Purification and characterization of intracellular toxin A of Clostridium difficile

艰难梭菌细胞经超声破碎后，经甲状腺球蛋白亲和层析（TGAC）、Mono Q阴离子交换层析和快速蛋白液相层析（FPLC）等方法纯化细胞内毒素A。在未结合TGAC的组分（2^9）中检测到高血凝（HA）活性，但在TGAC热洗脱液（2^0）中未检测到。用0.02 M Tris-HCl（pH 7.5）透析后，热洗脱液的低HA滴度显著增加至2^5。在第一个Mono Q-FPLC中观察到细胞毒性和HA活性之间的峰位置不一致。通过第二次Mono Q-FPLC获得无HA活性的细胞内毒素A。在非变性条件下，用聚丙烯酰胺凝胶电泳法测得胞内毒素A的分子量为580 kDa。该毒素引起细胞毒性、小鼠致死和肠毒性的最小剂量分别为0.83 ng、8.7 ng和5 μ g。对细胞内毒素A和细胞外毒素A进行血清学比较。在Ouchterlony试验中，抗细胞内毒素A和抗细胞外毒素A血清显示细胞内毒素A和细胞外毒素A的同一性。在中和试验中，抗细胞内毒素A和抗细胞外毒素A血清二者以与同源毒素几乎相同的滴度（1：128 - 1：256）交替中和异源毒素的细胞毒性、小鼠致死性和环反应。虽然细胞内毒素A缺乏HA活性，但抗细胞内毒素A中和细胞外毒素A的HA活性，其滴度与抗细胞外毒素A血清相同（1：16）。这些发现表明毒素A在细胞中合成为HA阴性形式的可能性，其具有与红细胞的几个结合位点，并且当其从细胞释放时或从细胞释放后转化为HA阳性形式。

After sonic disintegration of Clostridium difficile cells, intracellular toxin A was purified to homogeneity by thyroglobulin affinity chromatography (TGAC) followed by successive anion- exchange chromatography on Mono Q incorporated into fast protein liquid chromatography (FPLC) apparatus. High hemagglutination (HA) activity was detected in TGAC-unbound fractions (2^9), but not in TGAC thermal eluates (2^0). The low HA titer of the thermal eluates was markedly increased to 2^5 after dialysis against 0.02 M Tris-HCl (pH 7.5). A disparity in the position of the peak between cytotoxicity and HA activity was observed in the first Mono Q-FPLC. Intracellular toxin A without HA activity was obtained by second Mono Q-FPLC. The molecular weight of the intracellular toxin A was estimated by polyacrylamide gel electrophoresis to be 580 kDa in non-denaturing condition. The minimum doses of the toxin causing cytotoxicity, mouse lethality and enterotoxicity were 0.83 ng, 8.7 ng and 5 ug, respectively. Intracelluler toxin A and extracellular toxin A were compared serologically. In Ouchterlony test, anti-intracellular toxin A and anti-extracellular toxin A sera showed identity of the intracellular and extracellular toxin A. In neutralization tests, both anti-intracellular and anti-extracellular toxin A sera alternatively neutralized the heterologous toxins for cytotoxicity, mouse lethality and loop response at nearly the same titers (1:128 - 1:256) as did the homologous toxins. Although intracellular toxin A lacked HA activity, anti-intracellular toxin A neutralized HA activity of the extracellular toxin A at the same titer (1:16) as anti-extracellular toxin A serum. These findings suggest a possibility that toxin A is synthesized as HA-negative form in cells, which has a few binding sites to the erythrocytes, and converted to HA-positive form,when it is released from cells, or after release from cells.