Amino-acid residues involved in biological functions of the cytolytic enterotoxin from Aeromonas hydrophila

Some amino acid (aa) residues within the cytolytic enterotoxin (Act) of Aeromonas hydrophila essential for biological activity were identified. Act is a 52-kDa polypeptide, possessing hemolytic, cytotoxic and enterotoxic activities. By deletion analysis, generation of anti-peptide 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) blocked cytotoxic activity of Act, and aa Tyr²⁵⁶, Trp²⁷⁰ and Gly²⁷⁴ were essential for cytotoxicity. Within peptide 3 (aa 361-405), Trp³⁹⁴ and Trp³⁹⁶ were important for biological activities. Mutations in other regions of the toxin (e.g., Gly¹⁶⁹, Asp ¹⁷⁰, Gly¹⁷¹, Trp¹⁷², Asn^177,178, Asp¹⁷⁹ and His^144,209,355) also decreased biological activity. The reactivity of these mutant toxins with Ab in immunoblots was not altered. Data reported in this study suggested the role of some aa residues in biological function(s) of Act.