Division of Bacteriology, Department of Infectious Disease, Osaka Prefectural Institute of Public Health, Osaka, Osaka, Japan.
Infect Immun. 2014 Jun;82(6):2390-9. doi: 10.1128/IAI.01759-14. Epub 2014 Mar 24.
Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.
产气荚膜梭菌是食源性肠胃炎的病原体，其中产气荚膜梭菌肠毒素（CPE）被认为是一个重要因素。最近，我们经历了两起因疑似非产 CPE 产气荚膜梭菌引起的食源性肠胃炎爆发。在这里，我们报告了一种由产气荚膜梭菌分离株产生的新型肠毒素，BEC（产气荚膜梭菌二元肠毒素）。C. perfringens 菌株的培养上清液在兔回肠环和乳鼠试验中显示出积液活性。上清液中肠毒性物质的纯化和菌株基因组 DNA 的高通量测序揭示了 BEC，由 BECa 和 BECb 组成。BECa 和 BECb 与其他二元毒素家族成员（如产气荚膜梭菌iota 毒素）显示出有限的氨基酸序列相似性。becAB 基因位于 54.5-kb pCP13 样质粒上。单独的重组 BECb（rBECb）在乳鼠试验中具有积液活性。虽然 rBECa 单独没有肠毒性，但当同时给予乳鼠时，rBECa 增强了 rBECb 的肠毒性。与亲本菌株相比，破坏 becB 基因的突变体的肠毒性显著降低。rBECa 对纯化的肌动蛋白具有 ADP-核糖基转移酶活性。尽管我们尚未直接评估 BECb 是否将 BECa 递送入细胞，但只有当用 rBECa 和 rBECb 处理细胞时，Vero 细胞才会出现圆化。这些结果表明，BEC 是一种不同于 CPE 的产气荚膜梭菌新型肠毒素，产 BEC 的产气荚膜梭菌菌株可能是人类急性肠胃炎的病原体。此外，becAB 基因存在于不同谱系的产气荚膜梭菌分离株中几乎相同的质粒上，这表明毒素基因的获得涉及水平基因转移。
