Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella

Abstract

Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responses.

Keywords: BLS; TLR5; flagellin; scaffold.

Figure 1

BLS‐FliC131 chimeric protein conserves immunochemical reactivity. Western blot analysis using anti‐FliC and anti‐BLS monoclonal antibodies. Lane 1: BLS‐FliC131 chimera, Lane 2: FliC C131, Lane 3: BLS. MW: molecular weight marker.

Figure 2

Three‐dimensional structure of the decameric BLS (PDB code: 1T13), the flagellin 131 (PDB code: 3A5X), and a model of BLS‐FliC131 chimera. The structures are represented as surface and for BLS and flagellin also as cartoon. The expected MW values are indicated.

Figure 3

Static light scattering coupled to size exclusion chromatography. BLS (blue), FliC C131 (red), and BLS‐FliC131 (green) were subjected to SEC coupled to a light scattering instrument connected in tandem to a differential refractometer detector. The MW values estimated by the relation of scattering/RI signals are indicated above each curve. The values following after an asterisk correspond to the expected MW.

Figure 4

Far UV circular dichroism. Comparison of the far UV‐CD spectra of BLS (blue), FliC C131 (red), BLS‐FliC131 (green), and the sum of BLS and FliC C131 spectra (black dashed). Results are representative from three different experiments.

Figure 5

Thermal denaturation monitored by circular dichroism. The heat‐induced denaturation curve of BLS (blue), FliC C131 (red), BLS‐FliC131 (green). CD signal was measured at 220 nm. Dashed lines indicate the inflection points of each curve. Results are representative from two different experiments.

Figure 6

Reversibility of the thermal unfolding monitored by circular dichroism. CD spectra of BLS (blue), FliC131 (red), BLS‐FliC131 (green) at 25°C before (continuous line) and after (dashed lines) the thermal denaturation experiment. Results are representative from two different experiments.

Figure 7

Fusion protein keeps the capacity to trigger innate response either in vitro or in vivo. (A) Activity on Caco2‐luciferase reporter cell line. Cells were treated with different stimuli and luciferase activity was measured on cell lysate 6 h post‐stimulation. Results are expressed as fold‐increase with respect to non‐treated control. Results are representative from three different experiments. (***P < 0.005 two way ANOVA followed by Tuckey test). (B) Recruitment of neutrophils when administered either i.p. or i.n. C57BL/6J mice were treated with different stimuli and cells recovered by broncheoalveolar or peritoneal wash and analyzed by flow cytometry upon immunostaining. Results are expressed as percentage of polymorphonuclear cells (CD11c‐ CD11b+Ly6G+ GR1+). Results are representative from three different experiments. (***P < 0.005 two way ANOVA followed by Tuckey test).

Figure 8

BLS‐FliC131 has the capacity to trigger innate response activation in vivo in TLR4 and TLR5‐deficient mice. Animals were stimulated i.v. with different treatments. Cytokines were determined in serum samples 2 h upon treatment by ELISA. Results are representative from two different experiments. (***P < 0.005 two way ANOVA followed by Tuckey test).

Figure 9

Immunogenicity of BLS is enhanced in the BLS‐FliC131 fusion protein. Anti‐BLS antibodies were determined by indirect ELISA in animals immunized twice with the different antigenic mixtures. Results are expressed as geometric mean of each group (n = 5). Results are representative from two different experiments.