Clostridium perfringens Type B-D Virulence Plasmids

描述(由申请人提供)：B、C和D型产气荚膜梭菌分离株在医学、兽医和生物防御方面具有重要意义。由B-D型分离株产生的许多高致命性毒素(如B类选择毒素β毒素和epsilon毒素)都是由大质粒编码的。该项目的长期目标是了解这些大的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，以便改进针对自然或生物恐怖主义相关的人或动物感染的疫苗/疗法的设计。这项工作还将导致为涉及B-D分离株的自然或生物恐怖主义疾病暴发的分子流行病学或法医调查开发分型分析。这也对了解主要梭状芽胞杆菌肠道病原体的毒力进化具有重要意义。为了实现这些目标，将追求以下特定目标：i)确定哪些已知毒素导致B-D型分离物的致病性，目标A将使用我们最近开发的高效内含子突变方法，在B-D型背景下继续构建单个和多个毒素零突变体；ii)目标B将使用我们最近优化的动物模型比较这些毒素突变体与其亲本B-D型分离物的致病性，该动物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.)。(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的，Aim C将检查B、C和E型分离株的毒素质粒是否也是接合的(使用混合交配方法)，B-D型接合的毒素质粒是否可以在肠道中发生转移，这种转移可能有助于致病，产气荚膜梭菌是否能与另一种主要的难辨梭状芽胞杆菌接合交换毒素质粒或毒素基因，以及某些C型产气荚膜菌的毒素质粒是否彼此不相容；最后，目的D将利用脉冲场凝胶/Southern印迹技术评估B、C型分离株毒素质粒的基因多样性，并利用基因芯片技术评估B-D型分离株的质粒多样性。公共卫生相关性：B-D型产气荚膜梭菌分离株具有医疗、兽医和生物防御的重要性，因为它们能产生许多高度有效的毒素，如B类精选毒素epsilon毒素。为了获得关键信息，以开发针对自然或生物恐怖主义诱导的B-D感染的改进疫苗或治疗方法，该项目将评估个别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都是由大质粒编码的，我们将研究B-D型毒素质粒的多样性，以便为流行病学或法医学目的建立检测方法。

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.