The results of a study headed by scientists at Harvard Medical School (HMS) suggest that a toxin produced by the bacterium that causes anthrax may also have an unexpected benefit, in that it can silence multiple types of pain.
The research, in lab tests and in live rodents, revealed that this specific anthrax toxin works to alter signaling in pain-sensing neurons that carry a receptor for the toxin and when delivered in a targeted manner into neurons of the central and peripheral nervous system, can offer relief to animals in pain. The team also combined parts of the anthrax toxin with different types of molecular cargo to deliver the cargo into pain-sensing neurons.
The team suggests the technique could be used to design novel precision-targeted pain treatments that act on pain receptors, but without the widespread systemic effects of current pain-relief drugs, such as opioids. “This molecular platform of using a bacterial toxin to deliver substances into neurons and modulate their function represents a new way to target pain-mediating neurons,” said study senior investigator Isaac Chiu, PhD, associate professor of immunology in the Blavatnik Institute at Harvard Medical School.
The new findings also point to novel avenues for drug development beyond the traditional small-molecule therapies that are currently being designed across labs. “Bringing a bacterial therapeutic to treat pain raises the question ‘Can we mine the natural world and the microbial world for analgesics?’” Chiu said. “Doing so can increase the range and diversity of the types of substances we look to in search for solutions.”
Chiu, together with colleagues at HMS, in collaboration with industry scientists and researchers from other institutions, reported on their findings in Nature Neuroscience, in a paper titled, “Anthrax toxins regulate pain signaling and can deliver molecular cargoes into Antxr2+ DRG sensory neurons,” in which they concluded, “… we propose that anthrax toxin provides unique opportunities for targeting sensory neurons and merits further development as a platform for modulating their intracellular biology.”
Anthrax, which is caused by the bacterium Bacillus anthracis, has a scary reputation. Widely known to cause serious lung infections in humans and unsightly, albeit painless, skin lesions in livestock and people, the anthrax bacterium has even been used as a bioweapon.
The sensation of pain is triggered by nociceptive somatosensory neurons in response to harmful thermal, mechanical, and chemical stimuli, and “the identification of new strategies to selectively target and silence nociceptive neurons may contribute to the development of improved pain therapeutics,” the authors wrote. Opioids remain the most effective pain medication, but they have dangerous side effects—most notably their ability to rewire the brain’s reward system, which makes them highly addictive, and their propensity to suppress breathing, which can be fatal. So there is an acute need to expand the current therapeutic arsenal for pain management.
“There’s still a great clinical need for developing non-opioid pain therapies that are not addictive but that are effective in silencing pain,” said study first author Nicole Yang, PhD, HMS research fellow in immunology in the Chiu Lab. “Our experiments show that one strategy, at least experimentally, could be to specifically target pain neurons using this bacterial toxin.”
Naturally occurring toxins are a rich source of evolutionarily selected molecular agents that target neuronal function, the authors noted. For example, “We and others have recently found that bacterial products can act on sensory neurons to modulate pain or cough during pathogenic infection,” the investigators noted. Researchers in the Chiu lab have long been interested in the interplay between microbes, and the nervous and immune systems. Past work led by Chiu demonstrated that other disease-causing bacteria can also interact with neurons, and alter their signaling to amplify pain. Yet only a handful of studies so far have looked at whether certain microbes could minimize or block pain. This is what Chiu and Yang set out to do.
As the authors explained, anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF), which form two bipartite toxins: lethal toxin (LT; PA + LF) and edema toxin (ET; PA + EF). For their newly reported study, the investigators started out by trying to determine how pain-sensing neurons may be different from other neurons in the human body. To do so, they first turned to gene-expression data. One of the things that caught their attention was that pain fibers had receptors for anthrax toxins, whereas other types of neurons did not. In other words, the pain fibers were structurally primed to interact with the anthrax bacterium. “Anthrax toxins are composed of PA, which binds to ANTXR2, and the protein cargoes EF and LF,” they further wrote. “We describe a striking pattern of ANTXR2 expression in the nervous system, where it is mostly absent in central nervous system (CNS) neurons but enriched in Nav1.8+DRG neurons.”
The team’s studies found that pain silencing occurs when sensory neurons of dorsal root ganglia—nerves that relay pain signals to the spinal cord—connect with the two specific proteins made by the anthrax bacterium. The experiments revealed that when one of the bacterial proteins, PA, binds to the ANTXR2 nerve cell receptors it forms a pore that serves as a gateway for two other bacterial proteins, EF and LF, to be ferried into the nerve cell. The research further demonstrated PA and EF together, collectively known as ET, alter the signaling inside nerve cells—in effect silencing pain. And as they pointed out, “The enriched expression of ANTXR2 in DRG nociceptive neurons compared with CNS neurons offers an opportunity for selective targeting.”
In a series of experiments, the researchers confirmed that the anthrax toxin altered signaling in human nerve cells in dishes, but that it also did so in live animals.
Injecting the toxin into the lower spines of mice produced potent pain-blocking effects, preventing the animals from sensing high-temperature and mechanical stimulations. “We found that intrathecally administered ET targets DRG neurons in vivo and silences thermal and mechanical pain modalities across multiple mouse models,” they wrote. Importantly, other vital signs, such as heart rate, body temperature, and motor coordination, were not affected in the toxin-treated animals. This observation underscored that the technique was highly selective and precise in targeting pain fibers and blocking pain without widespread systemic effects.
Injecting mice with the anthrax toxin also alleviated symptoms of pain resulting from inflammation and pain caused by nerve cell damage, which are commonly experienced in the aftermath of traumatic injury and from certain viral infections such as herpes zoster or shingles, or as a complication of diabetes and cancer treatment.
Additionally, the researchers observed that as the pain diminished, the treated nerve cells remained physiologically intact—a finding that indicates the pain-blocking effects were not due to injury of the nerve cells but rather stemmed from the altered nerve signaling.
Beyond their roles in bacterial pathogenesis, anthrax toxins have been utilized as a delivery system for transporting functional molecular cargo into the cytoplasm of other cell types, such as cancer cells, the authors noted. So, in a final step, the team designed a carrier vehicle from anthrax proteins and used it to deliver other pain-blocking substances into nerve cells. One of these substances was botulinum toxin, yet another potentially lethal bacterium known for its ability to alter nerve signaling. That approach, too, blocked pain in mice. The experiments demonstrated that this could be a novel delivery system for targeting pain.
“In the present study, we described ANTXR2 as a receptor for anthrax toxins on nociceptive sensory neurons and identified B. anthracis ET as a modulator of neuronal signaling and pain,” the team concluded “The anthrax toxin system delivered multiple types of non-native proteins into sensory neurons to block pain, demonstrating potential as a protein delivery platform,” And while they acknowledged that the exact mechanism by which intrathecal ET produces analgesic effects isn’t yet understood, “… collectively our data show that ET targets ANTXR2 on primary nociceptive neurons to induce intrinsic changes at the transcriptional and signaling levels, leading to inhibition of synaptic transmission to second-order neurons in the spinal cord.”
Yang further commented, “We took parts of the anthrax toxin and fused them to the protein cargo that we wanted it to deliver. In the future, one could think of different kinds of proteins to deliver targeted treatments.” The scientists caution that as development work progresses, the safety of the toxin treatment would need careful monitoring, especially given that the anthrax protein has been implicated in disrupting the integrity of the blood-brain barrier during infection.
The new findings raise another interesting question, they suggested. Evolutionarily speaking, why would a microbe silence pain? Chiu thinks that one explanation—a highly speculative one, he added—may be that microbes have developed ways to interact with their host in order to facilitate their own spread and survival. In the case of anthrax, that adaptive mechanism may be through altered signaling that blocks the host’s ability to sense pain and therefore the microbe’s presence. This hypothesis could help explain why the black skin lesions that the anthrax bacterium sometimes forms are notably painless, Chiu added.