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

将产气荚膜梭菌(A型NCTC 8237)的α、β毒素基因分别克隆到pHY300PLK中，在枯草芽孢杆菌ISW 1214(Spoo^)中表达，各菌株产生的α、β毒素水平与产气荚膜梭菌基本一致。用巨噬细胞系SV-BP-1检测了这两株菌株在巨噬细胞内的存活能力。巨噬细胞内产生theta毒素的菌株数量增加，而产生α毒素的菌株数量减少。结果表明，theta毒素具有抗吞噬细胞决定簇的功能，而α毒素不起作用。通过测定对小鼠的致死剂量来检验两株菌株的毒力。当α毒素产生菌被注射到腹膜腔内时，其毒力比产生theta毒素的菌强。另一方面，前者在静脉注射时的毒性低于后者。还构建了携带这两种毒素基因的质粒，携带该质粒的枯草杆菌产生大量的theta毒素，但产生的α毒素很少。然而，产生这两种毒素的菌株对小鼠的致死率与联合使用的两种产生α和theta的菌株相同，这表明如果这两种毒素由单个细胞产生到细菌细胞周围的微环境中，则两种毒素的协同作用可能会增强。对其编码基因进行了克隆和测序。核苷酸序列分析表明，该毒素属于锌金属蛋白酶，与其他细菌蛋白水解酶和胶原酶同源。

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.