"About one percent of all hospital patients develop a C. difficile infection — they're treated with antibiotics to the point that benign gut bacteria are knocked out, and because C. difficile is resistant to antibiotics, it's able to proliferate," said University of Texas Medical Branch at Galveston associate professor Tor Savidge, lead author of a paper on the discovery to be published in Nature Medicine. "Then it releases these toxins that trigger colonic disease."
The toxins wreak havoc on cell structural proteins and biochemical communications networks, eventually killing the cell. But in order to do this damage, the toxins first have to get into the cell, and that means passing through the protective membrane that surrounds it. It's there that Savidge and his collaborators — a multidisciplinary team of researchers from UTMB, UCLA, Case Western Reserve University, Tufts University and the Commonwealth Medical College — may have found a way to stop them.
On the molecular scale, C. difficile toxin proteins are quite large — big enough that they have to "cleave" so that a smaller piece can slip through the membrane and into the cell. This cleavage is accomplished by a built-in molecular guillotine called a cysteine protease, which activates when the toxin encounters a molecule called InsP6 that is present at much higher levels inside the cell than outside.
"It's sort of like a sensor mechanism that detects when it's in a cell — the toxins say, InsP6 is here, it's time to cleave," Savidge said. "But we've identified a previously unknown protective response that activates after the toxins have induced gut inflammation, in which the host uses a process called nitrosylation to shut down the cysteine protease and prevent cleavage." A toxin that's unable to cleave stays stuck in the cell membrane, incapable of attacking the cell.
The researchers used test-tube, cell culture, patient specimens and animal model experiments, along with computer simulations of molecular interactions, to thoroughly explore this response — and to successfully devise a way to mimic it for therapeutic purposes. "Think of these toxins as missiles that the bacteria is producing to go off and detonate inside the cell," Savidge said. "One way to defend against missiles is to send out signals that trick them into either disarming their sensory mechanisms or get them to prematurely detonate."
Cell culture and mouse experiments demonstrated that a combination of GSNO (the nitrosylating agent and the "disarming" part of Savidge's analogy) and InsP6 (the "premature detonation" part) worked to prevent damage from C. diff. In fact, the combination therapy worked so well that the team is now preparing to test it in a clinical trial sponsored by UTMB's Institute for Translational Sciences.
"Identification of new treatment modalities to treat this infection would be a major advance," said Dr. Charalabos Pothoulakis, director of UCLA's Inflammatory Bowel Disease Center and a co-author on the study. "If we are successful with this approach, we may be able to treat other bacterial diseases in a similar way."