1887

Microbiology

Volume 151, Issue 9

Identification and expression of a host-recognized antigen, FspA, from Free

Abstract

is the aetiological agent of rainbow trout fry syndrome, an economically important disease of immature salmonid fish for which there is no vaccine. Convalescent serum from the host, rainbow trout (), reacted strongly with a ∼20 kDa, -specific protein antigen (subsequently named FspA) from . Protein-enriched, detergent-partitioned samples were separated by two-dimensional gel electrophoresis and the protein target was excised, proteolytically cleaved and the resulting peptides analysed by MS. Quadrupole-time-of-flight MS was used to generate a fragmented peptide spectrum. The resulting peptide sequences were then used to design degenerate PCR primers to amplify the gene () of interest: 612 bp encoding 203 aa, including a putative 19 aa N-terminal signal sequence which predicted a processed 19 303·6 Da protein. FspA proved to be unique and only homologous to two unspecified sequences reported from , although weakly homologous to a adhesin. An amplified gene fragment (537 bp, encoding 179 aa) was further cloned into an expression vector, expressed as a ∼30 kDa N-terminal fusion protein and found to retain its strong reactivity with host serum antibodies. These results suggest that the surface-localized FspA may be an important subunit vaccine candidate antigen against .

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): dPCR, degenerate PCR and Q-TOF, quadrupole time-of-flight
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2005-09-01
2021-10-18
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References

  1. Agarwal S., Hunnicutt D. W., McBride M. J. 1997; Cloning and characterization of the Flavobacterium johnsoniae ( Cytophaga johnsonae ) gliding motility gene, gldA . Proc Natl Acad Sci 94:12139–12144 [CrossRef]
     [Google Scholar]
  2. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
     [Google Scholar]
  3. Ames G. F.-L. 1974; Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. J Biol Chem 249:634–644
     [Google Scholar]
  4. Anderson N. G., Anderson N. L. 1978a; Analytical techniques for cell fractions. XXI. Two-dimensional analysis of serum and tissue proteins: multiple isoelectric focusing. Anal Biochem 85:331–340 [CrossRef]
     [Google Scholar]
  5. Anderson N. L., Anderson N. G. 1978b; Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis. Anal Biochem 85:341–354 [CrossRef]
     [Google Scholar]
  6. Bannai H., Tamada Y., Maruyama O., Nakai K., Miyano S. 2002; Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18:298–305 [CrossRef]
     [Google Scholar]
  7. Baudin-Laurencin F., Castric J.-C., Vigneulle M., Tixernat G. 1989; La myxobacteriose viscerale de la truie arc-en-ciel Salmo gardneri R: une forme nouvelle de la maladie de l'eau froide a Cytophaga psychrophila . Bull Acad Vet Fr 62:147–157
     [Google Scholar]
  8. Berkelman T., Stenstedt T. 1998 2-D Electrophoresis: Principles and Methods Uppsala, Sweden: Amersham Biosciences;
     [Google Scholar]
  9. Bernardet J.-F., Baudin-Laurencin F., Tixernat G. 1988; First identification of “ Cytophaga psychrophila ” in France. Bull Eur Ass Fish Pathol 8:104–105
     [Google Scholar]
  10. Borg A. F. 1960; Studies on myxobacteria associated with diseases in salmonid fishes. J Wildl Dis 8:1–85
     [Google Scholar]
  11. Bruno D. W. 1992; Cytophaga psychrophila (=‘ Flexibacter psychrophilus ’) (Borg), histopathology associated with mortalities among farmed rainbow trout, Oncorhynchus mykiss (Walbaum) in the U.K. Bull Eur Ass Fish Pathol 12:215–216
     [Google Scholar]
  12. Chen X., Wu R. 1997; Direct amplification of unknown genes and fragments by uneven polymerase chain reaction. Gene 185:195–199 [CrossRef]
     [Google Scholar]
  13. Collinson S. K., Kay W. W, Emödy L., Müller K.-H., Trust T. J. 1991; Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis . J Bacteriol 173:4773–4781
     [Google Scholar]
  14. Crump E. M., Perry M. B., Clouthier S. C., Kay W. W. 2001; Antigenic characterization of the fish pathogen Flavobacterium psychrophilum . Appl Environ Microbiol 67:750–759 [CrossRef]
     [Google Scholar]
  15. Crump E. M., Perry M. B., Gale S., Crawford E., Kay W. W. 2003; Lipopolysaccharide O-antigen antibody-based detection of the fish pathogen Flavobacterium psychrophilum . J Mol Microbiol Biotechnol 6:182–190 [CrossRef]
     [Google Scholar]
  16. Cunningham T. M., Walker E. M., Miller J. N., Lovett M. A. 1988; Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114. J Bacteriol 170:5789–5796
     [Google Scholar]
  17. Davis H. S. 1946 Care and diseases of trout. US Department of the Interior Research Report no. 12 Washington, DC: US Government Printing Office;
     [Google Scholar]
  18. Hsueh P. R., Hsiue T. R., Wu J. J., Teng L. J., Ho S. W., Hsieh W. C., Luh K. T. 1996; Flavobacterium indologenes bacteremia: clinical and microbiological characteristics. Clin Infect Dis 23:550–555 [CrossRef]
     [Google Scholar]
  19. Iida Y., Mizokami A. 1996; Outbreaks of coldwater disease in wild ayu and pale chub. Fish Pathol 31:157–164 [CrossRef]
     [Google Scholar]
  20. Inglis V., Roberts R. J., Bromage N. R. 1993 Bacterial Diseases of Fish Oxford: Blackwell Scientific Publications;
     [Google Scholar]
  21. Kaattari S. L., Piganelli J. D. 1996; The specific immune system: humoral defense. In The Fish Immune System: Organism, Pathogen, and Environment pp 207–254 Edited by Iwama G. K., Nakanishi T. Toronto: Academic Press;
     [Google Scholar]
  22. Kinter M., Sherman N. E. 2000 Protein Sequencing and Identification Using Tandem Mass Spectrometry New York: Wiley-Interscience;
     [Google Scholar]
  23. Kuzyk M. A., Burian J., Machander D., Dolhaine D., Cameron S., Thornton J. C., Kay W. W. 2001; An efficacious recombinant subunit vaccine against the salmonid rickettsial pathogen Piscirickettsia salmonis . Vaccine 19:2337–2344 [CrossRef]
     [Google Scholar]
  24. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:154–159 [CrossRef]
     [Google Scholar]
  25. LaFrentz B. R., LaPatra S. E., Jones G. R., Cain K. D. 2004; Protective immunity in rainbow trout Oncorhynchus mykiss following immunization with distinct molecular mass fractions isolated from Flavobacterium psychrophilum . Dis Aquat Organ 59:17–26 [CrossRef]
     [Google Scholar]
  26. Lehman J., Mock D., Sturenberg F. J., Bernardet J.-F. 1991; First isolation of Cytophaga psychrophila from a systematic disease in eel and cyprinids. Dis Aquat Organ 10:217–220 [CrossRef]
     [Google Scholar]
  27. Lorenzen E. 1994 Studies on Flexibacter Psychrophilus in Relation to Rainbow Trout Fry Syndrome (RTFS) Copenhagen, Denmark: Royal Veterinary and Agricultural University;
     [Google Scholar]
  28. Lorenzen E., Dalsgaard I., From J., Hansen E. M., Hørlyck V., Korsholm H., Mellergaard S., Olesen N. J. 1991; Preliminary investigations of fry mortality syndrome in rainbow trout. Bull Eur Ass Fish Pathol 11:77–79
     [Google Scholar]
  29. Massias B., Dumetz F., Urdaci M. C., Le Henaff M. 2004; Identification of P18, a surface protein produced by the fish pathogen Flavobacterium psychrophilum . J Appl Microbiol 97:574–580 [CrossRef]
     [Google Scholar]
  30. Merle C., Faure D., Urdaci M. C., Le Henaff M. 2003; Purification and characterization of a membrane glycoprotein from the fish pathogen Flavobacterium psychrophilum . J Appl Microbiol 94:1120–1127 [CrossRef]
     [Google Scholar]
  31. Müller K.-H., Trust T. J., Kay W. W. 1989; Fimbriation genes of Salmonella enteritidis . J Bacteriol 171:4648–4654
     [Google Scholar]
  32. Neuhoff V., Arold N., Taube D., Ehrhardt W. 1988; Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255–262 [CrossRef]
     [Google Scholar]
  33. Nielsen H., Engelbrecht J., Brunak S., von Heijne G. 1997; Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6 [CrossRef]
     [Google Scholar]
  34. Ostland V. E., McGrogan D. G., Ferguson H. W. 1997; Cephalic osteochondritis and necrotic scleritis in intensively reared salmonids associated with Flexibacter psychrophilus . J Fish Dis 20:443–451 [CrossRef]
     [Google Scholar]
  35. Pearson W. R. 1998; Empirical statistical estimates for sequence similarity searches. J Mol Biol 276:71–84 [CrossRef]
     [Google Scholar]
  36. Pennington K., McGregor E., Beasley C. L., Everall I., Cotter D., Dunn M. J. 2004; Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue. Proteomics 4:27–30 [CrossRef]
     [Google Scholar]
  37. Rahman M. H., Kuroda A., Dijkstra J. M., Kiryu I., Nakanishi T., Ototake M. 2002; The outer membrane fraction of Flavobacterium psychrophilum induces protective immunity in rainbow trout and ayu. Fish Shellfish Immunol 12:169–179 [CrossRef]
     [Google Scholar]
  38. Sambrook J., Maniatis T., Fritsch E. F. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  39. Santos Y., Huntly P. J., Turnbull A., Hastings T. S. 1992; Isolation of Cytophaga psychrophila ( Flexibacter psychrophilus ) in association with rainbow trout mortality in the United Kingdom. Bull Eur Ass Fish Pathol 12:209–210
     [Google Scholar]
  40. Sarti M., Giorgetti G., Manfrin A. 1992; Method for the rapid diagnosis of visceral myxobacteriosis in reared trout in Italy. Bull Eur Ass Fish Pathol 12:53
     [Google Scholar]
  41. Schmidtke L. M., Carson J. 1995; Characteristics of Flexibacter psychrophilus isolated from Atlantic salmon in Australia. Dis Aquat Organ 21:157–161 [CrossRef]
     [Google Scholar]
  42. Siegman-Igra Y., Schwartz D., Soferman G., Konforti N. 1987; Flavobacterium group IIb bacteremia: report of a case and review of Flavobacterium infections. Med Microbiol Immunol 176:103–111
     [Google Scholar]
  43. Toranzo A. E., Barja J. L. 1993; Fry mortality syndrome (FMS) in Spain. Isolation of the causative bacterium Flexibacter psychrophilus . Bull Eur Ass Fish Pathol 13:30–31
     [Google Scholar]
  44. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76:4350–4354 [CrossRef]
     [Google Scholar]
  45. Vancanneyt M., Segers P., Hauben L., Hommez J., Devriese L. A., Hoste B., Vandamme P., Kersters K. 1994; Flavobacterium meningosepticum , a pathogen in birds. J Clin Microbiol 32:2398–2403
     [Google Scholar]
  46. Wakabayashi H., Horiuchi M., Bunta T., Hoshiai G. 1991; Outbreaks of cold-water disease in coho salmon in Japan. Fish Pathol 26:211–212 [CrossRef]
     [Google Scholar]
  47. Wiklund T., Kaas K., Lonnstrom L., Dalsgaard I. 1994; Isolation of Cytophaga psychrophila ( Flexibacter psychrophilus ) from wild and farmed rainbow trout ( Oncorhynchus mykiss ) in Finland. Bull Eur Ass Fish Pathol 14:44–46
     [Google Scholar]
  48. Xie H. X., Nie P., Sun B. J. 2004; Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G4. J Fish Dis 27:719–729 [CrossRef]
     [Google Scholar]
  49. Yang Y., Merriam J. J., Mueller J. P., Isberg R. R. 1996; The psa locus is responsible for thermoinducible binding of Yersinia pseudotuberculosis to cultured cells. Infect Immun 64:2483–2489
     [Google Scholar]
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Identification and expression of a host-recognized antigen, FspA, from Flavobacterium psychrophilum
Microbiology 151, 3127 (2005); https://doi.org/10.1099/mic.0.28140-0
/content/journal/micro/10.1099/mic.0.28140-0
/content/journal/micro/10.1099/mic.0.28140-0
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