Clostridium perfringens Type B-D Virulence Plasmids

产气荚膜梭菌 B-D 型毒力质粒

• 批准号: 7163698
• 负责人: Bruce A Mc Clane
• 金额: $ 33.7万
• 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 2009-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7163698
• 关键词: Address; Animal Model; Antibiotic Resistance; Antibiotics; Attention; Bacteria; Biochemical; Biological Assay; Bioterrorism; Class; Clostridium perfringens; Clostridium perfringens epsilon toxin; Complement; Cultured Cells; DNA; Development; Disease; Enteral; Enterotoxins; Event; Facility Construction Funding Category; Forensic Medicine; Gene Transfer; Generations; Genes; Genome; Goals; Hand; In Vitro; Infection; Injection of therapeutic agent; Insertional Mutagenesis; Intestines; Intravenous; Investigation; Knock-out; Medical; Modeling; Molecular; Parents; Partner in relationship; Pathogenesis; Plasmids; Process; Pulsed-Field Gel Electrophoresis; Range; Research; Series; Study Section; Systemic disease; Techniques; Testing; Toxic effect; Toxin; Variant; Virulence; biodefense; improved; in vitro Assay; in vivo; intravenous injection; mutant; pathogen; promoter; research study; therapeutic vaccine; tool; transmission process

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.

B，C和D分离株类型梭状芽胞杆菌具有明显的医学，兽医和生物底面 重要性。 B-D类型表达的几种毒素（例如选择代理“ B”列表Epsilon毒素和β毒素） 分离株由存在于大质粒上的基因编码。该项目的长期目标是完全 了解那些小研究的质粒如何有助于B-D型分离株的毒力。开始 朝着这一目标迈进，将追求以下特定目标：i）造成的等源性单一和 B-D型背景中使用插入诱变方法中的双重毒素敲除突变体，2） 比较这些新结构的同基因突变体与父母的毒力（以及 补充突变体菌株）；这将使用体内和体外方法来完成 评估肠道毒力（肠环模型），全身毒力（静脉注射培养物 上清液}以及模仿整个疾病频谱的二次挑战的影响（即 3）使用AIM＃1毒素突变体，该突变体将带有标记的毒力质粒 在混合交配实验中，抗生素耐药性决定因素，以评估B-D型毒力是否是否 质粒可以通过共轭之间转移C.浓度分离菌分离株，4）传导表型/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型分离株至 确定它们产生多少β-和/或Epsilon toxin（根据适当），测试这些毒素是否存在 表达差异与启动子差异有关，确定某种B-D分离株是否产生 随着生物学活性改变的β-或Epsilon toxin变体（适当），并检查的多样性 B-D质质粒基因组使用脉冲场凝胶电泳和微阵列方法，5） 通过插入失活方法研究非毒素毒力质粒功能；如果AIM＃3确认 该类型的B-D毒力质粒可以通过共轭转移，AIM＃5最初将靶向推定的DNA 在这些质粒上转移基因。期望这些研究为开发提供关键信息 改善了针对B-D型感染的疫苗/治疗剂，并用于将分子测定到亚型开发分子测定 这些分离株，对于B-D型分离株故意的法医调查的必要条件 在生物恐怖事件中发行。