Archives of Microbiology

, Volume 194, Issue 9, pp 795–802 | Cite as

A TonB-dependent outer membrane receptor of Pseudomonas fluorescens: virulence and vaccine potential

Short Communication


Pseudomonas fluorescens is a Gram-negative bacterium and a common aquaculture pathogen. In this study, we identified from a pathogenic P. fluorescens strain a TonB-dependent outer membrane receptor, TdrA, as a secreted protein and examined its function and vaccine potential. TdrA is composed of 746 residues and possesses conserved structural domains of TonB-dependent outer membrane receptors. Quantitative real-time reverse transcriptase-PCR analysis showed that expression of tdrA was upregulated under conditions of iron starvation and during infection of host cells. Consistently, iron depletion induced increased production of TdrA protein in the outer membrane. Compared to the wild type, a tdrA-knock out mutant (1) was unable to grow in the absence of iron, (2) exhibited drastically attenuated overall bacterial virulence, and (3) was impaired in the ability to establish lethal infection in host tissues. Purified recombinant TdrA (rTdrA), when used as a subunit vaccine to immunize flounder, was able to induce strong protective immunity, including production of serum-specific antibodies that resulted in effective protection against lethal-dose P. fluorescens challenge. Together, these results indicate that TdrA is an outer membrane receptor and a protective immunogen that is likely to be involved in iron acquisition and, as a result, required for optimal bacterial virulence.


Pseudomonas fluorescens TonB-dependent receptor Virulence Vaccine 


  1. Afonina G et al (2006) Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA protects against infection in the swine model of chancroid. Infect Immun 74:2224–2232PubMedCrossRefGoogle Scholar
  2. Alvarez B, Alvarez J, Menendez A, Guijarro JA (2008) A mutant in one of two exbD loci of a TonB system in Flavobacterium psychrophilum shows attenuated virulence and confers protection against cold water disease. Microbiology 154:1144–1151PubMedCrossRefGoogle Scholar
  3. Amend DF (1981) Potency testing of fish vaccines. International symposium on fish biologics: serodiagnostics and vaccines. Dev Biol Stand 49:447–454Google Scholar
  4. Andrews SC, Robinson AK, Rodríguez-Quiñones F (2008) Bacterial iron homeostasis. FEMS Microbiol Rev 27:215–237CrossRefGoogle Scholar
  5. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  6. Braun V (2006) Energy transfer between biological membranes. ACS Chem Biol 1:352–354PubMedCrossRefGoogle Scholar
  7. Chimento DP, Kadner RJ, Wiener MC (2003) The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation. J Mol Biol 332:999–1014PubMedCrossRefGoogle Scholar
  8. Datta S, Crosa JH (2011) Identification and characterization of a novel outer membrane protein receptor required for hemin utilization in Vibrio vulnificus. Biometals. doi:10.1007/s10534-011-9501-y PubMedGoogle Scholar
  9. Deslandes V, Denicourt M, Girard C, Harel J, Nash JH, Jacques M (2010) Transcriptional profiling of Actinobacillus pleuropneumoniae during the acute phase of a natural infection in pigs. BMC Genomics 11:98PubMedCrossRefGoogle Scholar
  10. Fusco WG et al (2010) Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA with adjuvant monophosphoryl lipid A protects swine from a homologous but not a heterologous challenge. Infect Immun 78:3763–3772PubMedCrossRefGoogle Scholar
  11. Geng X, Ding S, Sun H, Xie J (2006) Immunogenicity and protective effects of Pseudomonas fluorescens vaccines on grass carp. J Southwest Agric Univ (Natural Science) 28: 120–123 (in Chinese, with English abstract)Google Scholar
  12. Hu YH, Zheng WJ, Sun L (2010) Identification and molecular analysis of a ferritin subunit from red drum (Sciaenops ocellatus). Fish Shellfish Immunol 28:678–686PubMedCrossRefGoogle Scholar
  13. Jiao XD, Cheng S, Hu YH, Sun L (2010) Comparative study of the effects of aluminum adjuvants and Freund’s incomplete adjuvant on the immune response to an Edwardsiella tarda major antigen. Vaccine 28:1832–1837PubMedCrossRefGoogle Scholar
  14. Koebnik R (2005) TonB-dependent trans-envelope signaling: the exception or the rule? Trends Microbiol 13:343–347PubMedCrossRefGoogle Scholar
  15. Krewulak KD, Vogel HJ (2011) TonB or not TonB: is that the question? Biochem Cell Biol 89:87–97PubMedCrossRefGoogle Scholar
  16. Leduc I et al (2011) Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid. Infect Immun 79:3168–3177PubMedCrossRefGoogle Scholar
  17. López JR et al (2011) Pseudomonas baetica sp. nov., a novel fish pathogen isolated from wedge sole, Dicologoglossa cuneata (Moreau). Int J Syst Evol Microbiol doi. doi:10.1099/ijs.0.030601-0 Google Scholar
  18. Milton DL, O’Toole R, Hörstedt P, Wolf-Watz H (1996) Flagellin A is essential for the virulence of Vibrio anguillarum. J Bacteriol 178:1310–1319PubMedGoogle Scholar
  19. Neilands JB (1995) Siderophores: structure and function of microbial iron transport compounds. J Biol Chem 270:26723–26726PubMedGoogle Scholar
  20. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  21. Shu XH, Jin XL, Xiao KY, Jin H (1997) Studies on the pathogenic bacteria of the Rotten-skin and Red-leg disease of the bullfrog. Life Sci Res 1:54–59 (in Chinese with English abstract)Google Scholar
  22. Stork M et al (2010) An outer membrane receptor of Neisseria meningitidis involved in zinc acquisition with vaccine potential. PLoS Pathog 6:e1000969PubMedCrossRefGoogle Scholar
  23. Sun K, Wang HR, Zhang M, Xiao Z, Sun L (2009a) Genetic mechanisms of multi-antimicrobial resistance in a pathogenic Edwardsiella tarda strain. Aquaculture 289:134–139CrossRefGoogle Scholar
  24. Sun K, Zhang WW, Hou JH, Sun L (2009b) Immunoprotective analysis of VhhP2, a Vibrio harveyi vaccine candidate. Vaccine 27:2733–2740PubMedCrossRefGoogle Scholar
  25. Sun Y, Liu CS, Sun L (2011) A multivalent killed whole-cell vaccine induces effective protection against Edwardsiella tarda and Vibrio anguillarum. Fish Shellfish Immunol 31:595–599PubMedCrossRefGoogle Scholar
  26. Swain P, Nayak SK, Sahu A, Meher PK, Mishra BK (2003) High antigenic cross-reaction among the bacterial species responsible for diseases of cultured freshwater fishes and strategies to overcome it for specific serodiagnosis. Comp Immunol Microbiol Infect Dis 26:199–211PubMedCrossRefGoogle Scholar
  27. Swain P, Behura A, Dash S, Nayak SK (2007) Serum antibody response of Indian major carp, Labeo rohita to three species of pathogenic bacteria; Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens. Vet Immunol Immunopathol 117:137–141PubMedCrossRefGoogle Scholar
  28. Tauseef I et al (2011) Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence. Microbiology 157:1446–1456PubMedCrossRefGoogle Scholar
  29. Tong SL, Li H, Miao HZ (1997) The establishment and partial characterization of a continuous fish cell line FG-9307 from the gill of flounder Paralichthys olivaceus. Aquaculture 56:327–333CrossRefGoogle Scholar
  30. Wang GX, Bai ZT, Zhang XQ, Zhang LL (1999) Study on the Redskin disease in Andrias daridianus. J Northeast Agric Univ 27:71–74Google Scholar
  31. Wang HR, Hu YH, Zhang WW, Sun L (2009) Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine. Vaccine 27:4047–4055PubMedCrossRefGoogle Scholar
  32. Wiener MC (2005) TonB-dependent outer membrane transport: going for Baroque? Curr Opin Struct Biol 15:394–400PubMedCrossRefGoogle Scholar
  33. Yan BF (2005) Improvements on the preventive methods of Saprolegniasis and Redskin diseases. Chin Aquacult 1:49–50 (in Chinese)CrossRefGoogle Scholar
  34. Zhang WW, Sun L (2007) Cloning, characterization and molecular application of a beta-agarase gene from Vibrio sp. strain V134. Appl Environ Microbiol 73:2825–2831PubMedCrossRefGoogle Scholar
  35. Zheng WJ, Sun L (2011) Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder (Paralichthys olivaceus). Fish Shellfish Immunol 30:638–645PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.Key Laboratory of Experimental Marine Biology, Institute of OceanologyChinese Academy of SciencesQingdaoPeople’s Republic of China
  2. 2.Hangzhou Key Laboratory for Animal Adaptation and Evolution, College of Life and Environmental SciencesHangzhou Normal UniversityHangzhouPeople’s Republic of China
  3. 3.Graduate University of the Chinese Academy of SciencesBeijingPeople’s Republic of China

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