ABSTRACT
In order to clarify the enteropathogenicity of Plesiomonas shigelloides, we investigated a cytotoxin produced by the P-1 strain isolated from patients suffering from diarrhea. The cytotoxicity of the culture filtrate of the strain reached a maximum in culture at 37 degrees C after 12 h shaken in BHI medium. The cytotoxin in the cultures was purified by (NH4)2SO4 precipitation, and Sephacryl S-100, Mono Q HR, and Superdex 200 HR column chromatographies. An approximate 340-fold purification was achieved, with a recovery of about 1.4%, from the culture supernatant. The cytotoxin is heat-stable, and is a complex of three major proteins (LPS-binding proteins with molecular weights of 32, 40, and 48 kDa), with lipopolysaccharide (LPS) giving a total a molecular weight of more than 600 kDa. The ratio of protein to LPS in the cytotoxin was 6-5. The cytotoxic activity was reduced by about 80% by proteinase K treatment or when incubated with anti-cholera toxin antibody (Anti-CT). Western blotting of the cytotoxin with Anti-CT demonstrated the presence of two anti-cholera toxin-reactive protein (ACRP) bands with molecular weights of 40 kDa (a major single protein band) and 48 kDa. The N-terminal amino acid sequence (20 residues) of the 40 kDa protein was 75% identical to Pasteurella multocida cell membrane proteins. The cytotoxin gave a positive reaction in the suckling mouse assay whereas LPS alone hardly exhibited any cytotoxic or enterotoxigenic activity. In conclusion, P. shigelloides produces a cytotoxin that consists of a complex of protein and LPS with the former component exhibiting both cytotoxicity and enteropathogenicity. This cytotoxin has the potential to have an important role in the enteropathogenicity of P. shigelloides.
