Production and immunological characterisation of recombinant flagellin C of <i>Pseudomonas aeruginosa</i>

Abstract

SCIENTIFIC RELEVANCE. Pseudomonas aeruginosa is one of the main causative agents of focal and diffuse suppurative inflammations in immunocompromised individuals. The multiple antimicrobial resistance of Р. aeruginosa has created an urgent need to develop effective preventive vaccines against this bacterium and to enhance their protective properties by selecting adjuvants. A promising strategy is to use flagellin, a P. aeruginosa flagellum component inducing the innate immune system through interaction with toll-like receptor 5 (TLR5) and activating the T-cell immune response, as a component or adjuvant in vaccine development.AIM. This study aimed to produce recombinant flagellin C (FliC) of P. aeruginosa and investigate its immunogenicity, adjuvanticity, and protective properties.MATERIALS AND METHODS. The fliC nucleotide sequence was obtained by PCR on template DNA of P. aeruginosa PA-103 and was inserted into the pQE-30 plasmid for subsequent expression in Escherichia coli M15. Recombinant FliC purification involved two stages: isolation of inclusion bodies and their dissolution in buffers containing urea and guanidine hydrochloride. Mice were immunised by two intraperitoneal injections with a two-week interval. The immunisation used purified recombinant FliC at a dose of 50 μg per animal and its combination with the Klebsiella pneumoniae surface antigen in a 1:1 ratio. Serum samples from immunised mice were tested for specific antibodies to recombinant FliC by enzyme-linked immunosorbent assay (ELISA). The protective properties of FliC were assessed by intraperitoneal challenge of mice with cultures of P. aeruginosa PA-103 and K. pneumoniae 204.RESULTS. The authors obtained the producing strain, generated recombinant FliC, and purified the protein to a 97.6% purity. The analysis of serum samples from immunised mice by ELISA and the protection assessment in challenge experiments showed that the purified recombinant FliC protein had immunogenic properties. Furthermore, the experimental challenge of FliC-immunised mice with P. aeruginosa confirmed that recombinant FliC had protective properties, as evidenced by the protection index of 3.0. Recombinant FliC exhibited adjuvant properties, as demonstrated by the effectiveness index of 6.1 observed in the experimental challenge of mice immunised with the combination of recombinant FliC and the surface antigen of K. pneumoniae.CONCLUSIONS. The purified recombinant FliC protein of P. aeruginosa demonstrated protective activity in mice challenged with P. aeruginosa and adjuvant properties when combined with the K. pneumoniae surface antigen, increasing the immunogenicity of the latter. The use of recombinant FliC holds promise for the creation of a candidate vaccine against P. aeruginosa infection.