Open AccessIsolation and characterization of a cytotoxin produced by Plesiomonas shigelloides P‐1 strain
Author(s) -
Okawa Yoshio,
Ohtomo Yuko,
Tsugawa Hitoshi,
Matsuda Yuko,
Kobayashi Hidemitsu,
Tsukamoto Teizo
Publication year - 2004
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/j.femsle.2004.08.026
Subject(s) - plesiomonas shigelloides , toxin , microbiology and biotechnology , molecular mass , lipopolysaccharide , vibrionaceae , biology , enterotoxin , cytotoxicity , biochemistry , bacteria , escherichia coli , in vitro , enzyme , gene , genetics , endocrinology
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 °C after 12 h shaken in BHI medium. The cytotoxin in the cultures was purified by (NH 4 ) 2 SO 4 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 .