Experiments involving live animals were conducted in accordance with the “Regulations for the Administration of Affairs Concerning Experimental Animals” promulgated by the State Science and Technology Commission of Shandong Province. The study was approved by the ethics committee of Institute of Oceanology – Chinese Academy of Sciences.

Pseudomonas fluorescens TSS is a pathogenic fish isolate that has been reported previously (Wang et al., 2009). Escherichia coli BL21(DE3) and DH5α were purchased from TransGen Biotech (Beijing, China). E. coli S17-1 λpir was purchased from Biomedal. All strains were grown in Luria-Bertani broth (LB) at 37°C (for E. coli) or 28°C (for TSS). Where indicated, 2,2′-dipyridyl (Sigma, St. Louis, MO, USA), tetracycline, and chloramphenicol were added at the concentrations of 600 μM, 20 and 50 μg/ml, respectively.

Two-dimensional gel electrophoresis, MALDI-TOF/TOF-MS, and protein identification were performed as reported previously (Liu et al., 2015).

The sequence of P. fluorescens fha has been reported previously (GenBank accession no. WP_014719704.1). The amino acid sequence was analyzed using the BLAST program at the National Center for Biotechnology Information (NCBI) and the Expert Protein Analysis System. Domain search was performed with the conserved domain search program of NCBI. Subcellular localization prediction was performed with the PSORTb v.3.0 server.

In order to create TSSfha, we first created the plasmid p705T, which was used for gene mutagenesis, as follows: the tetracycline-resistance gene of p704T (Hu et al., 2009) was cut out by SmaI/ScaI double digestion and inserted into pGP704 (Miller and Mekalanos, 1988) at the ScaI site. To construct the Pf_Fha-defective strain TSSfha, the internal fragment of Pf_Fha (positions 241–408) was amplified by PCR with the primer pairs F (5′-AGATCTGTGGTGTTGAACAACGCCT-3′, underlined se-quence, BglII site) and R (5′-AGATCTATCGGCCGCCTGGCCGAA-3′, underlined sequence, BglII site). The PCR product was inserted into the suicide plasmid p705T at the compatible BglII site, resulting in p705Fha. S17-1 λpir was transformed with p705Fha, and the transformant was conjugated with P. fluorescens TSS as described previously (Sun et al., 2009). The transconjugant was selected on LB agar plates supplemented with tetracycline and chloramphenicol, and one of the resistant clones was named TSSfha. Mutation of Pf_Fha in TSSfha was confirmed by PCR analysis. In addition, single-copy plasmid insertion in TSSfha was further confirmed by the quantitative real-time PCR (qRT-PCR) method described previously (Zhang et al., 2014).

To construct TSSΔfha, in-frame deletion of a 582 bp segment (positions 40–621) of Pf_Fha was performed by overlap extension PCR as follows: the first overlap PCR was performed with the primers F2 (5′-CCCGGGAACTGGCCTACAAAGACGT-3′, underlined sequence, SmaI site) and R2 (5′-CGACCTTCCTGGGGTGAAAGGTGGA-3′), the second overlap PCR was performed with the primers F3 (5′-CACCCCAGGAAGGTCGCCTCAGTGCTCG-3′) and R3 (5′-CCCGGGGGTGATGCTGCGTTGTTCG-3′, underlined sequence, SmaI site), and the fusion PCR was performed with the primer pair F2/R3. The PCR products were inserted into the suicide plasmid p7TS (Wang et al., 2009) at the SmaI site, resulting in p7TSFha. p7TSFha was introduced into S17-1 λpir (Biomedal, Spain) by transformation. The transformant S17-1 λpir/p7TSFha was conjugated with TSS. The transconjugants were selected first on LB plates supplemented with tetracycline and chloramphenicol and then on LB plates supplemented with 12% sucrose and chloramphenicol. The colonies that appeared on the plates were analyzed by PCR, and the PCR products were subjected to sequence analysis to confirm deletion of Pf_Fha.

FG-9307 cells, a cell line established from Japanese flounder gill cells (Tong et al., 1997), were cultured at 22°C in 96-well cell culture plates (∼10⁵ cell/well) with L-15 medium (GIBCO, Invitrogen, Carlsbad, CA, USA) as described previously (Tong et al., 1997). TSS and TSSfha were cultured in LB medium to an OD₆₀₀ of 0.8 and were resuspended in L-15 medium to 1 × 10⁷ CFU/ml. One hundred microliter of bacterial suspension was added to FG cells cultured as above. The plates were incubated at 28°C for 1, 2, and 4 h, followed by washing three times with PBS. FG cells were then lysed with 1% Triton X-100, and 50 μl lysate was plated in triplicate on LB agar plates. The plates were incubated at 28°C for 48 h, and the colonies that appeared on the plates were enumerated. The genetic identity of the colonies was verified by PCR and sequence analysis of selected PCR products. The experiment was performed three times.

For autoaggregation analysis, TSS and TSSfha were cultured at 28°C in test tubes containing LB broth in a shaking incubator to an OD₆₀₀ of 0.8. The tubes were removed from the shaker, and 100 microliters of cell suspension were added into 96-well microplates and incubated overnight. Meanwhile, the static cultures in the test tubes were monitored for sedimentation for 48 h. Bacterial autoaggregation in microplates and glass tubes were then examined. For microscopy, TSS and TSSfha were cultured as above, 100 microliters of cell suspension were added into a 12-well plate with embedded coverslips for 8 h at 28°C. Autoaggregation of cells on coverslips were photographed with a scanning electron microscope (S-3400N, Hitachi, Japan; Wang and Sun, 2015). For extracellular matrix analysis, TSS and TSSfha were cultured as above, 100 microliters of cell suspension were added into a 12-well culture plate with embedded coverslips for 20 h at 28°C. The cells were then observed with a scanning electron microscope. All experiments were performed three times.

To measure motility, TSS, TSSfha, and TSSΔfha were cultured in LB medium to an OD₆₀₀ of 1.0, and 5 μl cell suspension were spotted onto the center of fresh swimming plates containing LB medium plus 0.3% (w/v) agar. The plates were then incubated at 28°C. Two days later, the motility of the bacteria was assessed by examining the diameter of the motility halo on the soft agar. To measure flagella formation, the bacteria were cultured in LB agar plates at 28°C for 20 h and examined with a transmission electron microscope (JEM-1200, Jeol, Japan) as reported previously (Sun and Sun, 2016). The assays were performed three times.

Quantitative biofilm formation on polystyrene surfaces (96-well microtiter plates) was investigated as reported previously (Wang et al., 2013a). Briefly, 10⁷ CFU of TSS, TSSfha, and TSSΔfha were placed into a sterile 96-well flat-bottomed tissue culture plate and incubated at 28°C for 12 h. After incubation, the plates were washed to remove unbound cells, and the bound cells were stained with crystal violet. Quantification of the bound cells was performed by measuring the dissolved crystal violet at OD₅₇₀ after the addition of 100 μl 30% acetic acid. To quantify biofilm formation in glass tubes, the bacteria were cultured as above and the cultures in tubes were incubated under static conditions for 48 h at 28°C. Then the culture mediums were removed and the glass tubes were washed three times with PBS. Subsequently, 5 ml of 1% crystal violet dye was added to glass tube for 15 min at room temperature. Following, the tubes were washed three times with phosphate buffered saline (PBS). After addition of 5 ml of 30% acetic acid for 15 min at room temperature to solubilize the crystal violet, the glass tubes were allowed to dry and then photographed. The experiment was performed three times.

TSS and TSSfha cultured as above were resuspended in PBS to 2 × 10⁹, 2 × 10⁸, 2 × 10⁷, and 2 × 10⁶ CFU/ml respectively. Turbot red blood cells were collected, washed three times in PBS, and resuspended to a final concentration of 2% (v/v). Fifty microliters of bacterial suspension or PBS (control) was mixed with 50 μl of turbot erythrocytes in a 96-well microtiter V-bottom plate. The plate was incubated for 1 h at room temperature. Hemagglutination was detected by visual inspection as reported previously (Astaneh et al., 2014). A small pellet of erythrocytes at the bottom of the well after incubation was considered negative as against positive reactions exhibiting an even sheet of erythrocytes across the wells. The experiment was performed three times.

Serum survival analysis was performed as reported previously (Wang et al., 2013b).

Clinically healthy turbot (average13.6 g) were purchased from a local fish farm. The fish were maintained as reported previously (Sun and Sun, 2015). For tissue dissemination and colonization analysis, TSS, TSSfha, and TSSΔfha were cultured in LB medium to an OD₆₀₀ of 0.8. The cells were washed with PBS and resuspended in PBS to 10⁸ CFU/ml. Turbot were divided randomly into four groups (N = 15) and infected by intramuscular (i.m.) injection with 50 μl of TSS, TSSfha, TSSΔfha, or PBS. Kidney and spleen were taken from the fish at 12, 24, and 48 h post-infection (5 fish/time point). The tissues were homogenized in PBS with an OSE-20 electrictissue homogenizer (Tiangen, Beijing, China). The homogenates were diluted serially in PBS and plated in triplicate on LB agar plates. After incubation at 28°C for 48 h, the colonies that appeared on the plates were counted. The genetic identity of the colonies was verified as above. For mortality assay, turbot were infected with TSS, TSSfha, and PBS as above, and the fish were monitored for mortality over a period of 20 days. All experiments were performed three times.

The plasmids pEtFha, which expresses recombinant Pf_Fha (rFha), was constructed as follows. Pf_Fha containing the hemag-glutination activity domain (positions 1–684) was amplified by PCR with the primer pairs F (5′-GATATCATGCCGACTACTCCACACAG-3′, underlined sequence, EcoRV site) and R (5′-GATATCGAAGTCGACCTGATTGCGG-3′, underlined sequence, EcoRV site). The PCR product was ligated with the T-A cloning vector pEASY-T1 Simple (TransGen Biotech, Beijing, China), and the recombinant plasmid was digested with EcoRV to retrieve the Pf_Fha-containing fragment, which was inserted into pET259 (Hu et al., 2010) at the SwaI site, resulting pEtFha. To purify rFha and the control protein rTrx, E. coli BL21(DE3; TransGen Biotech, Beijing, China) was transformed with pEtFha and pET32a (Novagen, San Diego, CA, USA), the latter plasmid expresses Trx; the transformants were cultured in LB medium at 37°C to mid-logarithmic phase, and isopropyl-α-D-thiogalactopyranoside was added to the culture to a final concentration of 1 mM. After growing at 16°C for an additional 16 h, the cells were harvested by centrifugation, and His-tagged rFha and rTrx were purified using Ni-NTA Agarose (Qiagen, Valencia, CA, USA) as recommended by the manufacturer. The purified proteins were reconstituted, removed of endotoxin, and concentrated as described previously (Wang and Sun, 2015). Mouse antibody against rFha was prepared as reported previously (Sun and Sun, 2015). Control antibody from pre-immune mouse was also prepared. The antibody was purified using rProtein G Beads (Solarbio, Beijing, China). The specificity of the rFha antibody was determined by Western immunoblot as reported previously (Wang and Sun, 2015).

To detect Pf_Fha on TSS, the bacteria were cultured in LB medium supplemented with 2,2′-dipyridyl as indicated above to OD₆₀₀ 0.9; the cells were collected by centrifugation, washed with PBS, and resuspended in PBS to 1 × 10⁸ CFU/ml. One hundred microliters of bacterial suspension (∼1 × 10⁷ CFU) were seeded on a glass slide pre-treated with 0.001% polylysine (Sigma, St. Louis, MO, USA). For fluorescence microscopy, the samples were blocked with 1% bovine serum albumin for 2 h at 30°C and incubated with mouse anti-rFha antibody or control antibody from pre-immune mouse (1/500 dilution) for 1 h at 30°C. The cells were washed three times with PBST (0.05% Tween 20 in PBS) and incubated with goat anti-mouse IgG (1/1000 dilution; Bioss, Beijing, China) coupled to fluorescein isothiocyanate (FITC) for 1 h at 30°C. The cells were washed with PBST and stained with 4′,6-diamidino-2-phenylindole (DAPI) according to the instructions of the manufacturer (Bioss, Beijing, China). The cells were then analyzed with a Zeiss fluorescence microscope (Carl Zeiss Imager A2, Jena, Germany).

To examine interaction between rFha and FG cells, the cells were seeded on 0.001% polylysine-treated glass coverslips in 12-well cell culture plates; the cells were incubated with rFha or rTrx (100 μg/ml) at 22°C for 2 h and treated with paraformaldehyde (Sigma, St. Louis, MO, USA) at 22°C for 30 min. The cells were then treated with 1% bovine serum albumin at 4°C for overnight. The cells were incubated with mouse-anti His antibody (1/1000 dilution; Bioss, Beijing, China) for 1 h at 37°C. Subsequently, cells were washed three times with PBST and incubated with FITC-coupled goat-anti mouse IgG (1/1000 dilution; Bioss, Beijing, China) for 1 h at 37°C. After washing three times with PBST, the cells were stained with DAPI and subjected to microscopy as described above.

Turbot (as described above) were divided randomly into three groups (N = 15). TSS was cultured in LB medium to an OD₆₀₀ of 0.8. The cells were washed and resuspended in PBS to 10⁸ CFU/ml. One milliliter bacterial cells were mixed with 5 μl rFha antibody, control antibody, or PBS (control) and incubated at 28°C for 1 h. After incubation, 50 μl of the mixture was inoculated into turbot via i.m. injection. Kidney and spleen were taken from the fish (five at each time point) at 12, 24, and 48 h post-bacterial infection, and bacterial recovery from the tissues was determined as above.

rFha was resuspended in PBS to a concentration of 200 μg/ml and mixed at an equal volume with aluminum hydroxide as described previously (Jiao et al., 2010). As a control, PBS was mixed similarly with aluminum hydroxide without protein. Turbot (as described above) were divided randomly into two groups (N = 55) and injected intraperitoneally with 50 μl of the protein mixture or the PBS control. At 1 month post-immunization, the fish were challenged with TSS at the dose of 5 × 10⁶ CFU/fish. The fish were monitored for mortality for a period of 20 days. Dying fish were randomly selected for the examination of bacterial recovery from liver, kidney, and spleen. Relative percent of survival (RPS) was calculated according to the following formula: RPS = {1 – (% mortality in immunized fish/% mortality in control fish)}× 100. The immunization experiment was performed two times.

With the exception of the immunization trial which was performed twice, all other experiments were performed three times, and statistical analyses were carried out with the SPSS 17.0 package (SPSS, Inc., Chicago, IL, USA). Except for the survival analysis in the immunization experiment and the in vivo infection experiment, for which logrank test was used, analysis of variance (ANOVA) was used for all other analyses. In all cases, the significance level was defined as P < 0.05.

In an effort to identify secreted proteins of TSS regulated by iron, TSS was cultured in the presence and absence of the iron chelator 2,2′-dipyridyl, and the extracellular proteins were examined by two-DE analysis. A total of 15 differentially expressed proteins were identified, of which six were significantly upregulated (ratio ≥ 2, P ≤ 0.05) in the presence of 2,2′-dipyridyl (Figure 1; Supplementary Table S1). Pf_Fha was one of the upregulated proteins (Figure 1). Sequence analysis showed that Pf_Fha contains an N-terminal signal peptide (residues 1–32) and a hemagglutination activity domain (residues 50–170; Supplementary Figure S1).

In vivo infection assay showed that following inoculation into turbot, TSS disseminated into and multiplied in kidney and spleen, in which the numbers of TSS increased rapidly with time (Figure 8A). In contrast, in turbot inoculated with TSSfha, the bacterial numbers recovered from kidney and spleen were significantly lower than that from TSS-infected fish at 12, 24, and 48 h post-infection. Similar results were obtained in repeated infection analyses with TSS, TSSfha, and TSSΔfha in parallel, which showed that at 12, 24, and 48 h, the amounts of TSSΔfha in kidney and spleen were comparable to those of TSSfha and were significantly lower than those of TSS (Supplementary Figure S8). Consistent with these observations, the survival rate of TSSfha-infected fish (66.7%) was significantly higher than that of TSS-infected fish (6.7%; Figure 8B). To further investigate the importance of Pf_Fha to host infection, turbot were infected with TSS in the presence of anti-rFha antibody, and bacterial dissemination in and colonization of kidney and spleen were subsequently determined. The results showed that at 12, 24, and 48 h post-infection, bacterial recoveries from the fish infected with TSS plus rFha antibody were significantly lower than those from the fish infected with TSS alone or with TSS plus control antibody (Figure 9).

Since, as shown above, Pf_Fha was a secreted protein essential to host infection, we examined the immunoprotective potential of rFha in a model of turbot. For this purpose, turbot were vaccinated with rFha and challenged with TSS at 1 month post-vaccination. The fish were subsequently monitored for mortality and survival. The results showed that the survival rate of the vaccinated fish was 45.7%, which was significantly higher than that of the control fish (14.3%; Figure 10). The RPS of the vaccinated fish was 36.7%.