Mycobacteria Use Angiotensin Receptor Pathway to Produce Painless Infections
Signaling through the angiotensin II type 2 receptor (AT2R) is responsible for the surprisingly painless nature of lesions found in the early stages of the necrotizing tropical disease Buruli ulcer, according to findings reported June 19 in Cell. The mouse study, led by Laurent Marsollier and Priscille Brodin of the French National Institute of Health and Medical Research (INSERM) Angers and Lille sites, respectively, adds to a growing pile of evidence (see PRF related conference story) that AT2R is an analgesic target.
The mycobacteria responsible for Buruli ulcer secrete mycolactone, a toxin that is essential for invasion and immune modulation of the host, and also causes a diminished sensation of pain in the lesions. In the current study, the authors explored the mechanism underlying the toxin’s analgesic effect and found that mycolactone-induced signaling through AT2Rs and the cyclooxygenase pathway alters potassium channel activity and leads to neuronal hyperpolarization.
“The discovery of this mechanism, which limits pain in the cutaneous lesions during the early stages of the [Buruli ulcer] disease, opens up new possibilities in the search for new drugs to prevent pain,” said Brodin.
Bacterial analgesic
Buruli ulcer is caused by Mycobacterium ulcerans, a member of the same family of bacteria responsible for leprosy and tuberculosis. The lack of pain in the early-stage lesions, which often delays treatment until later stages of infection that require radical surgical intervention, was thought to result from mycolactone-mediated nerve destruction (En et al., 2008). However, Brodin and her colleagues were skeptical of the link between nerve loss and the painlessness, so they set out to directly examine the mechanism underlying the analgesia in a mouse model of M. ulcerans infection, she said.
Co-first authors Estelle Marion, Ok-Ryul Song, and Thierry Christophe injected the mycobacteria into the footpad of mice, then later applied noxious heat and measured the latency to move the foot. Reminiscent of the human disease, the animals exhibited hypoesthesia (loss of sensation) to the painful stimulus that lasted for one to two months post-injection.
A model of the signaling cascade triggered by mycolactone. Upon binding to the AT2R receptor, coupled to Gαi, mycolactone triggers a signaling cascade leading to PLA2 activation and inducing the synthesis of arachidonic acid. Arachidonic acid is then metabolized by COX-1 into PGE2, leading to the opening of TRAAK channels. AA, arachidonic acid; PTGS1, COX-1; PGE2, prostaglandin E2; TRAAK, potassium channel subfamily K member 4. Credit: Marion et al., 2014, used with permission from Elsevier.
However, despite obvious signs of M. ulcerans infection, there was no macroscopic or ultrastructural evidence of nerve damage. Similar results were obtained with direct injection of purified mycolactone.
If not nerve damage, what could be the cause of the diminished sensation? Marion and colleagues reasoned that the toxin could be interfering with nociceptive signaling, so they measured the voltage change in neurons derived from rat pheochromocytoma (PC12) cells after application of the toxin in vitro. Using both electrophysiological recordings of the membrane potential and a fluorescent voltage-sensitive dye, the researchers observed a gradual hyperpolarization of the membrane potential that was reversed after toxin removal. Experiments with the potassium channel blockers TEA and BaCl2 revealed that the mycolactone-mediated hyperpolarization was due to potassium efflux.
Role of angiotensin receptor
To investigate how mycolactone produces hyperpolarization-induced hypoesthesia, the researchers screened a small interfering RNA (siRNA) library of 8,000 genes in macrophage-like cells for the ability to block mycolactone-induced hyperpolarization (the cells were chosen for their high efficiency of siRNA silencing). Three genes emerged as possibilities, but two (IL23R and ICAM1) were eliminated due to a lack of association with any known nociceptive pathways. The remaining gene, angiotensin II type 2 receptor (AGTR2), is the target of EMA401, a compound that has been shown to improve neuropathic pain in a Phase 2 clinical trial of post-herpetic neuralgia (Rice et al., 2014).
Consistent with a role for AT2R in the analgesia, mycolactone did not hyperpolarize neurons from AT2R knockout mice. An AT2R antagonist similarly abolished the mycolactone-induced hyperpolarization in wild-type neurons. Marion and colleagues delved deeper into the pathway in a re-analysis of the siRNA screening data by using less stringent criteria and taking available literature into account. Using this approach, along with subsequent pharmacological and silencing experiments, the authors developed a model of mycolactone signaling. According to this model, binding of the toxin to AT2R activates phospholipase A2 (PLA2) and produces arachidonic acid (AA), which is then metabolized to prostaglandin E2 (PGE2) by the enzyme COX-1, causing potassium efflux through KCNK4 (TRAAK) K+ channels, hyperpolarization, and subsequent hypoesthesia.
In a final demonstration that AT2R was necessary for hypoesthesia, Marion and colleagues injected mycolactone into the footpads of AT2R knockout mice and found that the mice did not display analgesia. Treating wild-type M. ulcerans-infected mice with an inhibitor of AT2R had a similar effect.
AT2R: to block or bolster?
The current findings demonstrate a mechanism by which a natural analgesic exerts its effect—knowledge that could be harnessed to improve the treatment of pain.
However, although [the study] is exciting, it’s not what you would expect, said Peter Smith, University of Kansas Medical Center, Kansas City, US, who was not involved with the work.
The published data on the analgesic effect of AT2Rs suggest that inhibition, not stimulation, of the receptor is responsible for the alleviation of pain, Smith explained. In addition to the clinical trial of antagonist EMA401, other antagonists of AT2R have been shown effective in rat models’ prostate cancer bone pain and inflammatory pain (Muralidharan et al., 2014; Chakrabarty et al., 2013).
Smith suggested that the presence of multiple signaling pathways, or the possibility that these compounds are only partial agonists but more powerful antagonists, could explain the disparate results.
The findings also raise the question of where activation of AT2Rs occurs following footpad injections. The molecular studies were performed in PC12 and macrophage-like cells rather than in sensory neurons, so it is possible that the receptors could be located on non-neuronal cells, suggested Jan Danser, Erasmus MC, Rotterdam, the Netherlands, and Praveen Anand, Imperial College London, UK, in an accompanying editorial.
They also note the apparent contradiction that low levels of mycolactone (similar to the levels found in tissue after infection) are sufficient to produce hyperpolarization in vitro; yet the level necessary to stimulate AT2Rs is much higher. In addition, the mouse findings will, of course, require validation in humans.
Clearly, more evidence is needed to confirm that mycolactone truly is a bona fide AT2 receptor agonist at relevant in vivo levels, conclude Danser and Praveen.
Regardless of the details, the data converge on AT2Rs. We all agree that AT2R is a target for pain modulation … the question is, Do we want to stimulate it or inhibit it? said Smith.
Allison Curley, PhD, is a neuroscientist-turned-science writer in Pittsburgh, Pennsylvania, US.
Image: Adapted from Cell, Marion et al., 2014, with permission from Elsevier.
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