AMINO-ACID-RESIDUES INVOLVED IN BIOLOGICAL FUNCTIONS OF THE CYTOLYTICENTEROTOXIN FROM AEROMONAS-HYDROPHILA

Some amino acid (aa) residues within the cytolytic enterotoxin (Act) o f Aeromonas hydrophila essential for biological activity were identifi ed. Act is a 52-kDa polypeptide, possessing hemolytic, cytotoxic and e nterotoxic activities. By deletion analysis, generation of anti-peptid e Ab, and site-directed mutagenesis we showed that two regions in Act (aa 245-274 and 361-405) were very important for biological functions. As shown by competitive inhibition assays, peptide 2 (aa 245-274) blo cked cytotoxic activity of Act, and aa Tyr(256), Trp(270) and Gly(274) were essential for cytotoxicity. Within peptide 3 (aa 361-405), Trp(3 94) and Trp(396) were important for biological activities. Mutations i n other regions of the toxin (e.g., Gly(169), Asp(170), Gly(171), Trp( 172), Asn(177,178), Asp(179) and His(144,209,355)) also decreased biol ogical activity. The reactivity of these mutant toxins with Ab in immu noblots was not altered. Data reported in this study suggested the rol e of some aa residues in biological function(s) of Act.