ROLE OF A CYTOTOXIC ENTEROTOXIN IN AEROMONAS-MEDIATED INFECTIONS - DEVELOPMENT OF TRANSPOSON AND ISOGENIC MUTANTS

Transposon and marker exchange mutagenesis were used to evaluate the r ole of Aeromonas cytotoxic enterotoxin (Act) in the pathogenesis of di arrheal diseases and deep wound infections. The transposon mutants wer e generated by random insertion of Tn5-751 in the chromosomal DNA of a diarrheal isolate SSU of Aeromonas hydrophila, Some of the transposon mutants had dramatically reduced hemolytic and cytotoxic activities, and such mutants exhibited reduced virulence in mice compared to wild- type Aeromonas when injected intraperitoneally (i.p.), Southern blot d ata indicated that transposition in these mutants did not occur within the cytotoxic enterotoxin gene tact). The transcription of the act ge ne was affected drastically in the transposon mutants, as revealed by Northern blot analysis. The altered virulence of these transposon muta nts was confirmed by developing isogenic mutants of the wild-type Aero monas by using a suicide vector. In these mutants, the truncated act g ene was integrated in place of a functionally active act gene. The cul ture filtrates from isogenic mutants were devoid of hemolytic, cytotox ic, and enterotoxic activities associated with Act These filtrates cau sed no damage to mouse small intestinal epithelium, as determined by e lectron microscopy, whereas culture filtrates from wild-type Aeromonas caused complete destruction of the microvilli. The 50% lethal dose of these mutants in mice was 1.0 x 10(8) when injected i,p,, compared to 3.0 x 10(5) for the wild-type Aeromonas, Reintegration of the native act gene in place of the truncated toxin gene in isogenic mutants resu lted in complete restoration of Act's biological activity and virulenc e in mice. The animals injected with a sublethal dose of wild-type Aer omonas or the revertant, but not the isogenic mutant, had circulating toxin-specific neutralizing antibodies. Taken together, these studies clearly established a role for Act in the pathogenesis of Aeromonas-me diated infections.