A low-molecular-mass cytotoxin produced by isolated previously from patients with antibiotic-associated haemorrhagic enterocolitis was purified, and its biological and chemical properties were elucidated. The toxin inhibited the syntheses of DNA and RNA by HEp-2 cells dose-dependently, whereas protein synthesis was only slightly inhibited, as measured by the incorporation of radioactive precursors. When synchronously cultured HEp-2 cells were examined in the presence of cytotoxin, inhibition of DNA synthesis occurred promptly within 5 h, but cell-rounding, the earliest visible morphological change, was not observed until 6 h after exposure. The intracellular levels of ATP decreased with an approximately similar time course. These results suggest that cytotoxicity toward HEp-2 cells is primarily due to the inhibitory effect of the cytotoxin on nucleic acid synthesis, possibly on DNA synthesis. Cell rounding and cell death were induced even in the absence of the cytotoxin after incubation with the cytotoxin for 6 h. The cytotoxin was heat-labile, cytotoxic activity decreasing to 50% of the initial level on heating at 70°C for 20 min. Plasmids were extracted from three strains of producing the cytotoxin and analysed by agarose gel electrophoresis. Two strains possessed plasmids of different sizes, but one strain possessed no plasmid, indicating that the cytotoxin is probably chromosomally encoded. Analysis by NMR and FAB-mass-spectrometry revealed that the molecular mass of the cytotoxin should be 217.1062 Da (exact mass), its molecular formula being CHON.


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