Department of Pathology, Microbiology, and Immunology, Chemical and Physical Biology Program, Department of Surgery, Epithelial Biology Center, Department of Medicine, and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18674-9. doi: 10.1073/pnas.1313658110. Epub 2013 Oct 28.
Clostridium difficile infection (CDI) is a leading cause of health care-associated diarrhea and has increased in incidence and severity over the last decade. Pathogenesis is mediated by two toxins, TcdA and TcdB, which cause fluid secretion, inflammation, and necrosis of the colonic mucosa. TcdB is a potent cytotoxin capable of inducing enzyme-independent necrosis in both cells and tissue. In this study, we show that TcdB-induced cell death depends on assembly of the host epithelial cell NADPH oxidase (NOX) complex and the production of reactive oxygen species (ROS). Treating cells with siRNAs directed against key components of the NOX complex, chemical inhibitors of NOX function, or molecules that scavenge superoxide or ROS confers protection against toxin challenge. To test the hypothesis that chemical inhibition of TcdB-induced cytotoxicity can protect against TcdB-induced tissue damage, we treated colonic explants with diphenyleneiodonium (DPI), a flavoenzyme inhibitor, or N-acetylcysteine (NAC), an antioxidant. TcdB-induced ROS production in colonic tissue was inhibited with DPI, and both DPI and NAC conferred protection against TcdB-induced tissue damage. The efficacy of DPI and NAC provides proof of concept that chemical attenuation of ROS could serve as a viable strategy for protecting the colonic mucosa of patients with CDI.
艰难梭菌感染（CDI）是医疗保健相关腹泻的主要原因，在过去十年中其发病率和严重程度有所增加。发病机制由两种毒素 TcdA 和 TcdB 介导，它们导致液体分泌、结肠黏膜炎症和坏死。TcdB 是一种有效的细胞毒素，能够在细胞和组织中诱导非酶依赖性坏死。在这项研究中，我们表明 TcdB 诱导的细胞死亡依赖于宿主上皮细胞 NADPH 氧化酶（NOX）复合物的组装和活性氧物质（ROS）的产生。用针对 NOX 复合物关键成分的 siRNA、NOX 功能的化学抑制剂或清除超氧化物或 ROS 的分子处理细胞，可对毒素攻击提供保护。为了检验化学抑制 TcdB 诱导的细胞毒性可以防止 TcdB 诱导的组织损伤的假设，我们用二苯并碘（DPI）——一种黄素酶抑制剂，或 N-乙酰半胱氨酸（NAC）——一种抗氧化剂处理结肠外植体。DPI 抑制 TcdB 诱导的结肠组织 ROS 产生，DPI 和 NAC 都能防止 TcdB 诱导的组织损伤。DPI 和 NAC 的功效提供了一个概念验证，即化学抑制 ROS 可以作为保护 CDI 患者结肠黏膜的可行策略。
