Regulation of toxin gene expression in Clostridium difficile

艰难梭菌毒素基因表达的调控

• 批准号: 9180099
• 负责人: Craig D Ellermeier
• 金额: $ 18.93万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9180099
• 关键词: Address; Animal Model; Binding Sites; Biological; Branched-Chain Amino Acids; Carbon; Cell Death; Cell Separation; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Clinical Trials; Clostridium difficile; DNA-Directed RNA Polymerase; Data; Diarrhea; Disease; Epidemic; Epithelial Cells; Family; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Gene Expression; Genes; Glucose; Glucosyltransferase; Glucosyltransferases; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Hospitals; Human; Individual; Infection; Inflammation; Lead; Life; Mediating; Messenger RNA; Methods; Monitor; Names; Nucleic Acid Regulatory Sequences; Nursing Homes; Nutrient; Nutritional status; Pathology; Phase; Physiological; Play; Population; Production; Proteins; Pseudomembranous Colitis; Public Health; Regulation; Regulator Genes; Regulon; Reporter; Reporter Genes; Reporting; Role; Severities; Sigma Factor; Site; Source; Testing; Thinking; Toxin; United States; Virulence; Virulent; combat; cost; design; innovation; insight; interest; killings; man; mutant; new therapeutic target; novel strategies; novel therapeutics; overexpression; pathogen; pathogenic bacteria; promoter; red fluorescent protein; response; rho; single cell analysis; tool; transcriptome sequencing

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.

项目摘要 艰难梭菌是医院获得性感染性腹泻的最常见原因， 仅在美国每年就有25万人感染，14,000人死亡。大部分的病理 与C.艰难梭菌感染由两种主要毒素TcdA和TcdB引起。多项研究 已经证明了TcdA和TcdB在引起C.艰难感染。以前的研究 已经确定毒素的表达是响应于营养限制而诱导的。此外，委员会认为， 已经确定了几个介导这种响应的因素，包括另一个sigma因子 （TcdR），一种响应GTP和支链氨基酸（CodY）的全局调节剂，以及一种全局调节剂。 调节器，响应容易代谢的碳源，如葡萄糖（CcpA）。这里我们描述 利用单细胞分析技术监测毒素基因在C.很难我们融合了基因 红色荧光蛋白（RFP）的启动子毒素A，这使我们能够监测毒素表达- 通过荧光显微镜和流式细胞术观察单细胞中的细胞周期。令人惊讶的是，tcdA-rfp表达 看起来是“”。在稳定期，当毒素表达最高时，约30%的细胞 是“TcdA-ON”，并且这些细胞的平均荧光强度比“TcdA-ON”中高约50倍。 OFF”人群。我们发现双稳态依赖于TcdR的水平。这些发现提出 几个重要的问题：如何控制双稳态？是否有其他基因在一个人的基因中被调节- ner？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）检验毒素产生的表达是介导的假设 通过控制TcdR水平的基因表达的全局调节剂和（B）鉴定共调节的基因 tcdA的。实现这一建议的目标将有助于更好地了解这些机制， 该毒素基因的表达在C.在单细胞水平上很难。