Clostridium perfringens Type B-D Virulence Plasmids

描述（由申请人提供）：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。由B- d型分离株产生的许多高致命性毒素（如β毒素和epsilon毒素，B类选择毒素）由大质粒编码。该项目的长期目标是了解这些大型毒素编码质粒及其编码毒素对B-D型分离株致病性的贡献，以便改进针对自然或与生物恐怖主义有关的人类或动物感染的疫苗/疗法的设计。这项工作还将导致发展分型分析，用于涉及B-D型分离物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。这对了解主要梭状肠病原体的毒力进化也有重要意义。为了实现这些目标，将追求以下具体目标：1)确定哪些已知毒素有助于B-D型分离物的致病性，Aim A将继续使用我们最近开发的高效内含子诱变方法，在B-D型背景中构建单个和多个毒素无效突变体；ii)目的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型B- d分离物的致病性，这些模型评估了特定的疾病方面，包括肠道致病性（使用兔子或山羊回肠环）或致病性（使用小鼠id或胃攻击模型）；当突变体显示出减弱的致病性时，将对它们进行补充，以确认相关毒素基因失活导致的衰减，iii)由于我们已经证明D型分离株的epsilon毒素编码质粒是共轭的，Aim C将检查B型、C型和E型分离株的毒素质粒是否也是共轭的（使用混合交配方法）。B-D型共轭毒素质粒是否能在肠道内转移，这种转移是否有助于致病，产气荚膜梭菌是否能与另一种主要的梭状芽孢杆菌（Clostridium difficile）结合交换毒素质粒或毒素基因，某些产气荚膜梭菌毒素质粒是否彼此不相容；最后，iv) Aim D将使用脉冲场凝胶/Southern blot评估B型和C型分离株毒素质粒的基因型多样性，并使用微阵列方法评估B型和C型分离株毒素质粒的基因型多样性。公共卫生相关性：产气荚膜梭菌B- d型分离株在医学、兽医和生物防御方面具有重要意义，因为它们产生许多强效毒素，如epsilon毒素（B类选择毒素）。为了获得关键信息，以开发针对自然或生物恐怖主义引起的B-D型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型毒素分离物的许多毒素是由大质粒编码的，我们将研究B-D型毒素质粒的多样性，以便开发用于流行病学或法医目的的检测方法。

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.