Regulation of toxin gene expression in Clostridium difficile

项目摘要 艰难梭菌是引起医院获得性感染性腹泻的最常见原因，它是 仅在美国，每年就有25万人感染，1.4万人死亡。大部分的病理学 与艰难梭菌感染相关的是由两种主要毒素TcdA和TcdB引起的。多项研究 已经证明了TcdA和TcdB在导致艰难梭菌感染中所起的关键作用。以前的研究 已经确定毒素的表达是对营养限制的反应。此外， 已经确定了几个调节这种反应的因素，包括一个替代的西格玛因素。 (TcdR)，一个对GTP和支链氨基酸(Cody)做出反应的全球监管机构，以及一个全球 对葡萄糖(CCPA)等易代谢碳源做出反应的调节剂。在这里，我们描述 应用单细胞分析监测艰难梭菌毒素基因的表达。我们融合了基因 对于毒素A的启动子的红色荧光蛋白(RFP)，它使我们能够监测毒素的表达- 用荧光显微镜和流式细胞仪检测单细胞中的Sion。令人惊讶的是，TcdA-RFP表达式 似乎是“双稳”的。在稳定期，当毒素表达最高时，大约30%的细胞 是“TcdA-on”，这些细胞的平均荧光强度是“TcdA-on”的~50倍。 Off“人口。我们发现双稳态依赖于TcdR的能级。这些发现提出了 几个重要的问题：双稳是如何控制的？在一个双稳态的人身上有没有其他基因受到调控- 内尔？这种表型多样性的生物学意义是什么？在这里，我们建议解决 前两个问题通过(A)检验毒素产生的双稳态表达是中介的假设 由全球基因表达调控者控制TcdR水平和(B)识别共同调控的基因 与TCDA合作。实现这项建议的目标将有助于更好地理解这些机制 哪些毒素基因在艰难梭菌中的表达是在单细胞水平上控制的。

Project Summary Clostridium difficile, the most common cause of hospital-acquired infectious diarrhea, is responsible for 250,000 infections and 14,000 deaths each year in the United States alone. Much of the pathology associated with C. difficile infections is caused by two major toxins, TcdA and TcdB. Multiple studies have demonstrated the critical role TcdA and TcdB play in causing C. difficile infections. Previous studies have established that expression of the toxins is induced in response to nutrient limitation. Moreover, several factors that mediate this response have been identified, including an alternative sigma factor (TcdR), a global regulator that responds to GTP and branched chain amino acids (CodY), and a global regulator that responds to easily metabolized carbon sources like glucose (CcpA). Here we describe the use of single cell analysis to monitor expression of the toxin genes in C. difficile. We fused the gene for a red fluorescent protein (RFP) to the promoter for toxin A, which allowed us to monitor toxin expres- sion in single cells by fluorescence microscopy and flow cytometry. Surprisingly, tcdA-rfp expression appears to be “bistable.” In stationary phase, when toxin expression is highest, about 30% of the cells are “TcdA-ON” and the mean fluorescence intensity of these cells is ~50 fold higher than in the “TcdA- OFF” population. We find that bistability is dependent upon the levels of TcdR. These findings raise several important questions: How is bistability controlled? Are other genes regulated in a bistable man- ner? What is the biological significance of this phenotypic diversity? Here we propose to address the first two questions by (a) testing the hypothesis that bistable expression of toxin production is mediated by global regulators of gene expression controlling levels of TcdR and (b) identifying genes co-regulated with tcdA. Achieving the aims of this proposal will lead to a better understanding of the mechanisms by which toxin gene expression is controlled in C. difficile at the level of the single cell.