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.

Quantification and comparison of gene expression associated with iron regulation and metabolism in a virulent and attenuated strain of Flavobacterium psychrophilum

“…2016). In Pseudomonas fluorescens , these TonB receptors were upregulated under iron‐limiting conditions and TonB proteins are also being investigated in potential subunit vaccine candidates (Zhang et al., 2014). In the current study, the TBomr1 expression was found to be decreased in the B.17 strain at 96‐hr post‐iron‐limiting media exposure.…”

“…Bacterial strains that overproduce siderophores may be considered hypervirulent, and these siderophores can serve as toxins and immunomodulators (Holden & Bachman, 2015). Siderophores with attached iron are brought into the bacterial cell via outer membrane receptors (OMRs; Andrews et al., 2003; Zhang et al., 2014). The energy required to move this sequestered iron into the bacterial cell is provided by the TonB protein system, and it has been found that all Gram‐negative bacteria have at least one TonB protein (Beddek et al., 2004; Chu et al., 2010).…”

The Role of TonB Gene in Edwardsiella ictaluri Virulence

“…In the present study, catfish experiments demonstrated a 2.16-fold reduction in Ei Δ tonB virulence compared with wild-type E. ictaluri . Similarly, P. fluorescens mutants defective in the TonB-dependent outer membrane receptor (TDRs) tdr1, tdr2, and tdr3, which had 26.7, 22.3, and 24.5% mean percent mortalities, respectively, compared with 70% mortality caused by the parent strain in a turbot ( Seophthalmus maximus ) model fish (Zhang et al, 2014 ).…”

Pseudomonas fluorescens Filamentous Hemagglutinin, an Iron-Regulated Protein, Is an Important Virulence Factor that Modulates Bacterial Pathogenicity

“…Mutation of Pf Fha in TSS fha was confirmed by PCR analysis. In addition, single-copy plasmid insertion in TSS fha was further confirmed by the quantitative real-time PCR (qRT-PCR) method described previously (Zhang et al, 2014). …”