Institute of Veterinary Bacteriology, Vetsuisse, Universität Bern, Bern, Switzerland.
Vaccine. 2012 Aug 10;30(37):5500-5. doi: 10.1016/j.vaccine.2012.06.050. Epub 2012 Jun 27.
The purpose of this study was to determine the identity of the major toxin of Clostridium chauvoei, an important pathogen of cattle causing black leg and to determine its value as a protective antigen in vaccines against myonecrosis.
Genomic sequence analysis was used to determine potential virulence genes of C. chauvoei. Subsequently, the putative toxin candidate gene was cloned and expressed to obtain recombinant toxin. This toxin was investigated for its cytotoxic activity, hemolysis and its potential as a protective antigen in the guinea pig potency assay.
A novel protein toxin, named Clostridium chauvoei toxin A (CctA) that belongs to the family of β-barrel pore forming toxins of the leucocidin superfamily of bacterial toxins was discovered by whole genome sequence analysis. The corresponding gene cctA was found in all strains of C. chauvoei analyzed, isolated from various geographical areas over the globe during the last 50 years, but not in other pathogenic Clostridium species. Native CctA and recombinant rCctA produced in Escherichia coli in the form of a rCctA::NusA fusion protein or thrombin processed rCctA were highly cytotoxic for Embryonic Calf Nasal Epithelial (ECaNEp) cells and had high haemolytic activity against sheep erythrocytes in standard haemolysis assays. Polyclonal anti-rCctA rabbit antibodies fully neutralized the cytotoxic and haemolytic activity, not only of rCctA but also of supernatants from cultures of the various C. chauvoei strains, indicating that CctA is the main cytotoxic and haemolytic substance secreted by C. chauvoei. Using a standard vaccine release procedure, we demonstrated that vaccination of guinea pigs with CctA in the form of a fusion protein with the E. coli heat labile toxin B subunit (rCctA::LTB) as a peptide adjuvant protected the animals against challenge with spores of virulent C. chauvoei.
CctA is the major virulence factor of C. chauvoei and the main protective antigen in vaccines against blackleg.
本研究旨在确定引起牛黑腿的重要病原体梭状芽胞杆菌 chauvoei 的主要毒素的身份，并确定其作为肌肉坏死疫苗中保护性抗原的价值。
使用基因组序列分析来确定 C. chauvoei 的潜在毒力基因。随后，克隆并表达了假定的毒素候选基因，以获得重组毒素。在豚鼠效力测定中，研究了该毒素的细胞毒性、溶血作用及其作为保护性抗原的潜力。
通过全基因组序列分析发现了一种新型蛋白毒素，命名为梭状芽胞杆菌 chauvoei 毒素 A（CctA），属于细菌毒素白细胞毒素超家族的β-桶孔形成毒素家族。在过去 50 年中从全球不同地区分离的所有 C. chauvoei 菌株中都发现了相应的基因 cctA，但在其他致病性梭状芽胞杆菌物种中没有发现。天然 CctA 和在大肠杆菌中以 rCctA::NusA 融合蛋白形式或凝血酶处理的 rCctA 形式产生的重组 rCctA 对胚胎牛鼻上皮细胞（ECaNEp）具有高度细胞毒性，并在标准溶血试验中对绵羊红细胞具有高溶血活性。多克隆抗 rCctA 兔抗体完全中和了 rCctA 的细胞毒性和溶血活性，不仅中和了 rCctA，还中和了来自各种 C. chauvoei 菌株培养物的上清液，表明 CctA 是 C. chauvoei 分泌的主要细胞毒性和溶血物质。使用标准疫苗释放程序，我们证明了以与大肠杆菌不耐热毒素 B 亚单位（rCctA::LTB）融合蛋白的形式接种豚鼠 CctA 作为肽佐剂可保护动物免受强毒 C. chauvoei 孢子的攻击。
CctA 是 C. chauvoei 的主要毒力因子，也是黑腿疫苗的主要保护性抗原。
