Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States of America.
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.
PLoS Pathog. 2024 Sep 19;20(9):e1012568. doi: 10.1371/journal.ppat.1012568. eCollection 2024 Sep.
Clostridioides difficile is a spore-forming pathogen and the most common cause of healthcare-associated diarrhea and colitis in the United States. Besides producing the main virulence factors, toxin A (TcdA) and toxin B (TcdB), many of the common clinical strains encode the C. difficile transferase (CDT) binary toxin. The role of CDT in the context of C. difficile infection (CDI) is poorly understood. Inflammation is a hallmark of CDI and multiple mechanisms of inflammasome activation have been reported for TcdA, TcdB, and the organism. Some studies have suggested that CDT contributes to this inflammation through a TLR2-dependent priming mechanism that leads to the suppression of protective eosinophils. Here, we show that CDT does not prime but instead activates the inflammasome in bone marrow-derived dendritic cells (BMDCs). In bone marrow-derived macrophages (BMDMs), the cell binding and pore-forming component of the toxin, CDTb, alone activates the inflammasome and is dependent on K+ efflux. The activation is not observed in the presence of CDTa and is not observed in BMDMs derived from Nlrp3-/- mice suggesting the involvement of the NLRP3 inflammasome. However, we did not observe evidence of CDT-dependent inflammasome priming or activation in vivo. Mice were infected with R20291 and an isogenic CRISPR/Cas9-generated R20291 ΔcdtB strain of C. difficile. While CDT contributes to increased weight loss and cecal edema at 2 days post infection, the relative levels of inflammasome-associated cytokines, IL-1β and IL-18, in the cecum and distal colon are unchanged. We also saw CDT-dependent weightloss in Nlrp3-/- mice, suggesting that the increased weightloss associated with the presence of CDT is not a result of NLRP3-dependent inflammasome activation. This study highlights the importance of studying gene deletions in the context of otherwise fully isogenic strains and the challenge of translating toxin-specific cellular responses into a physiological context, especially when multiple toxins are acting at the same time.
艰难梭菌是一种产芽孢的病原体，也是美国最常见的与医疗保健相关的腹泻和结肠炎的病因。除了产生主要的毒力因子毒素 A（TcdA）和毒素 B（TcdB）外，许多常见的临床菌株还编码艰难梭菌转移酶（CDT）二元毒素。CDT 在艰难梭菌感染（CDI）中的作用还不太清楚。炎症是 CDI 的一个标志，已经报道了 TcdA、TcdB 和该生物体的多种炎性体激活机制。一些研究表明，CDT 通过一种 TLR2 依赖性的引发机制导致保护性嗜酸性粒细胞被抑制，从而导致这种炎症。在这里，我们表明 CDT 不会引发炎症体，但会在骨髓来源的树突状细胞（BMDCs）中激活炎症体。在骨髓来源的巨噬细胞（BMDMs）中，毒素的细胞结合和形成孔的成分 CDTb 单独激活炎症体，并依赖于 K+外排。在存在 TcdA 的情况下观察不到激活，并且在来自 Nlrp3-/- 小鼠的 BMDMs 中也观察不到激活，这表明 NLRP3 炎性体的参与。然而，我们没有在体内观察到 CDT 依赖性的炎症体引发或激活的证据。小鼠感染 R20291 并使用同源 CRISPR/Cas9 生成的 C. difficile R20291 ΔcdtB 菌株进行感染。虽然 CDT 有助于增加感染后 2 天的体重减轻和盲肠水肿，但盲肠和远端结肠中与炎症体相关的细胞因子，IL-1β和 IL-18 的相对水平没有改变。我们还观察到 Nlrp3-/- 小鼠中存在 CDT 依赖性的体重减轻，这表明与 CDT 存在相关的体重减轻不是 NLRP3 依赖性炎症体激活的结果。这项研究强调了在其他完全同源的菌株背景下研究基因缺失的重要性，以及将毒素特异性的细胞反应转化为生理背景的挑战，特别是当多种毒素同时作用时。
