SignificanceClostridioides difficile infection (CDI) is a major public health concern with over half a million cases in the United States annually resulting in ~30,000 deaths. Current therapies are inadequate and frequently result in cycles of recurrent infection. Using de novo protein design, w developed small protein inhibitors targeting two independent receptor binding sites on the toxin that drives pathology during CDI. We extend this approach to develop inhibitors of TcsL, a related toxin that causes highly lethal toxic shock with no effective treatments, and show that these inhibitors prolong survival in a murine lethal toxin challenge model.Clostridioides difficile remains a leading cause of hospital-acq... More
SignificanceClostridioides difficile infection (CDI) is a major public health concern with over half a million cases in the United States annually resulting in ~30,000 deaths. Current therapies are inadequate and frequently result in cycles of recurrent infection. Using de novo protein design, w developed small protein inhibitors targeting two independent receptor binding sites on the toxin that drives pathology during CDI. We extend this approach to develop inhibitors of TcsL, a related toxin that causes highly lethal toxic shock with no effective treatments, and show that these inhibitors prolong survival in a murine lethal toxin challenge model.Clostridioides difficile remains a leading cause of hospital-acquired infections, with its primary virulence factor, toxin B (TcdB), responsible for severe colitis and recurrent disease. The closely related toxin, TcsL, from Paeniclostridium sordellii , causes a rarer but often fatal toxic shock syndrome, particularly in gynecological and obstetric contexts. We report the de novo design of small protein minibinders that directly neutralize TcdB and TcsL by preventing their entry into host cells. Using deep learning and Rosetta-based approaches, we generated high-affinity minibinders that protect cells from intoxication with picomolar potency and, in the case of TcsL, prolonged survival following lethal toxin challenge in mice. The designed proteins against TcdB demonstrate exceptional stability in proteolytic and acidic environments, making them well-suited for oral delivery a valuable feature for treating C. difficile infections localized to the gastrointestinal tract. For TcsL, potent inhibitors were identified from 48 initial designs and 48 optimized designs, highlighting the potential of computational design for rapidly developing countermeasures against life-threatening bacterial toxins.
