Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B

The process of internalisation of Clostridium difficile toxin B into human lung fibroblasts was further studied, with the aim of elucidating the fate of endocytosed toxin. Development of the toxin-induced cytopathogenic effect was reversibly inhibited at 18 degrees C and in the presence of 200 mM KCl or 1-20 mM benzyl alcohol, i.e. at conditions when the fusion between endosomes and lysosomes is prevented. Fibroblasts treated with toxin at 37 degrees C but transferred to 18 degrees C within 10 min were also completely protected, whereas transfer to 18 degrees C later during the latency resulted in only partial protection. KCl was also protective upon addition after the toxin binding step. Inhibitors of lysosomal proteases, such as chymostatin, leupeptin and antipain, prevented the appearance of the cytopathogenic effect, when present during toxin exposure or added after the toxin binding step. Chinese hamster ovary cell mutants, defective in acidification of their endosomes, were resistant to toxin B, whereas wildtype cells were sensitive. The resistance was not overcome by applying a low extracellular pH. The results suggest that exposure to a low pH compartment is necessary but not sufficient for entry of active toxin B to the cytosol. In addition to a low pH, a fusion of toxin-containing endosomes with lysosomes and a further processing of the toxin by lysosomal proteases is required for cellular intoxication.