MOLECULAR AND BIOCHEMICAL-CHARACTERIZATION OF A HEAT-LABILE CYTOTONICENTEROTOXIN FROM AEROMONAS-HYDROPHILA

We report herein the DNA sequence analysis of the heat-labile cytotoni c enterotoxin gene (alt) from Aeromonas hydrophila and the biological function of the purified hyperproduced toxin (Alt). One large open-rea ding frame (ORF), comprised of 1104 bp, was detected at positions 804 to 1907 bp on a 4.0-kb San DNA fragment from Aeromonas. This ORF encod es for a protein having 368 amino acids (aa) with a computed molecular weight of 38 kDa. The aa sequence of the first 15 NH2-terminal residu es of the mature native Alt from A. hydrophila matched with the DNA-de rived aa sequence of the alt gene expressed in E. coil starting at pos ition 19, which was leucine. The first 18 aa residues of the Alt repre sented a putative signal sequence with alanine at its carboxy terminus . A BLAST search of the entire database showed 45-51% identity of the Alt, starting at position 158 with the carboxy half of the phospholipa se C (PLC) and lipase from A. hydrophila; however, the purified Alt ha d no lipase/PLC activity. The alt gene was hyperexpressed using gene f usion expression vector systems, and the recombinant Alt exhibited a s ize of 35-40 kDa. The pure recombinant Alt elongated Chinese hamster o vary cells and elicited fluid secretion in rat ligated intestinal loop s, indicating its enterotoxicity. Immunization of mice with recombinan t Alt resulted in a reduced fluid secretory response when challenged w ith Aeromonas. The biological activity of the recombinant Alt in E. co li was about 10-fold less, compared to native Alt from Aeromonas, indi cating differential processing of the toxin. The antibodies to native Alt neutralized the biological activity of the recombinant toxin, and these antibodies reacted with the same specificity to the native and r ecombinant Alt in immunoblots. The role of cyclic adenosine monophosph ate and prostaglandins in causing a fluid secretory response by Alt al so was demonstrated. (C) 1996 Academic Press Limited