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Cloning, Mapping and Identification of the Aeromonas Salmonicida Fsta

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Cloning, Mapping and Identification of the Aeromonas Salmonicida Fsta 魚 病 研 究 Fish Pathology,33(4),239-246,1998.10 Cloning,Mapping and Identificationof theAeromonas salmonicidafstA,fepA and irpAGenes, Encoding the 86,82 and 74 kDa Iron-regulated Outer Membrane Proteins,Respectively R.J. Collighan, A. J. Bennett and G. Coleman Department of Biochemistry,NottinghamUniversity Medical School,Queen'sMedical Centre, Clifton Boulevard, Nottingham,NG7 2UH,U.K. (Received February 23,1998) Three iromps(iron-regulatedouter membrane proteins)ofAeromonas salmonicidawere identifiedby the use of specificantibodies together with Southernhybridization analysis and limitednucleotide sequencing of their genes. The resultsof theseexperiments together with a searchof the internationaldatabase for homologous sequencesled to theiridentification as follows: 86 kDa iromp(FstA)as a Vibrioanguillarum Fat A homologue 82 kDa iromp(FepA)as an Escherichiacoli FepA homologue 74kDa iromp(IrpA)as an Escherichiacoli Cir homologue Key words : gene cloning,gene mapping,Aeromonas salmonicida,iron-regulated outer membrane protein,iromp Pathogenic bacteria require iron for growth in a host such are considered as potential vaccine components where the only source of this element is tightly com (Banerjee-Bhatnagar and Frasch, 1990; Robinson and plexed in a number of iron-binding proteins such as Melling, 1993). An earlier study by Chart and Trust haemoglobin, transferrin and lactoferrin. Two major (1983) detected an inducible siderophore-mediatedtrans mechanisms for acquiring iron from the host proteins port system in the fish pathogen,Aeromonas salmonicida, have been described. The first, present in Escherichia the causative agent of furunculosis in salmonid fish. coli requires the formation of soluble factors with a high Hirst et al. (1991) later identified the siderophore as a affinity for iron such that they are able to remove it from 2,3 diphenol-catechol derivative. the host iron-binding proteins. These compounds or Against this background, we have examined the for siderophores are recognised by receptors on the bacte mation of iron-regulated outer membrane proteins rial outer membrane and together with a series of iron (iromps) of A. salmonicida as part of a continuing transport proteins they are able to translocate the iron molecular biological study of genetically-engineered into the bacterial cytoplasm. By contrast, the second antigens as furunculosis vaccine components. In order mechanism, present in some Vibrio and Neisseria spe to pursue this objective it was necessary to clone and cies, enables the bacteria to remove iron directly from map the iromp genes and to identify the individual transferrin and lactoferrin by means of outer membrane proteins. In this regard it was considered relevant that receptors which recognise these iron complexes them well-conserved regions of homology have been found selves (Guerinot, 1994). In either case, under condi in many of the siderophore receptors on the outer mem tions of iron limitation the relevant genes are derepressed branes of Gram negative organisms (Braun and Hantke, and the bacteria produce iron-regulated proteins in their 1991) a finding which has been usefully applied to the outer membranes. In view of the association of these identification of the iromps of A. salmonicida. proteins with the ability of the organisms to survive they are regarded as important virulence factors and as Materials and Methods Correspondence: Dr G. Coleman, Department of Biochemistry, Bacteria and plasmids Nottingham University Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K. Aeromonas sal montci da (Unilever 2862) (Fyfe et Fax no. 0115-9422225 al., 1988), phagemids pTZ18R and pT7T3 and host 240 R. J. Collighan, A. J. Bennett and G. Coleman Escherichia coli DH5ƒ¿ (Mead et al., 1986) were used. resuspended in 100 ƒÊl of SDS-PAGE (polyacrylamide Plasmid pITS549 was the source of the E. coli fepA gene gel electrophoresis) loading buffer and 5 ƒÊl samples (Armstrong et al., 1989). separated by SDS-PAGE unless otherwise stated. Growth of the bacteria SDS polyacrylamide gel electrophoresis (SDS-PAGE) A. salmonicida (Unilever 2862) was grown in 3% Outer membrane protein preparations were separated (w/v) Tryptone Soya Broth (TSB, Oxoid). Batches of by SDS-PAGE (Laemmli, 1970). Unless otherwise medium (50 ml) contained in 250 ml conical flasks were stated 5 and 10 ƒÊl samples of outer membrane protein loopinoculated from a culture of the organism, stored preparations grown in the presence and absence of 2,2' at 4•Ž on 4% (w/v) Tryptone Soya Agar (TSA, Oxoid), dipyridyl were run in parallel on 14 cm 5%-15% (w/v) and incubated overnight, at 25•Ž, in a refrigerated gradient gels. "orbital" incubatorshaker (Gallenkamp & Co . Ltd.). Large-scale purification of individual iromps Outer membrane protein preparation The outer membrane protein fraction was purified One hundred milliliter 3% (w/v) TSB with or without from 4 litres of A. salmonicida broth culture grown 150 mM 2,2'-dipyridyl (an iron chelator) was inoculated overnight in the presence of 150 mM 2,2'dipyridyl as with 5 ml of overnight culture of the organism grown in described above. Aliquots of the outer membranes 3% (w/v) TSB at 25•Ž, with shaking, in a refrigerated were solubilised in gelloading buffer and subjected to incubator-shaker (Gallenkamp) which, in turn, was in SDS-PAGE on a 14 cm 5-15% gradient gel when a dis oculated from an agar slope (4% TSA) stored at 4•Ž. tinct separation of the individual iromps was achieved. Following growth overnight at 25•Ž, with shaking, in The separated iromp bands were visualised by immers the presence of air, the bacterial cells were harvested by ing the gel in 0.2M potassium chloride for up to 2 min centrifugation at 4,000 x g for 10 min, at 4•Ž, and with protein bands appearing opaque against a clear washed by resuspension in 20 ml of 20 mM TrisHC1/5 background. Individual iromp bands were excised mM EDTA, pH 7.2. The washed cells were collected from the gel, pooled together and homogenised. The by centrifugation as before and maintained at 4°C in all homogenised gel slices were suspended in 50 mM po further stages of the preparation unless otherwise stated. tassium acetate, pH 7.4, with 0.1% SDS and incubated The washed cells were resuspended in 8 ml of 20 mM with shaking for 2 h, at 37•Ž, gel fragments were then TrisHCl/5 mM EDTA, pH 7.2, containing 10 jig/ml removed by centrifugation at 5,000 •~ g for 15 min and DNase and 10 ƒÊ/ml RNase and treated for 45 s in a the supernatant carefully decanted. The gel debris was "Gallenkamp" sonic oscillator (using the medium probe) washed once more in the same way and the supernatant which lysed most of the cells and released the mem fractions pooled. The solutions containing purified branes as vesicles. After chilling on ice for 5 min the iromps were then dialysed against distilled water and lysate was centrifuged at 8,000 •~ g for 10 min, at 4•Ž, to finally vacuum desiccated. The purified iromps were remove whole cells and other debris. Then the super stored in this state at -20•Ž until required. natant fraction was carefully decanted into a 10 ml ultracentrifuge tube, avoiding the sediment. Production of polyclonal anti-iromp serum The resulting preparation was centrifuged at 30,000 x A preparation containing equal amounts of the three g for 45 min, at 4•Ž, to pellet the membrane vesicles A. salmonicida iromps was obtained using the previous and the supernatant fraction was decanted from the small purification protocol. One hundred microgram of the clear pellet which was resuspended in 4.5 ml of 20 mM preparation, in a volume of 500 µl, mixed with an equal TrisHC1/5 mM EDTA and 0.5 ml of 5% (w/v) "Sarkosyl" volume of Freund's incomplete adjuvant, were injected (sodium N-lauroylsarcosinate, Sigma). The preparation intramuscularly into rabbits at 2 week intervals over a 6 was then incubated at room temperature for 20 min, with week period. Test bleeds were carried out on days 14, frequent mixing, to selectively solubilise the inner mem 28 and 42 to assess the immune response to the iromps brane. The outer membrane was recovered by centrifu by Western blotting. Exsanguination was performed gation at 30,000 •~ g for 45 min at 4•Ž. on the 56th day after the initial inoculation. After leav After decanting the supernatant fraction and remov ing the blood to clot overnight at 4•Ž, the serum was ing all traces from the walls of the centrifuge tube, using decanted from the clot and was stored frozen at -20•Ž a piece of Kleenex tissue, the outer membrane was until required. Identification of A. salmonicida iromps 241 Western blotting with iromp antibody Southern hybridization analysis was carried out The procedure described by Towbin and Gordon using the iromp probes described above, and the DNA (1984) was followed using the polyclonal anti-iromp digest fragments identified were excised in bands of gel serum. and isolated by binding to glassmilk using a "Geneclean II" kit (Bio 101 Inc. from Stratech Scientific Ltd.). Amino acid sequence analysis The DNA fragments isolated were ligated into the The iromp running as an approximately 74 kDa band multiple cloning site of phagemid pTZ18R and the re on SDS-PAGE of an outer membrane protein preparation combinants transformed into competent CaCl2-treated was excised after transblotting onto "ProBlott" mem E. coli strain DH5ƒ¿ (Sambrook et al., 1989). The brane (Applied Biosystems Inc.) and loaded into a Pro transformed bacteria were plated out on L-broth agar tein Sequencer (model 473A, Applied Biosystems Inc.) containing 50 ƒÊg/ml ampicillin (Kaiser and Murray, for Nterminal amino acid analysis. An unambiguous 1985) to select for phagemidcontaining colonies whilst decapeptide was obtained, namely, ELPKANETMV.
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