Kroh Heather K, Jensen Jaime L, Wellnitz Sabine, Park Jeong Jin, Esadze Alexandre, Huynh Kevin W, Ammirati Mark, Han Seungil, Anderson Annaliesa S, Lacy D Borden, Gribenko Alexey
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Vaccine Research and Development, Pfizer Inc, Pearl River, NewYork, USA.
Infect Immun. 2025 Aug 22:e0013925. doi: 10.1128/iai.00139-25.
is a spore-forming, Gram-positive bacterium that can cause infections in subjects with weakened immune system or following antibiotic treatment. These infections may lead to pseudomembranous colitis and antibiotic-associated diarrhea in humans. As such, is a major cause of nosocomial illness worldwide. Major virulence factors of the bacterium are the large clostridium toxins A (TcdA) and B (TcdB)-high molecular mass proteins with intrinsic glucosyltransferase activity. Toxins bind to the intestinal epithelium and undergo endocytosis by the epithelial cells, followed by a conformational change triggered by the low pH of early endosomes. This conformational change leads to the exposure of hydrophobic segments, followed by membrane insertion, formation of pores, and translocation of the glucosyltransferase domain into the cellular cytoplasm. Once in the cytoplasm, the glucosyltransferase domain inactivates small GTPases of the Rho family of proteins, leading to the disruption of the cytoskeleton. In the current work, we describe the discovery and characterization of a panel of neutralizing mouse monoclonal antibodies capable of interfering with several steps of cellular intoxication by the toxins. The antibodies were produced using hybridoma technology. Neutralizing activity of the antibodies was confirmed using toxin neutralization assays, and functional assays were used to identify specific neutralization mechanisms. Binding epitopes of the antibodies were identified by hydrogen-deuterium exchange mass spectrometry and confirmed through negative-stain and cryo-electron microscopy. Together, our results show that full-length toxins and/or genetically- and chemically-modified toxoids can induce a wide spectrum of antibodies capable of neutralizing the toxins via a variety of mechanisms.
是一种形成芽孢的革兰氏阳性细菌，可在免疫系统较弱的个体或抗生素治疗后引起感染。这些感染可能导致人类伪膜性结肠炎和抗生素相关性腹泻。因此，它是全球医院感染的主要原因。该细菌的主要毒力因子是大梭菌毒素A（TcdA）和毒素B（TcdB）——具有内在葡萄糖基转移酶活性的高分子量蛋白质。毒素与肠上皮细胞结合并被上皮细胞内吞，随后由早期内体的低pH值引发构象变化。这种构象变化导致疏水片段暴露，随后插入膜中、形成孔，并将葡萄糖基转移酶结构域转运到细胞质中。一旦进入细胞质，葡萄糖基转移酶结构域就会使Rho家族蛋白质的小GTP酶失活，导致细胞骨架破坏。在当前的工作中，我们描述了一组能够干扰毒素细胞中毒几个步骤的中和小鼠单克隆抗体的发现和特性。这些抗体是使用杂交瘤技术产生的。通过毒素中和试验确认了抗体的中和活性，并使用功能试验来确定具体的中和机制。通过氢-氘交换质谱法确定了抗体的结合表位，并通过负染和冷冻电子显微镜进行了确认。总之，我们的结果表明，全长毒素和/或基因和化学修饰的类毒素可以诱导产生多种能够通过多种机制中和毒素的抗体。
