Roles of the alpha-,theta-,and k-toxins in histotoxicity of Clostridium perfringens

将编码产气荚膜梭菌（A型NCTC 8237）的α-和θ-毒素的基因分别克隆到pHY 300 PLK中，并在枯草芽孢杆菌ISW 1214（spoo）中表达。用巨噬细胞系SV-BP-1检测了这两株菌在巨噬细胞内的存活率，结果表明，θ毒素产生菌在巨噬细胞内的数量增加，而α毒素产生菌在巨噬细胞内的数量减少，表明θ毒素具有抗吞噬决定簇的功能，而α毒素则没有。当腹腔注射α-毒素产生菌株时，其毒性比θ-毒素产生菌株更强。另一方面，当静脉注射时，前者的毒性低于后者。同时构建了一个携带两种毒素基因的质粒，携带该质粒的枯草芽孢杆菌B产生大量的θ毒素，但产生极少量的α毒素。然而，产生这两种毒素的菌株表现出与组合使用的两种α-和θ-产生菌株相同的小鼠致死性，这表明如果它们由单个细胞产生到细菌细胞周围的微环境中，则可以增强这两种毒素的协同效应。发现并纯化了120-KDa产气荚膜梭菌胶原酶（k-毒素）。克隆其编码基因并测序。核苷酸序列分析表明，该毒素属于锌金属蛋白酶，与其他细菌蛋白酶和胶原酶同源。

Genes encoding for alpha-and theta-toxins of Clostridium perfringens(type A NCTC 8237)were separately cloned into pHY300PLK and expressed in Bacillus subtilis ISW 1214(spoo^).Levels of both the alpha- and theta-toxins produced by each strain were almost the same as those by C.perfringens. Viability of these two strains within a macrophage was examined using macrophage cell line SV-BP-1.The theta-toxin producing strain increased in number within the macrophage,while the alpha-toxin producing strain decreased similarly to a control.The result indicates that the theta-toxin functions as an antiphagocytic determinant but the alpha-toxin does not.Virulence of the two strains was examined by determining lethal dose against a mouse. When the alpha-toxin producing strain was injected interperitoneally,it was more virulent than the theta-toxin producing strain. On the other hand,the former was less virulent than the latter when injected intravenously. A plasmid carrying the two toxin genes was also constructed.B subtilis carrying this plasmid produced the theta-toxin in large amounts but the alpha-toxin in a very small amount. However,the strain producing the two toxins exhibited mouse lethality to the same extent as the two alpha- and theta-producing strains used in combination, suggesting that a synergistic effect of the two toxins may be enhanced if they are produced by a single cell into microenvironment surrounding a bacterial cell.A 120-KDa C.perfringens collagenase(k-toxin)was found and purified. Its encoding gene was cloned and sequenced. The nucleotide sequence shows that the toxin belongs to Zn metalloproteinase and is homologous to other bacterial proteinase and collagenase.