Clostridium perfringens Type B-D Virulence Plasmids

产气荚膜梭菌 B-D 型毒力质粒

• 批准号: 8288751
• 负责人: Bruce A Mc Clane
• 金额: $ 41.78万
• 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: 2003-07-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8288751
• 关键词: Animal Model; Animals; Anus; Attention; Attenuated; Bacteria; Biochemical; Biological Assay; Bioterrorism; Centers for Disease Control and Prevention (U.S.); Clostridium difficile; Clostridium perfringens; Clostridium perfringens epsilon toxin; Complement; DNA; DNA Microarray Chip; Development; Disease; Disease Outbreaks; Disease model; Enteral; Epidemiology; Evolution; Forensic Medicine; Funding; Gel; Genes; Goals; Goat; Health; Human; Individual; Infection; Intestines; Introns; Investigation; Ions; Knock-out; Knowledge; Laboratories; Lead; Mediating; Medical; Modeling; Molecular; Molecular Epidemiology; Mus; Mutagenesis; Oryctolagus cuniculus; Parents; Partner in relationship; Pathogenesis; Pathogenicity; Physiologic pulse; Plasmids; Production; Research; Role; Southern Blotting; Stomach; Techniques; Testing; Tetracycline Resistance; Therapeutic; Toxin; Vaccine Design; Vaccines; Virulence; Work; anthrax lethal factor; attenuation; biodefense; improved; in vivo; meetings; mutant; novel vaccines; pathogen; research study; therapeutic vaccine; tool; transmission process

DESCRIPTION (provided by applicant): Clostridium perfringens type B, C and D isolates have significant medical, veterinary and biodefense importance. Many highly lethal toxins (such as beta toxin and epsilon toxin, a class B select toxin) produced by type B-D isolates are encoded by large plasmids. The long-term goal of this project is to understand the contributions of these large toxin-encoding plasmids and their encoded toxins to the pathogenicity of type B-D isolates in order to improve the design of vaccines/therapeutics against natural or bioterrorism-related human or animal infections. This work will also lead to development of subtyping assays for molecular epidemiologic or forensic investigations of natural or bioterrorism disease outbreaks involving type B-D isolates. It also has significant implications for understanding the virulence evolution of the major clostridial enteropathogens. To accomplish these goals, the following specific aims will be pursued, i) to determine which known toxins contribute to the pathogenicity of type B-D isolates, Aim A will continue constructing single and multiple toxin null mutants in type B-D backgrounds, using our recently-developed, highly efficient intron mutagenesis approaches; ii) Aim B will compare the pathogenicity of those toxin mutants versus their parent type B-D isolates using our recently optimized animal models that evaluate specific disease aspects, including enteric pathogenicity (using rabbit or goat ileal loops) or lethality (using mouse i.d. or gastric challenge models); when mutants show attenuated pathogenicity, they will be complemented to confirm the attenuation specifically resulted from inactivation of the implicated toxin gene, iii) since we have shown the epsilon toxin-encoding plasmid of type D isolates is conjugative, Aim C will examine whether the toxin plasmids of type B, C and E isolates are also conjugative (using mixed mating approaches), whether type B-D conjugative toxin plasmid transfer can occur in the intestines where this transfer may contribute to pathogenesis, whether C. perfringens can conjugatively exchange toxin plasmids or toxin genes with Clostridium difficile, another major clostridial enteropathogen, and whether certain C. perfringens toxin plasmids are incompatible with one another; and, finally, iv) Aim D will evaluate the genotypic diversity of toxin plasmids in type B and C isolates using pulsed- field gel/Southern blot and plasmid diversity in type B-D isolates using microarray approaches. PUBLIC HEALTH RELEVANCE: Clostridium perfringens type B-D isolates have medical, veterinary, and biodefense importance because they produce a number of highly potent toxins such as epsilon toxin, a class B select toxin. To obtain critical information for developing improved vaccines or therapeutics against natural or bioterrorism-induced type B-D infections, this project will evaluate the contribution of individual known toxins to pathogenesis. In addition, since many toxins of type B-D isolates are encoded by large plasmids, we will study the diversity of the type B-D toxin plasmids in order to develop assays for epidemiologic or forensic purposes.

描述（由申请人提供）：产气荚膜梭菌 B、C 和 D 型分离株具有显着的医学、兽医和生物防御重要性。 B-D 型分离株产生的许多高度致命的毒素（例如 β 毒素和 ε 毒素，一种 B 类选择毒素）是由大质粒编码的。该项目的长期目标是了解这些大毒素编码质粒及其编码的毒素对 B-D 型分离株致病性的贡献，以改进针对自然或生物恐怖主义相关人类或动物的疫苗/疗法的设计感染。这项工作还将导致针对涉及 B-D 型分离株的自然或生物恐怖主义疾病爆发的分子流行病学或法医调查的亚型分析的发展。它对于了解主要梭菌肠道病原体的毒力进化也具有重要意义。为了实现这些目标，将追求以下具体目标，i) 确定哪些已知毒素有助于 B-D 型分离株的致病性，目标 A 将使用我们最近开发的技术，继续在 B-D 型背景中构建单个和多个毒素无效突变体，高效的内含子诱变方法； ii) 目标 B 将使用我们最近优化的动物模型来比较这些毒素突变体与其亲本 B-D 型分离株的致病性，该模型评估特定疾病方面，包括肠道致病性（使用兔或山羊回肠环）或致死性（使用小鼠 i.d. 或胃激发）模型）；当突变体表现出减弱的致病性时，将对其进行补充，以确认该减弱是由相关毒素基因失活引起的，iii) 由于我们已经证明 D 型分离株的 epsilon 毒​​素编码质粒是接合性的，Aim C 将检查该毒素是否B、C 和 E 型分离株的质粒也是接合的（使用混合交配方法），无论 B-D 型接合毒素质粒转移是否可以发生在这种转移可能有助于发病的肠道，产气荚膜梭菌是否可以与另一种主要梭菌肠道病原体艰难梭菌接合交换毒素质粒或毒素基因，以及某些产气荚膜梭菌毒素质粒是否彼此不相容；最后，iv) 目标 D 将使用脉冲场凝胶/Southern 印迹评估 B 型和 C 型分离株中毒素质粒的基因型多样性，并使用微阵列方法评估 B-D 型分离株中的质粒多样性。公共健康相关性：产气荚膜梭菌 B-D 型分离株具有医学、兽医和生物防御的重要性，因为它们产生许多高效毒素，例如 epsilon 毒​​素（一种 B 类选择毒素）。为了获得开发针对自然或生物恐怖主义诱发的 B-D 型感染的改进疫苗或疗法的关键信息，该项目将评估单个已知毒素对发病机制的贡献。此外，由于 B-D 型分离株的许多毒素是由大质粒编码的，因此我们将研究 B-D 型毒素质粒的多样性，以便开发用于流行病学或法医学目的的检测方法。