Molecular and biochemical characterization of a heat-labile cytotonic enterotoxin from Aeromonas hydrophila

Ashok K. Chopra, Johnny W. Peterson, Xin J. Xu, Dorian H. Coppenhaver, Clifford W. Houston

Research output: Contribution to journalArticle

53 Scopus citations

Abstract

We report herein the DNA sequence analysis of the heat-labile cytotonic enterotoxin gene (alt) from Aeromonas hydrophila and the biological function of the purified hyperproduced toxin (Alt). One large open-reading frame (ORF), comprised of 1104 bp, was detected at positions 804 to 1907 bp ona 4.0-kb Sall DNA fragment from Aeromonas. This ORF encodes 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 residues of the mature native Alt from A. hydrophila matched with the DNA-derived aa sequence of the alt gene expressed in E. coli starting at position 19, which was leucine. The first 18 aa residues of the Alt represented 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 phospholipase C (PLC) and lipase from A. hydrophila; however, the purified Alt had no lipase/PLC activity. The alt gene was hyperexpressed using gene fusion expression vector systems, and the recombinant Alt exhibited a size of 35-40 kDa. The pure recombinant Alt elongated Chinese hamster ovary cells and elicited fluid secretion in rat ligated intestinal loops, indicating its enterotoxicity. Immunization of mice with recombinant Alt resulted in a reduced fluid secretory response when challenged with Aeromonas. The biological activity of the recombinant Alt in E. coli was about 10-fold less, compared to native Alt from Aeromonas, indicating 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 recombinant Alt in immunoblots. The role of cyclic adenosine monophosphate and prostaglandins in causing a fluid secretory response by Alt also was demonstrated.

Original languageEnglish (US)
Pages (from-to)357-377
Number of pages21
JournalMicrobial Pathogenesis
Volume21
Issue number5
DOIs
StatePublished - Nov 1996

Keywords

  • DNA sequencing
  • alt
  • alt gene
  • gene fusion expression vector systems
  • protection studies
  • site-directed mutagenesis

ASJC Scopus subject areas

  • Microbiology
  • Infectious Diseases

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