Tomohiko Nomura

Purification and characterization of enterotoxin produced by Aeromonas sobria.

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.

Secretion of hemolysin of Aeromonas sobria as protoxin and contribution of the propeptide region removed from the protoxin to the proteolytic stability of the toxin

The sequence at the amino terminus region of the hemolysin of Aeromonas sobria is homologous with that of aerolysin of A. hydrophila. However, there is no homology between the two toxins in the sequence at the carboxy terminal region. It has been shown that aerolysin is secreted into culture supernatant as a protoxin. This proaerolysin is activated by the proteolytic removal of a carboxy terminal peptide. However, the role of the carboxy terminal region, which is removed in the activation process, has not been elucidated. In this study, we showed that hemolysin is also secreted as a protoxin into culture supernatant and that prohemolysin is cleaved by the protease of A. sobria between Ser‐446 and Ala‐447, resulting in the removal of a 42 amino acid peptide. The removal of the peptide converts the prohemolysin into active hemolysin. Subsequently, we mutated the hemolysin gene to delete the last several amino acid residues and expressed the genes in Escherichia coli, in order to examine the role of the carboxy terminal region of prohemolysin. The amounts of these mutant hemolysins accumulated in the periplasmic space of E. coli were very low compared with that of the wild‐type. This observation indicated that the carboxy terminal region of prohemolysin contributes to the proteolytic stability of the toxin.

Involvement of Ca2+ ‐Calmodulin‐Dependent Protein Kinase II in the Intestinal Secretory Action of Escherichia coli Heat‐Stable Enterotoxin II

Treating the mouse intestine with the calmodulin antagonist W‐7 and KN‐93, an inhibitor of Ca2+ ‐calmodulin‐dependent protein kinase II (CaMK II), reduced the sensitivity of the host to the action of Escherichia coli heat‐stable enterotoxin II (STII). CaMK II activity in mouse intestinal cells increased after exposure to STII. These results indicate that CaMK II is involved in the mechanism of action of STII.

Production of Serine Protease of Aeromonas sobria Is Controlled by the Protein Encoded by the Gene Lying Adjacent to the 3′ End of the Protease Gene

We cloned a protease gene of Aeromonas sobria and determined its nucleotide sequence. The protease is composed of 624 amino acid residues and its calculated molecular weight is 66,737.7. The amino acid sequence showed the characteristic features of a bacterial serine protease. We expressed the protease gene in Vibrio parahaemolyticus from which the synthesized protease is secreted into the culture medium as the mature form, and purified the mature protease by successive column chromatographies. The size of the mature protease is 65,000 daltons and the amino acid sequence analysis revealed that a 24‐amino acid peptide at the amino terminal of the precursor is removed from the mature protease. This peptide might function as a signal peptide in translocation across the inner membrane. Subsequently, we found that the protein, designated ORF2 protein, encoded by the gene lying adjacent to the 3′ end of the protease gene plays an important role in production of the protease. Mutation of the ORF2 gene did not affect transcription of the protease gene, but resulted in degradation of the protease in the cell. This shows that ORF2 protein is required for the successful production of the serine protease by cell.

Physicochemical and biological properties of an extracellular serine protease of Aeromonas sobria.

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature‐labile and had an optimal pH of 7.5. Metallo‐protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability‐enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability‐enhancing activity in skin. This suggests that a trypsin‐like protease from skin mediates the activity of the protease to enhance its vascular permeability.

Studies of the mechanism of action of the aerolysin-like hemolysin of Aeromonas sobria in stimulating T84 cells to produce cyclic AMP.

ABSTRACT We previously reported that the aerolysin-like hemolysin of Aeromonas sobria stimulates T84 cells to produce cyclic AMP, which then emerges in the culture medium. In order to clarify the mechanism of action of the hemolysin, we examined the involvement of adenosine nucleotide. The results show that the hemolysin stimulates T84 cells to release ATP, which is then converted to adenosine by ectonucleotidase. The adenosine generated might stimulate the P1 adenosine receptors of T84 cells to produce cyclic AMP.