1 Institute of Veterinary Bacteriology, University of Berne, Länggass-Strasse 122, Postfach, CH-3001 Berne, Switzerland
2 Centre for Fish and Wildlife Health, Institute of Animal Pathology, University of Berne, Länggass-Strasse 122, Postfach, CH-3001 Berne, Switzerland
Correspondence
Joachim Frey
joachim.frey{at}vbi.unibe.ch
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of the complete A. salmonicida subsp. salmonicida type III secretion locus in strain JF2267 is AJ616218.
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INTRODUCTION |
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Because of the negative impact A. salmonicida subsp. salmonicida has on the aquaculture industry, extensive work has been done to identify virulence factors produced by this bacterium. Recently, a functional type III protein secretion system has been identified in an A. salmonicida subsp. salmonicida field isolate (Burr et al., 2002, 2003
). Such secretion systems enable the movement or translocation of effector proteins, produced in the bacterial cytoplasm, into the cytosol of eukaryotic cells (see Ghosh, 2004
, for a recent review). Once within the eukaryotic cytosol, effector proteins are able to disrupt the cytoskeleton or interfere with cell signalling cascades (Cornelis, 2002
; Galán, 2001
; Zaharik et al., 2002
). As a result, type III secretion systems are important virulence factors of many Gram-negative bacteria including the pathogenic Yersinia spp., Salmonella spp., Shigella spp., enteropathogenic and enterohaemorrhagic Escherichia coli and Pseudomonas aeruginosa (Ghosh, 2004
).
Using marker-replacement mutagenesis, the type III secretion system of A. salmonicida subsp. salmonicida, has been found to be responsible for the secretion and translocation of the ADP-ribosylating toxin, AexT, into the cytosol of cultured fish cells (Burr et al., 2002, 2003
). Inactivation of the secretion system has also been shown to reduce the cytoxicity of A. salmonicida subsp. salmonicida towards fish cell lines. We were therefore interested to determine the role of the type III secretion system in the virulence of A. salmonicida subsp. salmonicida towards the fish host. In the current study, we examined the virulence of a defined type III secretion mutant of A. salmonicida subsp. salmonicida using an in vivo rainbow trout model and found that this sytem is essential for virulence of the bacterium. We also investigated the presence of type III secretion genes in atypical A. salmonicida strains and other aeromonad fish isolates.
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METHODS |
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DNA sequencing was performed with the dRhodamine Terminator Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's protocol. Sequences were determined on both strands. Reaction products were analysed on an ABI Prism 310 genetic analyser (Applied Biosystems) and sequence alignment and editing were performed using the software Sequencher (Gene Codes Corporation). Comparison of DNA sequences and their corresponding amino acid sequences with the Swiss Prot and EMBL databases were performed using BLAST (http://www.ncbi.nlm.nih.gov/blast/). The molecular mass and theoretical isoelectric point (pI) of the type III secretion proteins were calculated using ProtParam (http://www.expasy.org/tools/protparam.html).
Marker-replacement mutagenesis.
The A. salmonicida subsp. salmonicida gene ascV was inactivated by marker-replacement mutagenesis. A 1061 bp fragment from ascV was excised using the restriction enzymes KpnI and SpeI (Roche Diagnostics) and replaced with the kanamycin (Km) cassette from pSSVI186 that had been previously excised on a 1·3 kb KpnISpeI fragment. The inactivated ascV and flanking genes were then cloned into the mobilizable suicide vector pSUP202sac. The resulting plasmid was transformed into E. coli S17-1 and conjugated into A. salmonicida subsp. salmonicida strain JF2267 by filter mating (Simon et al., 1983). Double crossover mutants were selected directly by growth on Tryptic Soy Agar (Difco) containing 15 % (w/v) sucrose, 40 µg kanamycin ml1 and 20 µg chloramphenicol ml1 at 15 °C for 7 days. Chloramphenicol served to select against the E. coli donor as A. salmonicida subsp. salmonicida strain JF2267 is resistant to this antibiotic. The absence of the wild-type (wt) ascV gene and the insertion of the Km cassette were verified by PCR. Western blot analysis was also carried out to determine whether the
ascV mutant retained the ability to express S-layer protein (Belland & Trust, 1987
).
Fish maintenance.
Juvenile rainbow trout (Oncorhynchus mykiss Walbaum), raised in closed systems under specific pathogen-free (SPF) conditions, were used in all in vivo assays. Prior to the onset of experiments, 10 fish were screened for bacterial, viral and parasitic pathogens according to routine diagnostic procedures. Fish, measuring 1116 cm total length, were stocked in aerated 13 l volume PVC-tanks to a stocking densitiy of 24 kg m3. Flowthrough of tap water was set at 0·6 l min1. The fish were adapted to these conditions for 2 weeks before the experiments began and were fed a commercial fish diet at 1 % body weight per day.
Experimental infection.
Experimental infections were carried out with permission from the Ethical Committee for Animal Experiments, Canton Berne, Switzerland. One hundred and twenty rainbow trout were randomly distributed into tanks containing 10 fish per tank. Suspensions of the A. salmonicida subsp. salmonicida wt or ascV mutant strains were prepared in 10 mM PBS, pH 7·4. Fish (four tanks per group) were injected intraperitoneally (i.p.) with 1x105 c.f.u. of either one of the bacterial suspensions in 50 µl PBS or with PBS only. Mortality was recorded daily or, in cases of high mortality, twice daily for 3 weeks. The temperature range during the course of the experiment was 11·8±0·5 °C. All dead fish were investigated for the presence of A. salmonicida subsp. salmonicida as were at least three surviving fish from each tank at the end of the experiment. Material was collected from the liver and kidney of each fish using sterile loops and used to inoculate Columbia sheep blood agar (BioMérieux). The plates were incubated at 18 °C for up to 5 days and the identity of A. salmonicida subsp. salmonicida isolated from dead fish was confirmed by PCR.
Histology.
Rainbow trout were injected intramuscularly (i.m.) with either the A. salmonicida subsp. salmonicida wt or ascV mutant strains (five fish for each strain) using an infectious dose of 5x104 c.f.u. per fish. Four days following infection, live and moribund fish were sacrificed and examined for macroscopic alterations. For histological examination, muscle and organ tissues were fixed in 4 % buffered formalin, embedded in paraffin wax and sectioned according to routine histological procedures to produce 5 µm sections. Sections were then stained with haematoxylin and eosin and examined by light microscopy. At least 10 slides from each fish were examined.
Expression of green fluorescent protein.
The gfp gene, encoding green fluorescent protein, and its ribosome-binding site were excised from pBCgfp using the restriction endonucleases EcoRI and HindIII and subsequently cloned into pMMB66EH for expression. The resulting plasmid, pMMB66EH-gfp, was transformed into E. coli S17-1 and transferred to A. salmonicida subsp. salmonicida by filter-mating, as described above. Transconjugants were selected on LB agar containing ampicillin and chloramphenicol, and the presence of pMMB66EH-gfp was confirmed by PCR. Expression of gfp was induced by addition of IPTG to the culture medium at a final concentration of 1 mM.
Phagocytosis assay.
Heparinized blood, obtained from a naïve rainbow trout raised under SPF conditions, was combined with an equal volume of Eagle's minimum essential medium (EMEM, Gibco) before being layered onto a discontinuous Ficoll separating solution with a 1·077 gradient (Biochrom KG). Following centrifugation at 1200 g for 30 min, the layer at the interface, which contained peripheral blood leukocytes, was collected and washed three times with EMEM. The cells were then seeded into six-well culture plates (Falcon), containing glass cover slips coated with Matrigel (BD Biosciences) in 1 ml L-15 medium (Sigma) supplemented with 5 % fetal bovine serum and cyproxin (20 µg ml1), to a concentration of 1x106 cells ml1. Cells were incubated for 12 h at 15 °C to allow for attachment to the surface of the cover slips. Following incubation, the medium was replaced with 1 ml L-15 medium containing 5 % fetal bovine serum and 1 mM IPTG. The cultures were then infected with A. salmonicida subsp. salmonicida wt or ascV mutant strains. Four hours post-infection (p.i.), the cells were fixed in 4 % buffered formalin and the preparations were examined by fluorescence microscopy. Three independent trials, each in duplicate, were performed.
Dot blot hybridization.
Digoxigenin-11-dUTP (DIG)-labelled probes directed against type III secretion genes ascV and ascU of A. salmonicida subsp. salmonicida strain JF2267 were prepared by PCR using the following primer pairs: AslcrD-L (5'-GCCCGTTTTGCCTATCAA-3') and AslcrD-R (5'-GCGCCGATATCGGTACCC-3') for ascV, and AscU-5' (5'-CGATGAGCGGCGAGAAAAC-3') and AscU-3' (5'-AGCGTGCAGCCCTACCACTG-3') for ascU. PCR conditions were 3 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C and 30 s at 72 °C. Following PCR amplification, the probes were purified using the High Pure PCR Product Purification Kit (Roche) according to the manufacturer's instructions.
Total DNA was prepared using the EZNA bacterial DNA kit (Peqlab Biotechnologie) and denatured in 0·4 M NaOH, 10 mM EDTA. Denatured DNA was spotted onto positively charged nylon membranes (Roche) and fixed by baking at 80 °C under vacuum. Hybridization was performed overnight at 65 °C and membranes were washed under low-stringency conditions (Ausubel et al., 1999). Hybridization reactions were detected using anti-DIG antibodies and the chemiluminescent reagent CPD-Star (Roche) according to the manufacturer's protocol. DNA from A. salmonicida subsp. salmonicida strain JF2267 and E. coli DH5
served as positive and negative controls respectively.
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RESULTS AND DISCUSSION |
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Histological examination was carried out on the internal organs and muscle tissue of the infected fish. In fish that were injected with the wt isolate, strain JF2267, extensive tissue damage and large aggregates of bacteria could be seen in all organs examined, including the kidney, liver and heart (Fig. 4A, C and E), indicating that this strain established a systemic infection. Tissue from the posterior kidney revealed focal to coalescing necrosis of the interstitial tissue and, to a lesser extent, necrosis of single glomeruli and tubuli (Fig. 4A
). In the liver, sinusoids were distended and partially filled with granular, eosinophilic material (oedema fluid) (Fig. 4C
). Many cells with pycnotic nuclei were observed randomly distributed throughout the liver tissue (Fig. 4C
). The lymphoid tissue of the spleen was severely depleted and foci of necrosis could be seen. The mucosa of the intestine was multifocally or generally necrotic. The heart muscle displayed a massive separation of muscle fibres due to oedema (Fig. 4E
) and focal to coalescing myolysis could be seen in the body muscle tissue. In contrast, none of the fish that were infected with the
ascV A. salmonicida subsp. salmonicida mutant displayed signs of disease at 4 days p.i. Histological examination revealed no pathological changes in any organ (Fig. 4B, D and F
) and no bacterial cells were seen in the internal organs or muscle tissue surrounding the initial injection site. Furthermore, we were unable to re-isolate the bacterium from the internal organs of these fish. These findings indicate that the type III secretion system of A. salmonicida subsp. salmonicida is required by the bacterium to establish a systemic infection.
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Finally, type III secretion system genes were also found in fish isolates of Aeromonas encheleia, Aeromonas eucrenophila, Aeromonas hydrophila, Aeromonas media, Aeromonas sobria and Aeromonas veronii (Table 2). This is believed to be the first time such genes have been found in A. enceleia, A. eucrenophila and A. media, and in A. veronii isolates of non-human origin. In addition to finding ascV and ascU in such Aeromonas species isolated from fish, we also detected these genes in four isolates from water sources.
Conclusion
The type III secretion system of A. salmonicida subsp. salmonicida has been shown to be an important virulence factor. It enables the bacterium to avoid uptake by peripheral blood leukocytes, induce alterations in the internal organs and muscle tissue of the fish host and establish systemic infection. Furthermore, our findings suggest that type III secretion systems may also be important in the virulence of atypical A. salmonicida and other aeromonad fish pathogens.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (editors) (1999). Current Protocols in Molecular Biology. New York: Wiley.
Belland, R. J. & Trust, T. J. (1987). Cloning of the gene for the surface array protein of Aeromonas salmonicida and evidence linking loss of expression with genetic deletion. J Bacteriol 169, 40864091.[Medline]
Braun, M., Stuber, K., Schlatter, Y., Wahli, T., Kuhnert, P. & Frey, J. (2002). Characterization of an ADPribosyltransferase toxin (AexT) from Aeromonas salmonicida subsp. salmonicida. J Bacteriol 181, 18511858.[CrossRef]
Bullock, W. O., Fernandez, J. M. & Short, J. M. (1987). XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 5, 376378.
Burr, S. E., Stuber, K., Wahli, T. & Frey, J. (2002). Evidence for a type III secretion system in Aeromonas salmonicida subsp. salmonicida. J Bacteriol 184, 59665970.
Burr, S. E., Stuber, K. & Frey, J. (2003). The ADP-ribosylating toxin, AexT, from Aeromonas salmonicida subsp. salmonicida is translocated via a type III secretion pathway. J Bacteriol 185, 65836591.
Cornelis, G. R. (2002). The Yersinia Ysc-Yop type III weaponry. Nat Rev Mol Cell Biol 10, 742752.[CrossRef]
Fürste, J. P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M. & Lanka, E. (1986). Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48, 119131.[CrossRef][Medline]
Galán, J. E. (2001). Salmonella interactions with host cells: type III secretion at work. Annu Rev Cell Dev Biol 17, 5386.[CrossRef][Medline]
Ghosh, P. (2004). Process of protein transport by the type III secretion. Microbiol Mol Biol Rev 68, 771795.
Gudmundsdóttir, B. K. (1998). Infections by atypical strains of the bacterium Aeromonas salmonicida. Icel Agr Sci 12, 6172.
Kuhnert, P., Frey, J., Lang, N. P. & Mayfield, L. (2002). Phylogenetic analysis of Prevotella nigrescens, Prevotella intermedia and Porphyromonas gingivalis clinical strains reveals a clear species clustering. Int J Syst Evol Microbiol 52, 13911395.
Matthysse, A. G., Stretton, S., Dandie, C., McClure, N. C. & Goodman, A. E. (1996). Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiol Lett 145, 8794.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Sring Harbor Laboratory.
Sharples, G. J., Ingleston, S. M. & Lloyd, R. G. (1999). Holliday junction processing in bacteria: insights from the evolutionary conservation of RuvABC, RecG, and RusA. J Bacteriol 181, 55435550.
Simon, R., Priefer, U. & Puhler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology 1, 784791.[CrossRef]
Stuber, K., Frey, J., Burnens, A. P. & Kuhnert, P. (2003). Detection of type III secretion genes as a general indicator of bacterial virulence. Mol Cell Probes 17, 2532.[CrossRef][Medline]
Vipond, R., Bricknell, I. R., Durant, E., Bowden, T. J., Ellis, A. E., Smith, M. & MacIntyre, S. (1998). Defined deletion mutants demonstrate that the major secreted toxins are not essential for the virulence of Aeromonas salmonicida. Infect Immun 66, 19901998.
Viret, J. F. (1993). Meganuclease I-SceI as a tool for the easy subcloning of large DNA fragments devoid of selection marker. Biotechniques 14, 325326.[Medline]
Yu, H. B., Sriniasa Rao, P. S., Lee, H. C., Vilches, S., Merino, S., Thomas, J. M. & Leung, K. Y. (2004). A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis. Infect Immun 72, 12481256.
Zaharik, M. L., Gruenheid, S., Perrin, A. J. & Finlay, B. B. (2002). Delivery of dangerous goods: type III secretion in enteric pathogens. Int J Med Microbiol 291, 593603.[Medline]
Received 28 January 2005;
revised 2 March 2005;
accepted 7 March 2005.
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