Cytolethal Distending Toxin and Intestinal Homeostasis

我们的长期目标是了解病原体介导的遗传毒性是如何与 致病细菌、真菌和病毒影响微生物-宿主的相互作用和疾病的发病机制。我们是 研究细胞致死性扩张毒素(CDTS)，一个保守的，但分布广泛的胞内毒素家族- 作用基因毒素，由感染和破坏皮肤粘膜组织的病原菌分泌 身体内的不同部位，包括胃肠道。尽管越来越多的证据表明 对于CDT作为致病的决定因素，我们对CDT生物学理解的一个主要缺陷是确切的作用 这些遗传毒素在感染过程中。在这一应用中，我们建议进行研究以解决遗传毒性的影响 在由人类肠道病原体空肠弯曲菌(CJ-CDT)产生的CDT介导的肠道中， 已被证明在空肠弯曲菌(CJ)侵袭上皮屏障以及随后的 扩散到小鼠的血液、脾和肝脏。尽管入侵的机制还不太清楚， CJ-CDT介导的细胞毒性已初步在体外使用永生化细胞系进行了检测，并得到了广泛的应用 被认为是感染期间上皮屏障受损的主要驱动因素。然而，初步研究在 我们的实验室发现，在没有毒素介导的细胞的情况下，CJ-CDT改变了上皮屏障的完整性和功能 死亡，在小鼠衍生的肠道器官模型中，该模型概括了 肠上皮的隐窝/绒毛构筑。这些发现挑战了CJ-CDT的重要性 细胞毒性作为上皮屏障毒素依赖性改变的一种机制，提示可能 CJ-CDT介导的DNA损伤和/或宿主DNA损伤修复的关系及其机制 在肠上皮正常再生期间每隔3-5天调节细胞的增殖和分化 几天。根据我们实验室的初步数据，我们对这种潜在联系的总体模型是CJ- 依赖CDT的DNA损伤和随后的DNA损伤反应(DDR)促进肠屏障 通过调节细胞增殖和细胞谱系分配而导致的功能障碍，这两者对 再生。在这个探索性的R21应用程序中，我们解决了知识中的几个现有差距。在目标1中，我们 建议进行研究，以检验CJ-CDT依赖的遗传毒性对肠道的影响这一假设的预测 屏障功能障碍。我们将研究依赖CJ-CDT的DNA损伤/修复之间的因果关系 以及细胞增殖和分化的变化，并评估CJ-CDT对诱导 空肠弯曲菌感染中肠上皮屏障功能的研究在目标2中，我们将调查两者之间的关系 在CJ-CDT依赖的DNA损伤/修复和细胞内SNAI1水平降低之间，SNAI1是一个重要的 参与调节肠道谱系分配的转录因子。因为颠覆上皮屏障 诚信与人类疾病和病理有关，我们最终感兴趣的是扩展这些 研究CJ-CDT介导的遗传毒性对人类肠道健康的潜在负面影响。

Our long-term goal is to understand how pathogen-mediated genotoxicity, which is increasingly associated with pathogenic bacteria, fungi, and viruses, impacts microbe-host interactions and disease pathogenesis. We are studying the cytolethal distending toxins (CDTs), a conserved, but broadly distributed family of intracellular- acting genotoxins, which are secreted by pathogenic bacteria that infect and damage mucocutaneous tissues at different sites within the body, including the gastrointestinal tract. Despite increasing evidence of the importance of CDTs as determinants of pathogenesis, a major deficit in our understanding of CDT biology is the exact role of these genotoxins during infection. In this application, we propose studies to address the impact of genotoxicity at the intestine mediated by CDT produced by the human intestinal pathogen Campylobacter jejuni (Cj-CDT), which has been demonstrated to be important for C. jejuni (Cj) invasion of the epithelial barrier, and subsequent dissemination to the blood, spleen and liver in mice. Although the mechanism of invasion is poorly understood, Cj-CDT-mediated cytotoxicity, which has been examined primarily in vitro using immortalized cell lines, is widely believed to be a major driver of damage to the epithelial barrier during infection. However, preliminary studies in our lab revealed that Cj-CDT alters epithelial barrier integrity and function, in the absence of toxin-mediated cell death, within a murine-derived intestinal organoid model that recapitulates the dynamic regeneration of the crypt/villus architecture of the intestinal epithelium. These findings challenge the importance of Cj-CDT cytotoxicity as a mechanism of toxin-dependent changes at epithelial barriers, by suggesting possible associations between Cj-CDT-mediated DNA damage and/or host DNA damage repair and the mechanisms that regulate cell proliferation and differentiation during normal regeneration of the intestinal epithelium every 3-5 days. Based on preliminary data from our laboratory, our overall model of this potential association is that Cj- CDT-dependent DNA damage and subsequent DNA damage response (DDR) promote intestinal barrier dysfunction by modulating cell proliferation and cellular lineage allocation, both of which are critical for regeneration. In this exploratory R21 application, we address several existing gaps in knowledge. In Aim 1, we propose studies to test predictions of the hypothesis that Cj-CDT-dependent genotoxicity is causal for intestinal barrier dysfunction. We will investigate the causal relationship between Cj-CDT-dependent DNA damage/repair and alterations in cellular proliferation and differentiation, and assess the essentiality of Cj-CDT for inducing epithelial barrier function during Cj infection of intestinal organoids. In Aim 2, we will investigate the relationship between Cj-CDT-dependent DNA damage/repair, and the reduction in cellular levels of SNAI1, an important transcriptional factor involved in regulating intestinal lineage allocation. Because subversion of epithelial barrier integrity is associated with human disease and pathology, we are ultimately interested in extending these studies to investigate the potential negative impact of Cj-CDT-mediated genotoxicity on human intestinal health.