Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America.
PLoS Pathog. 2023 Oct 23;19(10):e1011496. doi: 10.1371/journal.ppat.1011496. eCollection 2023 Oct.
Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo.
艰难梭菌是美国抗生素相关性腹泻和医院感染的主要原因。艰难梭菌感染（CDI）的症状与两种同源蛋白毒素 TcdA 和 TcdB 的产生有关。这些毒素被认为是临床诊断以及开发新型预防和治疗策略的真正靶点。尽管有大量研究记录了这些努力，但在知识方面仍存在一些空白，这些空白可以通过创建新的研究工具来弥补。首先，我们现在认识到，虽然 TcdA 序列是保守的，但 TcdB 序列在循环临床分离株中可以有所不同。了解驱动广泛中和抗体反应的 TcdA 和 TcdB 表位，可以推进识别安全有效的毒素-蛋白嵌合体和片段用于疫苗开发的工作。此外，了解 TcdA 和 TcdB 在体内浓度的变化可以指导研究宿主和微生物组为重点的干预措施如何影响艰难梭菌的毒力潜力。我们已经开发了一组骆驼科动物衍生的纳米抗体，这些纳米抗体可以结合 TcdA 和 TcdB 的特定结构和功能域。我们注意到，许多强效的 TcdA 中和剂结合了输送结构域内的表位，这一发现可能反映了输送结构域在受体结合中的作用，或孔形成在将毒素酶结构域输送到细胞质中的保守作用。相比之下，TcdB 的中和表位则存在于多个结构域中。这些纳米抗体还被用于创建夹心 ELISA 测定法，以定量测定体外和感染小鼠的盲肠和粪便内容物中的 TcdA 和/或 TcdB。我们预计这些试剂和测定法将使研究人员能够监测 TcdA 和 TcdB 产生的动力学随时间的变化，以及各种实验干预措施对体内毒素产生的影响。
