title: Identification and analysis of three virulence-associated TonB-dependent outer membrane receptors of Pseudomonas fluorescens.
abstract: Pseudomonas fluorescens is a Gram-negative bacterium that can infect a wide range of farmed fish. However, very little is known about the virulence mechanism of P. fluorescens as a fish pathogen. In this study, we identified and analyzed 3 TonB-dependent outer membrane receptors (TDRs) from a pathogenic P. fluorescens strain isolated from fish. In silico analysis revealed that all 3 proteins (named Tdr1 to 3) possess structural domains typical of TDRs. Quantitative real time RT-PCR analysis showed that tdr1, tdr2, and tdr3 expressions were upregulated under iron-depleted conditions. Compared to the wild type, mutants defective in tdr1, tdr2, and tdr3 were retarded in growth to different extents. Infection in a turbot Scophthalmus maximus model showed that all 3 mutants were impaired in their ability to desseminate into and colonize host tissues. In addition, the tdr1 and tdr3 mutants exhibited significantly reduced virulence. When used as subunit vaccines, purified recombinant proteins of Tdr1, Tdr2, and, in particular, Tdr3 elicited significant protection in turbot against lethal P. fluorescens challenge. The vaccinated fish produced specific serum antibodies, which, when incubated with P. fluorescens, blocked infection of P. fluorescens in fish cells. Together these results indicate that Tdr1, Tdr2, and Tdr3 are iron-regulated factors that participate in bacterial virulence and induce protective immunity as subunit vaccines.
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, PR China.
authors.forename: Shu-ren
authors.initials: SR
authors.lastname: Zhang
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.forename: Li
authors.initials: L
authors.lastname: Sun
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001428:Bacterial Vaccines / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005393:Fish Diseases / Q000517:prevention & control; D005412:Flatfishes; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011552:Pseudomonas Infections / Q000517:prevention & control / Q000662:veterinary; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014774:Virulence
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D008565:Membrane Proteins; C057616:tonB protein, Bacteria