Purification and characterisation of intracellular toxin A of Clostridium difficile.
After sonic disintegration of Clostridium difficile cells, intracellular toxin A was purified to homogeneity by thyroglobulin affinity chromatography (TGAC) followed by anion-exchange (Mono Q) by fast protein liquid chromatography (FPLC). High haemagglutinating (HA) activity was detected in TGAC-unbound fractions (2(9)/50 microliters), but not in TGAC thermal eluates (2(0)/50 microliters). The low HA titre of the thermal eluates was markedly increased to 2(5)/50 microliters after dialysis against 0.02 M Tris-HCl (pH 7.5). A disparity in the position of the peaks containing cytotoxic and HA activity was observed in the first Mono Q-FPLC step. Intracellular toxin A without HA activity was obtained by a second Mono Q-FPLC step. The M(r) of the intracellular toxin A was estimated by polyacrylamide gel electrophoresis (PAGE) to be 580 kDa under non-denaturing conditions. The minimum doses of the toxin causing cytotoxicity, mouse lethality and enterotoxicity were 0.83 ng, 8.7 ng and 5 micrograms, respectively.
Meng
X Q
XQ
Department of Bacteriology, School of Medicine, Kanazawa University, Japan.
Kamiya
S
S
Yamakawa
K
K
Ogura
H
H
Nakamura
S
S
D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D016360:Clostridioides difficile; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D006384:Hemagglutination; D007928:Lethal Dose 50; D051379:Mice
D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile