| The cytotoxic enterotoxin (Act) of A. hydrophila is a 52-kDa polypeptide, possessing hemolytic, cytotoxic, and enterotoxic activities, and the toxin causes lethality in mice when injected intravenously. We demonstrated that one synthetic peptide (aa 245-274) blocked cytotoxic activity of Act on Chinese hamster ovary cells. Further, antibodies generated to synthetic peptides made to amino acid residues 245-274 and 361-405 of Act neutralized the biological activity of the toxin. Amino acid residues {dollar}rm Tyrsp{lcub}256{rcub}to Ser, Trpsp{lcub}270{rcub}to Leu, Glysp{lcub}274{rcub}to Ala, Trpsp{lcub}394{rcub}to Leu,{dollar} and {dollar}rm Trpsp{lcub}396{rcub} to Leu{dollar} were important for biological activities as determined by site-directed mutagenesis. Mutations in other regions of the toxin also decreased biological activity of Act.; Act initially was hyperexpressed using pET, pTRX, and pGEX vector systems in E. coli. The act gene was placed in a broad-host range vector pMMB66 under the control of a tac-promoter. This plasmid then was transferred into an act-gene deficient strain of Aeromonas. This strategy allowed secretion of Act into the medium. Purified mature toxin migrated as a 52-kDa polypeptide on an SDS-polyacrylamide gel that reacted with specific Act antibodies in immunoblots. The minimum amount of toxin needed to cause fluid secretion in rat ileal loops was 200 ng, and the LD{dollar}sb{lcub}50{rcub}{dollar} for mice was 27.5 ng when injected intravenously. Binding of the toxin to red blood cells was temperature dependent, with no binding occurring at 4{dollar}spcirc{dollar}C. However, at 37{dollar}spcirc{dollar}C the toxin bound to erythrocytes within 1-2 min. The mechanism of action of the toxin involved the formation of holes in erythrocyte membranes, and the size of the pores were estimated to be 1.14-2.8 nm in diameter. Calcium chloride prevented lysis of red blood cells by the toxin; however, it did not affect the binding and pore-forming capabilities of the toxin. We observed a dose-dependent reduction in hemoglobin release from erythrocytes when Act was preincubated with cholesterol, but not with myristylated cholesterol. None of the other phospholipids and gangliosides tested reduced the hemolytic activity of the Act. The toxin appeared to aggregate, when preincubated with cholesterol, thus preventing Act's capacity to form holes in the erythrocyte membrane. The binding of Act to {dollar}sp{lcub}14{rcub}{dollar}C-labeled cholesterol was confirmed by gel filtration.; Transposon mutagenesis on the chromosomal DNA of A. hydrophila was performed to study regulation of the act gene. Culture filtrates from five mutants exhibited drastically reduced biological activities. The effect of transposition appeared to be at a transcriptional level based on Northern blot analysis. None of the transposon mutants was lethal to mice when injected intraperitoneally compared to wild-type (wt) A. hydrophila. An isogenic mutant of wild-type A. hydrophila with a truncated act gene and no biological activity has been generated by homologous recombination. By the complementation experiment, a revertant of Aeromonas with regained Act activity also has been generated. Our data indicated that the isogenic mutant was less virulent compared to wild-type and revertant Aeromonas strains in a mouse model. (Abstract shortened by UMI.)... |
