Abstract：
OBJECTIVE: Clostridium perfringens causes food poisoning and gas gangrene, a serious wound-associated infection. C. perfringens cells adhere to collagen via fibronectin (Fn). We investigated whether the peptidoglycan hydrolase of C. perfringens, i.e., autolysin (Acp), is implicated in Fn binding to C. perfringens cells.
METHODS: This study used recombinant Acp fragments, human Fn and knockout mutants (C. perfringens 13 acp::erm and HN13 ΔfbpC ΔfbpD). Ligand blotting, Western blotting analysis, and complementation tests were performed. The Fn-binding activity of each mutant was evaluated by ELISA.
RESULTS: From an Fn-binding assay using recombinant Acp fragments, Fn was found to bind to the catalytic domain of Acp. In mutant cells lacking Acp, Fn binding was significantly decreased, but was restored by the complementation of the acp gene. There are three known kinds of Fn-binding proteins in C. perfringens: FbpC, FbpD, and glyceraldehyde-3-phosphate dehydrogenase. We found no difference in Fn-binding activity between the mutant cells lacking both FbpC and FbpD (SAK3 cells) and the wild-type cells, indicating that these Fn-binding proteins are not involved in Fn binding to C. perfringens cells.
CONCLUSIONS: We found that the Acp is an Fn-binding protein that acts as an Fn receptor on the surface of C. perfringens cells.
METHODS: This study used recombinant Acp fragments, human Fn and knockout mutants (C. perfringens 13 acp::erm and HN13 ΔfbpC ΔfbpD). Ligand blotting, Western blotting analysis, and complementation tests were performed. The Fn-binding activity of each mutant was evaluated by ELISA.
RESULTS: From an Fn-binding assay using recombinant Acp fragments, Fn was found to bind to the catalytic domain of Acp. In mutant cells lacking Acp, Fn binding was significantly decreased, but was restored by the complementation of the acp gene. There are three known kinds of Fn-binding proteins in C. perfringens: FbpC, FbpD, and glyceraldehyde-3-phosphate dehydrogenase. We found no difference in Fn-binding activity between the mutant cells lacking both FbpC and FbpD (SAK3 cells) and the wild-type cells, indicating that these Fn-binding proteins are not involved in Fn binding to C. perfringens cells.
CONCLUSIONS: We found that the Acp is an Fn-binding protein that acts as an Fn receptor on the surface of C. perfringens cells.
摘要：
目的：产气荚膜梭菌引起食物中毒和气体坏疽，严重的伤口感染.产气荚膜梭菌细胞通过纤连蛋白(Fn)粘附于胶原。我们以为产气荚膜梭菌细胞具有某种Fn受体。我们研究了产气荚膜梭菌的肽聚糖水解酶，即，自溶素（Acp），涉及Fn与产气荚膜梭菌细胞的结合。
方法：本研究使用重组Acp片段，人类Fn和敲除突变体(C.产气荚膜13acp::erm和HN13ΔfbpCΔfbpD)。配体印迹,蛋白质印迹分析,并进行了补充试验。通过ELISA评估每个突变体的Fn结合活性。
结果：从使用重组Acp片段的Fn结合测定，发现Fn与Acp的催化结构域结合。在缺乏Acp的突变细胞中，Fn结合显著降低,但通过acp基因的互补而恢复。产气荚膜梭菌中已知有三种Fn结合蛋白：FbpC，FbpD,和甘油醛-3-磷酸脱氢酶.我们发现缺乏FbpC和FbpD的突变细胞（SAK3细胞）与野生型细胞之间的Fn结合活性没有差异，这表明这些Fn结合蛋白不参与Fn与产气荚膜梭菌细胞的结合。
结论:我们发现Acp具有Fn结合蛋白,其在产气荚膜梭菌细胞表面上充当Fn受体。
方法：本研究使用重组Acp片段，人类Fn和敲除突变体(C.产气荚膜13acp::erm和HN13ΔfbpCΔfbpD)。配体印迹,蛋白质印迹分析,并进行了补充试验。通过ELISA评估每个突变体的Fn结合活性。
结果：从使用重组Acp片段的Fn结合测定，发现Fn与Acp的催化结构域结合。在缺乏Acp的突变细胞中，Fn结合显著降低,但通过acp基因的互补而恢复。产气荚膜梭菌中已知有三种Fn结合蛋白：FbpC，FbpD,和甘油醛-3-磷酸脱氢酶.我们发现缺乏FbpC和FbpD的突变细胞（SAK3细胞）与野生型细胞之间的Fn结合活性没有差异，这表明这些Fn结合蛋白不参与Fn与产气荚膜梭菌细胞的结合。
结论:我们发现Acp具有Fn结合蛋白,其在产气荚膜梭菌细胞表面上充当Fn受体。
