Immunization of zebrafish with VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs.

<p>Survival curves after i.p. injection of VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs at 150 μg/fish and challenge with <i>P</i>. <i>aeruginosa</i> PAO1 (4.9x10⁷ cfu/animal) (n = 15). Untreated zebrafish that had been infected with PAO1 at LD₅₀ were used as a mortality control. Significant differences were analyzed using the Log-rank test, **, <i>p<</i>0.01.</p

Rainbow trout specific primers for qPCR.

<p>Rainbow trout specific primers for qPCR.</p

Characterization of IB particles.

<p>A) FESEM images and B) size distribution of VP1GFP (ClpA^-), VP1GFP (KPM335) and iRFP-H6 (BL21(DE3)) IBs.</p

Immunization of zebrafish with VP1GFP (ClpA^-) IBs.

<p>Survival curves of zebrafish after i.p. injection of VP1GFP (ClpA^-) IBs at different doses (300, 150, 75, 50 and 25 μg/fish) and challenge with <i>P</i>. <i>aeruginosa</i> PAO1 (4.3x10⁷ cfu/animal) (<i>n</i> = 13). Untreated zebrafish that had been infected with PAO1 at LD₅₀ were used as a mortality control. Significant differences were analyzed using the Log-rank test; **, <i>p<</i>0.01; *, <i>p<</i>0.05.</p

Analysis of gene expression in RT-HKM cells stimulated with iRFP-H6 (BL21(DE3)) IBs and LPS.

<p>Cells were incubated with 10 μg/ml of iRFP-H6 (BL21(DE3)) IBs and 10 μg/ml LPS for 12 h and the gene expression was analyzed by qPCR. Values represent means ± SD (<i>n</i> = 3). Significant differences against control were analyzed using One-way ANOVA followed by Tukey’s post test; *, <i>p<</i>0.05; **, <i>p<</i>0.01; ***, <i>p<</i>0.001.</p

Immunization of zebrafish with VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs.

<p>Survival curves after i.p. injection of VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs at 150 μg/fish and challenge with <i>P</i>. <i>aeruginosa</i> PAO1 (4.9x10⁷ cfu/animal) (n = 15). Untreated zebrafish that had been infected with PAO1 at LD₅₀ were used as a mortality control. Significant differences were analyzed using the Log-rank test, **, <i>p<</i>0.01.</p

Lipid quantification and dose-response curves of NF-κB induction by IBs.

<p>A) Total lipid quantification of IB samples. Differences were analyzed using the T test, *, p<0.01. The IBs produced in <i>E</i>. <i>coli</i> strains with different LPS chemotypes were assayed with HEK™-Blue hTLR4 (B) and Null2 (C) cells for relative NF-κB induction. The absorbance values despicted in Fig 2B and 2C represent the means and standard deviations from three individual experiments. The IBs displayed negligible stimulation of the parental HEK™-Blue Null2 cell line (C), which indicates that NF-κB-dependent SEAP expression was specifically induced via the hTLR4/MD-2 signalling pathway in HEK™-Blue hTLR4 cells.</p

MOESM1 of Functional inclusion bodies produced in the yeast Pichia pastoris

Additional file 1. VP1GFP gene and protein sequences

MOESM2 of Functional inclusion bodies produced in the yeast Pichia pastoris

Additional file 2. Raw experimental data