Evidence for a type III secretion system in Aeromonas salmonicida subsp. salmonicida

Abstract

Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host-range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen.

FIG. 1.

Genetic organization of the virA operon of A. salmonicida subsp. salmonicida strain JF2267 (and that of the Rif^r derivative, JF2646). The virA locus of Yersinia species is shown for comparison. Genes that comprise the virA locus are represented by black boxes. Flanking genes are shown in white. The jagged box represents an incomplete gene sequence. Potential promoter sequences, represented by gray arrowheads, are found upstream of aopN and acrG. The KpnI and SpeI sites used for the inactivation of the ascV gene are indicated.

FIG. 2.

Secretion of AexT under low-calcium conditions. Bacterial cultures were grown overnight in TSB medium. Left lane, A. salmonicida subsp. salmonicida strain JF2646 (ascV⁺); right lane, A. salmonicida subsp. salmonicida ΔascV mutant JF2678. Horizontal lines mark the positions of molecular mass standards.

FIG. 3.

Toxicity of A. salmonicida subsp. salmonicida to RTG-2 cells. (A) RTG-2 cells inoculated with A. salmonicida subsp. salmonicida strain JF2646 (ascV⁺). (B) RTG-2 cells inoculated with isogenic ΔascV mutant JF2678. (C) RTG-2 cells inoculated with PBS only.