Mullard Robin M, Sheedlo Michael J
University of Minnesota, Department of Biochemistry, Molecular Biology, and Biophysics.
University of Minnesota Medical School, Department of Pharmacology.
bioRxiv. 2025 May 6:2025.05.06.652354. doi: 10.1101/2025.05.06.652354.
is a gram-positive, pathogenic bacterium and is currently the leading cause of hospital-acquired, infectious diarrhea in the United States. During infection, produces and secretes up to three toxins called Toxin A, Toxin B, and the transferase (CDT). While Toxin A and Toxin B are thought to drive the pathology associated with the disease, strains that produce CDT have been linked to increased disease severity, higher rates of infection recurrence, and increased incidence of mortality. A basic understanding of how CDT intoxicates host cells has emerged over the past two decades and includes a framework that relies on the oligomerization of the components that comprise CDT to promote cellular intoxication. Although several key steps of this process have been biochemically described, a clear, molecular description of toxin assembly has not been resolved. We have collected cryogenic electron microscopy (Cryo-EM) data of purified, recombinant CDT. From these data, we have generated several structural snapshots of the toxin, including a series of structures that correspond to intermediates that form during oligomerization. These structures provide insight into the mechanism underlying toxin assembly and highlight a role for structural plasticity during this process. We have also shown that these partially assembled toxins are equally potent in cytotoxicity assays supporting this model in a cellular context. Finally, we show that CDTb oligomers are stabilized by CDTa and assembly is triggered by hydrophobic molecules.
是一种革兰氏阳性病原菌，目前是美国医院获得性感染性腹泻的主要病因。在感染过程中，会产生并分泌多达三种毒素，即毒素A、毒素B和细胞致死性扩张毒素（CDT）。虽然毒素A和毒素B被认为是导致该疾病相关病理的原因，但产生CDT的菌株与疾病严重程度增加、感染复发率升高以及死亡率增加有关。在过去二十年中，人们对CDT如何使宿主细胞中毒有了基本的了解，其中包括一个依赖于构成CDT的成分寡聚化以促进细胞中毒的框架。尽管这一过程的几个关键步骤已在生化层面上得到描述，但毒素组装的清晰分子描述尚未得到解决。我们收集了纯化的重组CDT的低温电子显微镜（Cryo-EM）数据。从这些数据中，我们生成了该毒素的几个结构快照，包括一系列与寡聚化过程中形成的中间体相对应的结构。这些结构为毒素组装的潜在机制提供了见解，并突出了这一过程中结构可塑性的作用。我们还表明，这些部分组装的毒素在细胞毒性试验中同样具有效力，在细胞环境中支持了这一模型。最后，我们表明CDTb寡聚体由CDTa稳定，并且组装由疏水分子触发。
