Characterization of an ADP-Ribosyltransferase Toxin (AexT) from Aeromonas salmonicida subsp. salmonicida

Abstract

An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis of Salmonidae. This fish disease causes most severe losses in production farms of salmon and trout and leads to the use of large amounts of antibiotics in closed and open waters for prevention and therapy of furunculosis. To develop efficient strategies to prevent outbreaks of A. salmonicida subsp. salmonicida, it is essential to know the main mechanisms of pathogenicity of this pathogen. Several potential virulence factors of A. salmonicida subsp. salmonicida have been described thus far. They include the surface array layer protein (7); hemolysins ASH1, ASH3, and ASH4 (12); H-lysin (29); salmolysin (19); serine protease AspA (32); and the glycerophospholipid:cholesterol acyltransferase (GCAT) complexed with lipopolysaccharide (18). Recent reports demonstrate the role of the S layer in adhesion (11) of A. salmonicida subsp. salmonicida. The other potential virulence factors of A. salmonicida subsp. salmonicida that are currently known do not seem to play a primary role in pathogenesis. GCAT and aspA gene deletion mutants showed that neither GCAT nor aspA is essential ...

Identification of Aeromonas salmonicida in European perch from North Lithuanian rivers during mass mortalities in 2008

“…salmonicida [38]. This system is responsible for the translocation of the ADPribosylating toxin, AexT, into the cytosol of target fish cells and is associated with the pathogenicity of the bacterium [37,39,40].…”

“…A type III secretion system (TTSS) has also been identified in A. salmonicida subsp. salmonicida Strain JF2267, a strain originally isolated from Arctic char Salvelinus alpinus presenting typical furunculosis symptoms [37,38]. Studies relating to Aeromonas virulence have identified a type III secretion system in A. salmonicida subsp.…”

Contribution of the type III secretion system (TTSS) to virulence of Aeromonas salmonicida subsp. salmonicida

“…This is believed to be the first study that has investigated the in vivo behaviour of A. salmonicida TTSS effectors by conducting animal challenges with mutants deleted in three effector genes. A. salmonicida TTSS effectors are poorly characterized compared with those of other systems; only the functionality of AexT has been studied in any detail (Braun et al, 2002(Braun et al, , 2003b(Braun et al, , 2005. The in vivo behaviour of the three effector mutant strains mimicked the behaviour of a suite of effector mutant strains created in Y. enterocolitica O : 8 (Trülzsch et al, 2004).…”

“…Three TTSS effectors were also identified. One, AexT, was chromosomally located, had 100 % amino acid identity to a previously characterized translocated ADP-ribosylating cytotoxin from A. salmonicida strain JF2267 and had similarity to exoT of P. aeruginosa (Braun et al, 2002(Braun et al, , 2003b. The other two, located on plasmid pAsa5, were termed aop (Aeromonas outer protein) H and aopO, with similarity to Yersinia yopH and yopO/ypkA, respectively (Table 3).…”

The ADP-Ribosylating Toxin, AexT, fromAeromonas salmonicidasubsp.salmonicidaIs Translocated via a Type III Secretion Pathway

“…This study focuses on the recently identified ADP ribosyltransferase toxin A.salmonicida exoenzyme T, or AexT, which has recently been shown to play a role in the cytotoxicity of A. salmonicida subsp. salmonicida to fish cells (4).…”