Clostridium perfringens Type B-D Virulence Plasmids

超出所提供的空间。B型、C型和D型产气荚膜梭菌分离株具有重要的医学、兽医和生物防御意义。B- d型分离物表达的几种毒素（如选择剂“B”列表中的epsilon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这些很少被研究的质粒如何促进B-D型分离株的毒力。为了开始实现这一目标，将追求以下具体目标：1)使用插入诱变方法构建B-D型背景的等基因单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（和补充突变株）的毒力；这将通过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（静脉注射培养上清液）和模拟整个疾病谱系（即肠道和全身疾病）的十二指肠内攻击的影响，3)使用Aim #1毒素突变体，该突变体将携带带有抗生素抗性决定因素标记的毒力质粒。在混合交配实验中，评估B-D型毒力质粒能否通过接合在产气荚膜荚膜荚膜荚膜菌分离株之间转移；4)进行表型/基因型分析，评估这些分离株的多样性；这些研究将包括检查B-D型分离株，以确定它们产生多少β -和/或ε -毒素（视情况而定），测试这些毒素表达差异是否与启动子差异有关，确定某些B-D型分离株是否产生具有改变的生物活性的β -或ε -毒素变体（视情况而定），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。5)通过插入失活方法研究非毒素毒力质粒的功能；如果Aim #3确认B-D型毒力质粒可以通过接合转移，Aim #5将首先针对这些质粒上假定的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/治疗方法提供关键信息，并为开发对这些分离株进行分型的分子分析提供关键信息，如果在生物恐怖主义事件期间故意释放B-D型分离株，则需要进行法医调查。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================