Clostridium perfringens Type B-D Virulence Plasmids

描述(由申请人提供)：B、C和D型产气荚膜梭菌分离株具有重要的医学、兽医和生物防御重要性。B-D型分离株表达的几种毒素(如选择剂“B”表表型Esilon毒素和β毒素)是由存在于大质粒上的基因编码的。该项目的长期目标是充分了解那些鲜有研究的质粒如何对B-D型分离株的毒力做出贡献。为了开始朝着这个目标前进，将追求以下具体目标：1)利用插入突变方法构建B-D型背景下的单、双毒素等基因敲除突变体，2)比较这些新构建的等基因突变体与其亲本(和互补突变株)的毒力；这将使用体内和体外方法来评估肠道毒力(肠环模型)、全身毒力(静脉注射培养上清液)和模仿整个疾病谱的十二指肠内挑战的影响(即肠道和全身疾病)，3)在混合交配实验中使用AIM#1毒素突变体，它将携带标记有抗生素耐药性决定因素的毒力质粒，以评估B-D型毒力质粒是否可以通过接合在产气荚膜梭菌分离株之间转移，4)进行表型/基因分析以评估这些分离物的多样性；这些研究将包括检测B-D型分离株以确定它们产生多少β和/或epsilon-毒素(适当)，测试这些毒素表达差异是否与启动子差异有关，确定一些B-D型分离株是否产生生物活性改变的β-或epsilon毒素变体(适当)，并使用脉冲场凝胶电泳和微阵列方法检测B-D型质粒基因组的多样性，以及5)通过插入失活方法研究无毒素毒力质粒的功能；如果Aim#3证实B-D型毒力质粒可以通过接合转移，则Aim#5最初将针对这些质粒上可能的DNA转移基因。这些研究预计将为开发针对B-D型感染的改进疫苗/疗法以及开发分子分析以对这些分离物进行亚型鉴定提供关键信息，这是在生物恐怖主义事件期间故意释放B-D分离物进行法医调查所必需的。

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.