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

Abstract

Pseudomonas fluorescens is a common bacterial pathogen to a wide range of aquaculture animals including various species of fish. In this study, we employed proteomic analysis and identified filamentous hemagglutinin (FHA) as an iron-responsive protein secreted by TSS, a pathogenic P. fluorescens isolate. In vitro study showed that compared to the wild type, the fha mutant TSSfha (i) exhibited a largely similar vegetative growth profile but significantly retarded in the ability of biofilm growth and producing extracellular matrix, (ii) displayed no apparent flagella and motility, (iii) was defective in the attachment to host cells and unable to form self-aggregation, (iv) displayed markedly reduced capacity of hemagglutination and surviving in host serum. In vivo infection analysis revealed that TSSfha was significantly attenuated in the ability of dissemination in fish tissues and inducing host mortality, and that antibody blocking of the natural FHA produced by the wild type TSS impaired the infectivity of the pathogen. Furthermore, when introduced into turbot as a subunit vaccine, recombinant FHA elicited a significant protection against lethal TSS challenge. Taken together, these results indicate for the first time that P. fluorescens FHA is a key virulence factor essential to multiple biological processes associated with pathogenicity.

Keywords: Pseudomonas fluorescens; adhesion; filamentous hemagglutinin; motility; vaccine; virulence.

FIGURE 1

Representative 2-DE maps of the extracellular protein profiles of Pseudomonas fluorescens TSS cultured under different conditions. Extracellular proteins of P. fluorescens TSS cultured in the presence (B) and absence of 2,2′-dipyridyl (DP) (A) were subjected to 2-DE analysis. Numbers indicate protein spots with differential expression. The spot of Pf_Fha (number 6) is indicated.

FIGURE 2

Motility and flagella formation of Pseudomonas fluorescens TSS and TSSfha. (A) TSS (left) and TSSfha (right) were cultured in LB medium to an OD₆₀₀ of 1.0, and 5 μl cell suspensions were spotted onto the center of swimming plates containing LB medium plus 0.3% (w/v) agar. The plates were incubated at 28°C for 2 days. (B) TSS (left) and TSSfha (right) were cultured in LB agar plates and examined with a transmission electron microscope. Scale bar, 1 μm. The results represent one of three independent experiments.

FIGURE 3

Autoaggregation of Pseudomonas fluorescens TSS and TSSfha. (A) TSS (left) and TSSfha (right) were cultured in LB medium at 28°C to an OD₆₀₀ of 0.8; 100 microliters of cell suspensions were added into 96-well microplates and incubated overnight (Top), or the cell cultures were left standing for 2 days at room temperature without shaking (Bottom). (B) Autoaggregation observed by scanning electron microscope (SEM). TSS and TSSfha were cultured in LB medium at 28°C to an OD₆₀₀ of 0.8; cell suspensions were added onto coverslips and observed by SEM after incubating for 8 h at room temperature. The pictures represent one of three independent experiments.

FIGURE 4

Biofilm formation of Pseudomonas fluorescens TSS and TSSfha. (A) Image of crystal violet (CV)-stained biofilms formed inside glass tubes. Cells were grown in LB medium overnight at 28°C and then stood for 2 days at room temperature before staining with CV. Image shown is from one representative experiment of three independent experiments. (B) Quantitation of biofilm. Stained biofilms were dissolved in 30% acetic acid, and the optical density at OD₅₇₀ was recorded. Data are the means of three independent assays and presented as mean ± SEM. ^∗∗P < 0.01.

FIGURE 5

Production of extracellular matrix structure by Pseudomonas fluorescens TSS and TSSfha. TSS (A,B) and TSSfha (C,D) were cultured in LB medium at 28°C to an OD₆₀₀ of 0.8; cell suspensions were added onto coverslips and observed by a scanning electron microscope after incubation for 20 h at room temperature. The results represent one of three independent experiments.

FIGURE 6

Binding of rFha to FG-9307 cells. FG cells were incubated with rFha (A,B) or the control protein rTrx (D,E), and the cell-bound protein was detected with FITC-labeled antibody. The cells were stained with DAPI and observed with a fluorescence microscope. (C) A merge of (A) and (B); (F) A merge of (D) and (E). Magnifications: 20 × 10, scale bars: 50 μm.

FIGURE 7

Hemagglutination of Pseudomonas fluorescens TSS and TSSfha. Turbot red blood cells were incubated with PBS (control) or with TSS and TSSfha in various concentrations for 1 h at room temperature, and hemagglutination was observed. The results represent one of three independent experiments.

FIGURE 8

In vivo infectivity of Pseudomonas fluorescens TSS and TSSfha. (A) TSS and TSSfha were inoculated into turbot, and bacterial recovery from kidney (left) and spleen (right) was determined at different times. The results are the means of three independent experiments and presented as mean ± SEM. ^∗∗P < 0.01. (B) Turbot were infected with TSS and TSSfha, and the fish were monitored daily for mortality and survival for 20 days (only 12 days are shown in the figure). The results are the means of three independent experiments. Significance between the survivals of wild type- and mutant-infected fish was determined with logrank test. ^∗∗P < 0.01.

FIGURE 9

Effect of rFha antibody on Pseudomonas fluorescens infection. Turbot were infected with TSS in the presence or absence of rFha antibody, control antibody, or PBS (control). Bacterial recovery from kidney (A) and spleen (B) was determined at different hours post-infection. The results are the means of three independent experiments and presented as mean ± SEM. ^∗∗P < 0.01.

FIGURE 10

Protective effect of rFha as a vaccine. Turbot were vaccinated with or without (control) rFha and challenged with P. fluorescens TSS at 1 month post-vaccination. The fish were monitored daily for survival. Significances between the survivals of the vaccinated fish and the control fish were determined with logrank test. ^∗∗P < 0.01.