Regulation of the Cytotoxic Enterotoxin Gene in Aeromonas hydrophila : Characterization of an Iron Uptake Regulator

Abstract

The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of Aeromonas hydrophila is aerolysin related and crucial to the pathogenesis of Aeromonas infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (act), a portion of the act gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (phoA) of Escherichia coli. The act::phoA reporter gene was then introduced into the chromosome of A. hydrophila by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the act promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The act promoter exhibited optimal activity at pH 7.0 and at 37°C, and maximal PhoA activity was noted when the culture was aerated. Using a Vibrio cholerae iron uptake regulator gene (fur) as a probe, a 2.6-kb SalI/HindIII DNA fragment from an A. hydrophila chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the fur gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of V. cholerae. Complementation experiments demonstrated that the A. hydrophila fur gene could restore iron regulation in an E. coli fur-minus mutant. Using the suicide vector pDMS197, we generated a fur isogenic mutant of wild-type A. hydrophila SSU. Northern blot analysis data indicated that the repression in the transcription of the act gene by iron was relieved in the fur isogenic mutant. Further, iron regulation in the fur isogenic mutant of A. hydrophila could be restored by complementation. These results are important in understanding the regulation of the act gene under in vivo conditions.

The Type III Secretion System and Cytotoxic Enterotoxin Alter the Virulence of Aeromonas hydrophila

“…This effectively expands the range of environmental signals that influence target gene expression beyond population density (26). As we previously reported, Act is a type II-secreted cytotoxic enterotoxin (12), and the expression of the act gene was affected by different environmental stimuli, including its regulation through the fur regulatory circuitry (68). Act production increased as bacterial density increased (66), and thus, it is plausible that act gene expression is also under the control of QS, which is a topic worth pursuing in the future.…”

“…Briefly, the fosmid library was plated directly onto the nylon filters (Gibco BRL, Gaithersburg, MD) and placed on the surface of LB agar plates containing 12.5 g/ml chloramphenicol. Each filter with 150 to 200 colonies was prehybridized (2 h) and then hybridized (using 32 P-labeled ascV gene probe) in Quikhyb solution (Stratagene, La Jolla, CA) at 68°C for 3 h. The membranes were washed twice at 68°C in 2ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0) plus 0.1% sodium dodecyl sulfate (SDS) for 20 min and then twice in 1ϫ SSC plus 0.1% SDS for 20 min at 68°C (68). The blots were exposed to X-ray film at Ϫ70°C for 2 to 12 h. Three rounds of colony blot hybridization were performed to ensure purity of the clones.…”

“…32 P]dCTP-labeled ascV gene (2,166-bp) probe of A. hydrophila using colony blot hybridization (68). Briefly, the fosmid library was plated directly onto the nylon filters (Gibco BRL, Gaithersburg, MD) and placed on the surface of LB agar plates containing 12.5 g/ml chloramphenicol.…”

DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

“…This finding was compared to those in animals infected with act and aopB mutants alone (40 to 50% lethality) and thus signified that both the T2SS-associated Act and the T3SS played crucial roles in bacterial virulence (75). Further, we identified the role of the ferric uptake regulator (fur) and glucose-inhibited division gene (gidA) in modulating the biological effects of Act (73,74). In the search for additional genes that could alter bacterial virulence, we now report characterization of the DNA adenine methyltransferase (Dam) from A. hydrophila SSU that modulates the function of both T3SS and T2SS-associated Act.…”

Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

“…These ubiquitous bacteria produce a large number of virulence factors, many of which have been linked to Aeromonas-associated pathogenesis. Among them are matrix-binding proteins, elastases, proteases, cytotonic and cytotoxic enterotoxins, hemolysins, aldolase, chitinase, lipases/phospholipases, and the ability to form a capsule-like outer layer (10,30,35,45). Aeromonas spp.…”