Role of Various Enterotoxins in Aeromonas hydrophila- Induced Gastroenteritis: Generation of Enterotoxin Gene-Deficient Mutants and Evaluation of Their Enterotoxic Activity

Abstract

Three enterotoxins from the Aeromonas hydrophila diarrheal isolate SSU have been molecularly characterized in our laboratory. One of these enterotoxins is cytotoxic in nature, whereas the other two are cytotonic enterotoxins, one of them heat labile and the other heat stable. Earlier, by developing an isogenic mutant, we demonstrated the role of a cytotoxic enterotoxin in causing systemic infection in mice. In the present study, we evaluated the role of these three enterotoxins in evoking diarrhea in a murine model by developing various combinations of enterotoxin gene-deficient mutants by marker-exchange mutagenesis. A total of six isogenic mutants were prepared in a cytotoxic enterotoxin gene (act)-positive or -negative background strain of A. hydrophila. We developed two single knockouts with truncation in either the heat-labile (alt) or the heat-stable (ast) cytotonic enterotoxin gene; three double knockouts with truncations of genes encoding (i) alt and ast, (ii) act and alt, and (iii) act and ast genes; and a triple-knockout mutant with truncation in all three genes, act, alt, and ast. The identity of these isogenic mutants developed by double-crossover homologous recombination was confirmed by Southern blot analysis. Northern and Western blot analyses revealed that the expression of different enterotoxin genes in the mutants was correspondingly abrogated. We tested the biological activity of these mutants in a diet-restricted and antibiotic-treated mouse model with a ligated ileal loop assay. Our data indicated that all of these mutants had significantly reduced capacity to evoke fluid secretion compared to that of wild-type A. hydrophila; the triple-knockout mutant failed to induce any detectable level of fluid secretion. The biological activity of selected A. hydrophila mutants was restored after complementation. Taken together, we have established a role for three enterotoxins in A. hydrophila-induced gastroenteritis in a mouse model with the greatest contribution from the cytotoxic enterotoxin Act, followed by the Alt and Ast cytotonic enterotoxins.

Different Clinical Characteristics between Polymicrobial and Monomicrobial Aeromonas Bacteremia -A Study of 216 Cases

“…Aeromonas species produces an array of virulence factors, one of the most significant of which is a 52-kDa cytotoxic enterotoxin (Act) (49), which possesses several biological activities, including hemolysis, cytotoxicity, enterotoxicity and lethality to mice, and induced acute inflammatory response both in vitro and in vivo (50). In vitro, Act activates mitogen-activated protein kinase (MAPK) pathways and classical caspase-associated apoptosis in intestinal epithelial cells and macrophages (51)(52)(53). In addition, a recent study showed that with intramuscular bacterial inoculation in mice, A. hydrophila could recruit inflammatory cells around the inoculation site earlier than E. coli and K. pneumonia, and elicit a specific pathological change, myonecrosis (54).…”

Pic, an Autotransporter Protein Secreted by Different Pathogens in the Enterobacteriaceae Family, Is a Potent Mucus Secretagogue

“…To evaluate Pic activity on intestinal mucin in vivo, the rat ligated ileal loop model was used (9,42,48). Sprague-Dawley rats (n ϭ 4) of either sex and between 70 and 100 g in weight were used.…”

Prevalence of Hemolytic and Enterotoxigenic Aeromonas Spp. in Healthy and Diseased Freshwater Food Fishes as Assessed by Multiplex PCR

“…(Aguilera-Arreola et al, 2007). A correlation between the higher number of toxin genes harboured in an isolate of Aeromonas and its potential to induce diarrhea have been earlier reported (Sha et al, 2002;Chang et al, 2008) and their detection in food products is very essential. The results of the present study indicated that the detection and identification of toxigenic Aeromonas spp.…”

“…Exotoxins are major virulence factors of Aeromonas spp. that include a cytotoxic heat-labile enterotoxin (Act), also known as aerolysin/hemolysin (Chopra et al, 1993); a cytotonic heat-labile enterotoxin (Alt), also known as lipase, extracellular lipase, or phospholipase (Chopra et al, 1996); and a cytotonic heat-stable enterotoxin (Ast) (Sha et al, 2002). These toxins are encoded by the genes act, alt and ast, respectively (Sen and Rodgers, 2004).…”