Clostridium perfringens Type B-D Virulence Plasmids

B、C和D型产气荚膜梭菌分离株具有显著的医学、兽医和生物防御能力 重要性。几种由B-D型表达的毒素(如选择剂“B”列表中的埃西隆毒素和β毒素) 分离株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 了解这些鲜有研究的质粒对B-D型分离株的毒力有何影响。要开始 为了实现这一目标，将追求以下具体目标：一)构建同基因单一和 B-D型背景双毒素基因敲除突变体的插入突变方法，2) 比较这些新构建的等基因突变体与它们的双亲的毒力(和 互补突变株)；这将使用体内和体外方法来完成，这将 评估肠道毒力(肠环模型)、全身毒力(静脉注射培养物 上清液)和模拟整个疾病谱的十二指肠内挑战的影响(即，两者 肠道和全身疾病)，3)使用AIM#1毒素突变体，它将携带标记有 在混合交配实验中评估B-D型毒力是否具有抗生素耐药性决定因素 质粒可以通过接合在产气荚膜梭菌之间转移，4)进行表型/基因分型 分析以评估这些分离物的多样性；这些研究将涉及检查B-D型分离物以 确定它们产生多少β和/或epsilon毒素(视情况而定)，测试这些毒素是否 表达差异与启动子差异有关，决定了某些B-D分离株是否会产生 β或epsilon毒素变异体(视情况而定)具有改变的生物活性，并检查 B-D型质粒基因组脉冲场凝胶电泳法和微阵列方法，以及5) 通过插入失活方法研究无毒毒力质粒的功能；如果目标#3确认 B-D型毒力质粒可以通过接合转移，Aim#5最初将针对假定的DNA 将基因转移到这些质粒上。这些研究可望为制定 改进的B-D感染疫苗/治疗方法和亚型的分子检测方法 这些分离物，在B-D型分离物被故意 在一次生物恐怖主义活动中被释放。

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: i) contructing 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.