Clostridium perfringens Type B-D Virulence Plasmids

产气荚膜梭菌 B-D 型毒力质粒

• 批准号: 6676995
• 负责人: Bruce A Mc Clane
• 金额: $ 18.95万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6676995
• 关键词: Clostridium; antibiotics; bacterial cytopathogenic effect; bacterial genetics; bacterial toxicology; bacterial toxins; bioterrorism /chemical warfare; clostridial infection; disease /disorder model; drug resistance; enzyme linked immunosorbent assay; gel electrophoresis; gene targeting; genetic strain; genotype; goats; microorganism conjugation; microorganism culture; mutant; phenotype; plasmids; polymerase chain reaction; sheep; site directed mutagenesis; virulence

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.

描述（由申请人提供）：B、C 和 D 型产气荚膜梭菌分离株具有显着的医学、兽医和生物防御重要性。 B-D 型分离株表达的几种毒素（例如选择剂“B”列表中的 ε 毒素和 β 毒素）由大质粒上存在的基因编码。该项目的长期目标是充分了解这些很少研究的质粒如何影响 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）使用插入诱变方法在 B-D 型背景中构建同基因单毒素和双毒素敲除突变体，2）将这些新构建的同基因突变体的毒力与它们的毒力进行比较亲本（和补充突变株）；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（肠循环模型）、全身毒力（静脉注射培养物上清液）以及模拟整个疾病谱（即肠道和全身疾病）的十二指肠内攻击的影响), 3) 在混合交配实验中使用 Aim #1 毒素突变体，该突变体将携带带有抗生素抗性决定簇标记的毒力质粒，以评估是否为 B-D 型毒力质粒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/基因型分析以评估这些分离株的多样性；这些研究将涉及检查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相关，确定某些B-D型分离株是否产生β-或ε-毒素-具有改变的生物活性的毒素变体（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，以及5）研究非毒素毒力通过插入失活方法发挥质粒功能；如果目标 #3 确认 B-D 型毒力质粒可以通过接合转移，目标 #5 将首先针对这些质粒上假定的 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/疗法以及开发对这些分离株进行亚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。