions, such as antibiotic treatment or acidic pH. Thus, Heaton and Crosson hypothesized that the toxin-antitoxin system could trigger a protective sort of hibernation when the bacteria is confronted with dangerous conditions, like a turtle withdrawing into his shell.
“It’s a wild result actually,” Crosson said. “The toxin is turned on strongly by multiple stressors. The bacteriostasis result is clear. So what are the implications here? It’s a genetic system, and it’s inherent in this bacterium in which we know relapse of infection is a real problem. This system provides a mechanism for a bacterial cell to put itself into a static state.”
The inference is that Brucella’s toxin-antitoxin system could play a big hand in why brucellosis is so hard to treat and so frequently relapses in patients. Current experiments in the laboratory are studying this relationship, as well as looking for agents that could prevent Brucella and other bacterial pathogens from entering their protective dormant state.
“This toxin is found in a ton of different bacterial species and a ton of human pathogens,” Heaton said. “So we’re also doing high throughput screening to look for inhibitors of the toxin that could inhibit the ability of these cells to enter this static state. You could give a co-therapeutic with the antibiotics and bacteria would be more sensitive to that treatment if they can’t go into an arrest.”
Photo: This colorized image shows a mouse immune cell 48 hours after being infected with the bacterium Brucella abortus (red). Courtesy NIAID.
Heaton, B., Herrou, J., Blackwell, A., Wysocki, V., & Crosson, S. (2012). Molecular Structure and Function of the Novel BrnT/BrnA Toxin-Antitoxin System of Brucella abortus Journal of Biological Chemistry, 287 (15), 12098-12110 DOI: 10.1074/jbc.M111.332163