Construction and analysis of chromosomal Clostridium difficile mutants
Construction and analysis of chromosomal Clostridium difficile mutants
复制标题
DOI：
10.1111/j.1365-2958.2006.05315.x
复制
发表时间：
2006-09-01
影响因子：
3.6
通讯作者：
Rood, Julian I.
中科院分区：
文献类型：
--
作者：
O'Connor, Jennifer R.;Lyras, Dena;Rood, Julian I.
艰难梭菌是一种重要性和毒力日益增加的医院感染病原体，但我们研究艰难梭菌致病机制的分子机制的能力受到限制。由于缺乏对其进行遗传操作的工具，艰难梭相关疾病的治疗受到限制。我们现在已经开发了一种可重复的方法，用于染色体C的靶向插入失活。艰难基因。该方法依赖于大肠杆菌-产气荚膜梭菌穿梭载体在C.艰难梭菌，并且可以用作条件致死载体的形式以将基因构建体递送至染色体。我们已经使用这种方法插入两个假定的反应调节基因，rgaR和rgbR，它们编码的蛋白质与来自C.产气荚膜杆菌转录组学分析表明C.艰难梭菌RgaR蛋白正调控四个基因，包括一个推定的agrBD操纵子。RgaR蛋白也被纯化，并显示出与包含两个共有VirR盒的位点特异性结合，这些盒位于这些基因推定启动子的上游。这种方法的发展将大大提高我们的能力，使用分子方法来开发一个更好的理解的能力，C。很难引起疾病。
Clostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile-associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli-Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease.