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.

FIG. 1.

Genetic map of the gene encoding AexT and ORFX of A. salmonicida subsp. salmonicida in alignment with the corresponding genes of P. aeruginosa and Y. pestis. Maps were constructed from the current sequence data and from EMBL/GenBank accession no. AF288366 for A. salmonicida subsp. salmonicida AexT, L27629 for P. aeruginosa ExoS, L46800 for P. aeruginosa ExoT, and AF053946 for Y. pestis YopE. The last is also representative of the analogous genes of Y. pseudotuberculosis and Y. enterocolitica (8). Top line, scale in kilobase pairs; line below, physical map of the locus showing a few sites for restriction enzymes (arrows, locations of the oligonucleotide primers that were used for verification of the insertion of the Km^r cassette in the aexT mutant. Boxes with arrowheads, ORFs. Numbers indicate corresponding amino acid positions. The putative biglutamic acid active sites (grey boxes) are shown in detail. Black boxes, transcription activator (ExsA) binding site; black triangles, consensus sequences for the transcription promoter. Abbreviations: AS, A. salmonicida subsp. salmonicida; PA, P. aeruginosa; YP, Y. pestis; CP, Clostridium perfringens; EC, E. coli; VC, V. cholerae; Iota, iota toxin; LT, heat-labile toxin; CT, cholera toxin.

FIG. 2.

Toxicity of AexT-producing A. salmonicida subsp. salmonicida to RTG-2 fish cells. The cells were photographed 2 h after inoculation. (A) RTG-2 cells inoculated with AexT-producing (wt) A. salmonicida subsp. salmonicida strain JF2267. (B) RTG-2 cells inoculated with isogenic aexT mutant JF2580. (C) RTG-2 cells inoculated with 100 μl of PBS buffer. (D) Strain JF2267 in culture medium without fish cells.

FIG. 3.

Biosynthesis of AexT by A. salmonicida subsp. salmonicida. The immunoblots were reacted with anti-AexT antibodies and contained RTG-2 cells inoculated with JF2267 (lanes 1), RTG-2 cells inoculated with mutant JF2580 (lanes 2), RTG-2 cells with PBS (lanes 3), and A. salmonicida subsp. salmonicida JF2267 in culture medium (lanes 4). Lanes c, purified recombinant AexT-His as a control; lanes st, molecular mass standard. (A) Pellets containing cells and bacteria. (B) Supernatants.

FIG. 4.

Expression of AexT by A. salmonicida subsp. salmonicida in low-Ca²⁺ medium and serological cross-reactions with ExoS and ExoT. Bacterial cultures were grown in Ca²⁺-depleted TSB medium. Lane 1, A. salmonicida subsp. salmonicida wt strain JF2267; lane 2, A. salmonicida subsp. salmonicida aexT mutant JF2580; lane 3, P. aeruginosa strain ATCC 27853. Culture supernatants were concentrated 20-fold and analyzed on immunoblots with anti-AexT antibodies. Lane c, purified recombinant AexT-His as a control; lane st, molecular mass standard. The identity of the band at 30 kDa reacting with P. aeruginosa ATCC 27853 (lane 3) is not determined.