The ADP-ribosylating toxin, AexT, from Aeromonas salmonicida subsp. salmonicida is translocated via a type III secretion pathway - PubMed (original) (raw)

The ADP-ribosylating toxin, AexT, from Aeromonas salmonicida subsp. salmonicida is translocated via a type III secretion pathway

Sarah E Burr et al. J Bacteriol. 2003 Nov.

Abstract

AexT is an extracellular ADP ribosyltransferase produced by the fish pathogen Aeromonas salmonicida subsp. salmonicida. The protein is secreted by the bacterium via a recently identified type III secretion system. In this study, we have identified a further 12 open reading frames that possess high homology to genes encoding both structural and regulatory components of the Yersinia type III secretion apparatus. Using marker replacement mutagenesis of aopB, the A. salmonicida subsp. salmonicida homologue of yopB in Yersinia, we demonstrate that the bacterium translocates the AexT toxin directly into the cytosol of cultured fish cells via this type III secretion pathway. An acrV mutant of A. salmonicida subsp. salmonicida displays a calcium-blind phenotype, expressing and secreting significant amounts of AexT even in the presence of CaCl2 concentrations as high as 10 mM. This acrV mutant is also unable to translocate AexT into the cytosol of fish cells, indicating AcrV is involved in the translocation process. Inactivation of either the aopB or acrV gene in A. salmonicida subsp. salmonicida (resulting in an inability to translocate AexT) is accompanied by a loss of cytotoxicity that can be restored by trans complementation. Finally, we present data indicating that preincubation of the wild-type bacteria with antibodies directed against recombinant AcrV-His protein provides fish cells protection against the toxic effects of the bacterium.

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Figures

FIG. 1.

FIG. 1.

Genetic map of the type III secretion genes found in A. salmonicida strain JF2267. The solid boxes represent genes identified in this study. Potential promoter sequences, represented by arrowheads, are found preceding the ascN, aopN, and acrG genes. (In the case of ascN, two potential promoter sequences have been identified.) The restriction sites used in the marker replacement of the acrV and aopB genes and the direction of the kanamycin cassette replacement are indicated. All genes have been designated based solely on sequence similarity to Yersinia (ysc, lcr, and yop) TTSS genes.

FIG. 2.

FIG. 2.

Type III-dependent translocation of AexT. (A and B) Bacterial cultures were grown overnight in TSB medium, and the presence of the proteins AexT (A) and AcrV (B) was analyzed by SDS-PAGE and Western blotting using polyclonal antibodies. WT, A. salmonicida strain JF2646 (aopB+); aopB, isogenic Δ_aopB_ mutant, strain JF2724. Lane C in panel A contains recombinant AexT-His protein, whereas lane C in panel B contains recombinant AcrV-His. (C) After infection of EPC cells with A. salmonicida strain JF2646 (WT) or A. salmonicida strain JF2724 (aopB), cytosolic fractions of EPC were prepared using Triton X-100 lysis. The Triton-insoluble (containing bacterial cells, unlysed EPC cells, and nuclei) and -soluble (containing EPC cytosolic proteins and membrane proteins) fractions were analyzed by SDS-PAGE and Western blotting using polyclonal anti-AexT antibodies. Lane C contains recombinant AexT-His protein.

FIG. 3.

FIG. 3.

Cytotoxicity of A. salmonicida to EPC cells. Phase-contrast micrographs of EPC cells 5 h after infection with A. salmonicida strain JF2646 (aopB+) (A); the isogenic Δ_aopB_ mutant, strain JF2724 (B); strain JF2953 (Δ_aopB_/pMMB66HE_aopB_+) (C); or PBS only (D). The multiplicity of infection was 20:1.

FIG. 4.

FIG. 4.

Type III-dependent expression and secretion of AexT. Expression (A) and secretion (B) of AexT under low-calcium conditions are shown. Bacterial cultures were grown overnight in TSB medium supplemented with millimolar concentrations of CaCl2 as indicated. The presence of AexT in the cell pellets (A) and culture supernatants (B) was analyzed by SDS-PAGE and Western blotting using polyclonal anti-AexT antibodies. WT, A. salmonicida strain JF2646 (acrV+); acrV, isogenic Δ_acrV_ mutant, strain JF2684.

FIG. 5.

FIG. 5.

Role of AcrV in A. salmonicida cytotoxicity. (A to C) Phase-contrast micrographs of EPC cells 5 h after infection with A. salmonicida strain JF2646 (acrV+) (A), isogenic Δ_acrV_ mutant strain JF2684 (B), or strain JF2953 (Δ_acrV_/pMMB66HE_acrV_+) (C). The multiplicity of infection was 20:1. (D) After infection with strain JF2684, cytosolic fractions of EPC cells were prepared using Triton X-100 lysis. The Triton-insoluble and -soluble fractions were analyzed by SDS-PAGE and Western blotting using polyclonal anti-AexT antibodies.

FIG. 6.

FIG. 6.

Anti-AcrV antibodies protect against A. salmonicida cytotoxicity. (A and B) Phase-contrast micrographs of EPC cells 5 h following infection with the wt isolate A. salmonicida strain JF2267 in the presence of preimmune IgG (A) or anti-AcrV IgG (B). (C) Cells treated with anti-AcrV IgG in PBS only as a control. The multiplicity of infection was 2:1.

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