Role of a cytotoxic enterotoxin in Aeromonas-mediated infections: development of transposon and isogenic mutants

Abstract

Transposon and marker exchange mutagenesis were used to evaluate the role of Aeromonas cytotoxic enterotoxin (Act) in the pathogenesis of diarrheal diseases and deep wound infections. The transposon mutants were 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 data indicated that transposition in these mutants did not occur within the cytotoxic enterotoxin gene (act). The transcription of the act gene was affected drastically in the transposon mutants, as revealed by Northern blot analysis. The altered virulence of these transposon mutants was confirmed by developing isogenic mutants of the wild-type Aeromonas by using a suicide vector. In these mutants, the truncated act gene was integrated in place of a functionally active act gene. The culture filtrates from isogenic mutants were devoid of hemolytic, cytotoxic, and enterotoxic activities associated with Act. These filtrates caused no damage to mouse small intestinal epithelium, as determined by electron 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 resulted in complete restoration of Act's biological activity and virulence in mice. The animals injected with a sublethal dose of wild-type Aeromonas 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-mediated infections.

FIG. 1

Flow diagram showing construction of various recombinant plasmids. Recombinant plasmid pXHC95 contained a 2.8-kb BamHI DNA fragment from chromosomal DNA of A. hydrophila SSU with the act gene (II). A kanamycin resistance gene cartridge from plasmid pUCK4 was introduced within the act gene to generate recombinant plasmid pXHC97.1 before ligation of the truncated act gene in the suicide vector pJQ200 to generate plasmid pXHC97.2 (III). A 2.8-kb BamHI DNA fragment containing the act gene also was subcloned in another suicide vector, pMW1823, to generate plasmid pXHC97.3 (I) for the purpose of generating a revertant of A. hydrophila with parental biological activity of Act. MCS, multiple cloning site.

FIG. 2

Southern blot analysis of the genomic DNAs from wild-type and transposon mutants of A. hydrophila. The genomic DNA (15 μg) was digested with the SalI and BamHI restriction enzymes and subjected to Southern blot analysis. The probe used was a 1.4-kb XbaI/SalI DNA fragment (³²P labeled), which depicts the coding region of the act gene. The blot was prehybridized, hybridized (5 × 10⁶ cpm/ml), and washed as described in Materials and Methods. The lanes contained digested DNAs from the transposon mutants 353 (lane 1), 325 (lane 2), 312 (lane 3), 225 (lane 4), and 42 (lane 5) and from wild-type A. hydrophila SSU (lane 6) as a positive control.

FIG. 3

Northern blot analysis of the total RNAs isolated from the wild-type and transposon mutants of A. hydrophila. The total RNA was isolated by using a total RNA isolation kit (Qiagen). A 439-bp ³²P-labeled DNA fragment, which represented the 5′ end of the toxin structural gene, was used as a probe in these blots. The blot was prehybridized, hybridized, and washed as described in Materials and Methods. The blots were exposed to X-ray film at −70°C for 12 h. The lanes contained RNAs from mutant cultures 353 (lane 1), 325 (lane 2), 312 (lane 3), 225 (lane 4), and 42 (lane 5) and from wild-type A. hydrophila SSU (lane 6).

FIG. 4

Electron microscopy of intestinal tissues of mice injected with culture filtrates from wild-type Aeromonas and its isogenic mutant. Mouse ligated loops were placed in fixative and cut into 1-mm pieces. Ultrathin sections were stained and photographed in a Philips 201 electron microscope. (A) After administration of Act (contained in culture filtrate) from wild-type Aeromonas, enterocytes were completely destroyed. (B) Mouse loops challenged with culture filtrate from a double-crossover mutant. Normal enterocytes with intact brush borders surround the lumen. Mucus is being emptied from a goblet cell into the lumen in the upper layer of cells. Bars, 1 μm.

FIG. 5

Southern blot analysis of the chromosomal DNAs from A. hydrophila SSU and its isogenic mutants. Total DNAs (15 μg) from A. hydrophila and its mutants were digested with the BamHI restriction enzyme and subjected to Southern blot analysis. The probes used were a 439-bp XbaI/SalI DNA fragment, which depicts part of the coding region of the act gene (A), a 1.2-kb kanamycin resistance gene cassette (B), and a 4.9-kb pJQ200 suicide vector (C). The blots were probed and washed as described in Materials and Methods. (A) Digested DNAs from E. coli with suicide vector and truncated act gene (lane 1), double-crossover mutants of A. hydrophila (lanes 3 and 4), a single-crossover mutant of A. hydrophila (lane 5), and wild-type A. hydrophila (lane 6). (B and C) Digested DNAs from double-crossover mutants of A. hydrophila (lanes 1 and 2), single-crossover mutants of A. hydrophila (lanes 3 and 4), and wild-type A. hydrophila (lanes 5 and 6).