Clostridium perfringens Type B-D Virulence Plasmids

描述（由申请方提供）：产气荚膜梭菌B、C和D型分离株具有重要的医学、兽医和生物防御意义。由B-D型分离株表达的几种毒素（例如，选择剂“B”列出的毒素和β毒素）由存在于大质粒上的基因编码。该项目的长期目标是充分了解这些很少研究的质粒如何促进B-D型分离株的毒力。为了实现这一目标，本研究的具体目标如下：1）利用插入突变技术构建B-D型背景下的等基因单毒素和双毒素敲除突变体; 2）比较这些新构建的等基因突变体与其亲本的毒力（和互补突变株）;这将使用体内和体外方法来完成，这些方法将评估肠道毒力（肠环模型）、全身毒力（培养物上清液的静脉内注射）和模拟整个疾病谱的十二指肠内攻击的作用（即，肠道和全身性疾病），3）在混合交配实验中使用Aim #1毒素突变体，其将携带用抗生素抗性决定簇标记的毒力质粒，以评估B-D型毒力质粒是否可以在C. 4）进行表型/基因型分析以评估这些分离物的多样性;这些研究将包括检查B-D型分离株，以确定有多少β-和/或ε-毒素（适当时）它们产生，测试那些毒素表达差异是否与启动子差异有关，确定某些B-D型分离株是否产生β或ε毒素变体（适当时）改变生物活性，并使用脉冲场凝胶电泳和微阵列方法检查B-D型质粒基因组的多样性，和5）通过插入失活方法研究无毒毒力质粒功能;如果目标#3证实B-D型毒力质粒可以通过接合转移，则目标#5将首先靶向这些质粒上的推定DNA转移基因。预计这些研究将为开发针对B-D型感染的改进疫苗/治疗剂以及开发对这些分离株进行分型的分子测定提供关键信息，这对于在生物恐怖主义事件期间故意释放B-D型分离株的情况下进行法医调查是必要的。

DESCRIPTION (provided by applicant): Clostridium perfringens type B, C, and D isolates have significant medical, veterinary, and biodefense importance. Several toxins (e.g. select agent "B" list epsilon toxin and beta toxin) expressed by type B-D isolates are encoded by genes present on large plasmids. The long-term goal of this project is to fully understand how those little-studied plasmids contribute to the virulence of type B-D isolates. To start progressing towards this goal, the following specific aims will be pursued: 1) constructing isogenic single and double toxin knock-out mutants in type B-D backgrounds using insertional mutagenesis approaches, 2) comparing the virulence of these newly-constructed isogenic mutants against their parents (and complemented mutants strains); this will be accomplished using in vivo and in vitro approaches that will evaluate enteric virulence (intestinal loop models), systemic virulence (intravenous injections of culture supernatants} and effects of an intraduodenal challenge mimicking the entire disease spectrum (i.e., both enteric and systemic disease), 3) using Aim #1 toxin mutants, which will carry virulence plasmids tagged with antibiotic resistance determinants, in mixed mating experiments to evaluate whether type B-D virulence plasmids can transfer between C. perfringens isolates via conjugation, 4) conducting phenotypic/genotypic analyses to evaluate the diversity of these isolates; these studies will involve examining type B-D isolates to determine how much beta- and/or epsilon-toxin (as appropriate) they produce, testing whether those toxin expression differences are related to promoter differences, determining if some type B-D isolates produce beta- or epsilon-toxin variants (as appropriate) with altered biologic activities, and examining the diversity of type B-D plasmid genomes using pulsed-field gel electrophoresis and microarray approaches, and 5)investigating non-toxin virulence plasmid functions by insertional inactivation approaches; if Aim #3 confirms that type B-D virulence plasmids can transfer via conjugation, Aim #5 will initially target putative DNA transfer genes on these plasmids. These studies are expected to provide critical information for developing improved vaccines/therapeutics against type B-D infections and for developing molecular assays to subtype these isolates, as necessary for forensic investigations in the event that type B-D isolates are deliberately released during a bioterrorism event.