Purification and Characterization of Enterotoxin Produced by Aeromonas sobria

Abstract

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.

Fluid Secretion Caused by Aerolysin-Like Hemolysin of Aeromonas sobria in the Intestines Is Due to Stimulation of Production of Prostaglandin E 2 via Cyclooxygenase 2 by Intestinal Cells

“…The overall homology in amino acid sequence between the hemolysin and aerolysin was 68.5% identity (14). The two toxins have similar modes of action.…”

“…The hemolysin was purified from a culture supernatant of A. sobria strain 357 by successive column chromatographies as described previously (14). The purified hemolysin gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.…”

“…Both hemolysin and aerolysin possess enterotoxic activity (1,14,29). In a previous paper, we reported that hemolysin stimulates the production of cyclic AMP (cAMP) in T84 cells (human colon carcinoma cell line) and that the cAMP thus produced emerges into the extracellular space (15).…”

“…We purified and characterized the enterotoxin from the culture supernatant of A. sobria isolated from a patient with diarrhea and demonstrated that it possessed hemolytic activity in addition to enterotoxic activity (14,26).…”

“…The two toxins have similar modes of action. Both act to form small pores in the cell membrane to which they have bound and to generate the osmotic gradient that develops as a result of cellular injury (6,14,41).…”