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An aerolysin-related cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses multiple biological activities, which include its ability to lyse red blood cells, destroy tissue culture cell lines, evoke a fluid secretory response in ligated intestinal loop models, and induce lethality in mice. The role of Act in the virulence of the organism has been demonstrated. In this study, we evaluated the potential of Act to induce production of proinflammatory cytokines associated with Act-induced tissue injury and Act's capacity to activate in macrophages arachidonic acid (AA) metabolism that leads to production of eicosanoids (e.g., prostaglandin E 2 [PGE 2 ]). Our data indicated that Act stimulated the production of tumor necrosis factor alpha and upregulated the expression of genes encoding interleukin-1␤ (IL-1␤) and IL-6 in the murine macrophage cell line RAW264.7. Act also activated transcription of the gene encoding inducible nitric oxide synthase. Act evoked the production of PGE 2 coupled to the cyclooxygenase-2 (COX-2) pathway. AA is a substrate for PGE 2 , and Act produced AA from phospholipids by inducing group V secretory phospholipase A 2 . We also demonstrated that Act increased cyclic AMP (cAMP) production in macrophages. cAMP, along with PGE 2 , could potentiate fluid secretion in animal models because of infiltration and activation of macrophages resulting from Act-induced tissue injury. After Act treatment of RAW cells, we detected an increased translocation of NF-B and cAMP-responsive element binding protein (CREB) to the nucleus using gel shift assays. Act also upregulated production of antiapoptotic protein Bcl-2 in macrophages, suggesting a protective role for Bcl-2 against cell death induced by proinflammatory cytokines. The increased expression of genes encoding the proinflammatory cytokines, COX-2, and Bcl-2 appeared correlated with the activation of NF-B and CREB. This is the first report of the detailed mechanisms of action of Act from A. hydrophila.Aeromonas spp. recently have been placed in the family Aeromonadaceae. They cause both intestinal and nonintestinal infections in humans (12), and, unlike gastroenteritis, which generally occurs in young children, these nonintestinal infections are often fatal and involve adults (36). Aeromonas spp. have been cultured from both freshwater and salt water and from many foods. These bacteria have emerged as important human pathogens and are being isolated in an increased incidence from patients with traveler's diarrhea (3,11,28,29,41,44,70). Aeromonas spp. produce an array of virulence factors, and the pathogenesis of Aeromonas infections is therefore complex and multifactorial (2). These virulence factors include hemolysins, cytotoxins, enterotoxins, proteases, lipases/phospholipases, leucocidin, endotoxin, fimbriae or adhesins, and the capacity to form an S-layer (17,45,47). Aeromonas hydrophila has been shown to be invasive for HEp-2 cell monolayers, and the bacterial cells adhere to human erythrocytes (6, 26). Two distinct families of type IV ...

The structural gene and regulatory element for a cytolytic enterotoxin of a diarrheal isolate, SSU, of Aeromonas hydrophila was cloned and its DNA sequence was determined. A complementary, mixed synthetic oligonucleotide based on the first 10 NH2-terminal amino acid residues of the Aeromonas cytolytic enterotoxin was used as a probe to screen a genomic library constructed in bacteriophage EMBL3. Cell lysates of Escherichia coli (lambda CH4), containing the cytolytic enterotoxin gene, lysed rabbit red blood cells and destroyed Chinese hamster ovary cells, caused fluid secretion in rat ileal loops, and were lethal to mice when injected intravenously. All biological activities associated with the cytolytic enterotoxin were neutralized by rabbit homologous polyclonal antibodies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent Western blot analysis of the cell lysate of E. coli (lambda CH4) revealed a protein band of approximately 52 kDa, using antisera to the cytolytic enterotoxin or antibodies generated against a synthetic peptide to the toxin. DNA sequence analysis of a 2.8-kb SalI-BamHI fragment revealed the presence of one large open reading frame (1479 bp) that would encode a protein of 54.5 kDa, a precursor form of the cytolytic enterotoxin, with a 23 amino acid leader peptide. Despite a significant amount of homology at the DNA and amino acid levels between our cytolytic enterotoxin and two aerolysins of Aeromonas species, variation in the restriction maps of these three toxin genes was prominent. Likewise, considerable divergence in DNA sequence was observed upstream of the structural genes for the reported aerolysins and our cytolytic enterotoxin, suggesting that these structurally similar toxin molecules may be regulated differently. Finally, our data showed that the cytolytic enterotoxin from a diarrheal isolate, SSU, of A. hydrophila exhibited characteristics that were unique compared with those of the reported aerolysins.

By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.

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