Bioinformatics Analysis And Mucosal Immune Protection Study Of Stenotrophomonas Maltophilia Outer Membrane Protein A

Background:Stenotrophomonas maltophilia(S.maltophilia),a multidrug-resistant opportunistic pathogen,is associated with nosocomial and community-acquired infections and exists widely in nature.The incidence of S.maltophilia hospital-acquired infections is increasing,particularly in the immunocompromised patient popolution.Preventive and therapeutic strategies for such infections are greatly needed.Vaccination with outer membrane protein antigens has been shown to be efficacious against bacterial infection in a number of studies.However,there are very few studies to evaluate the protective effects of immunization with S.maltophilia outer membrane protein.Our previous study has shown that Omp A of S.maltophilia is highly immunogenic and may be used to develop vaccines against S.maltophilia infection.However,whether Omp A in S.maltophilia could be an effective vaccine against S.maltophilia infection remains unclear.In the current study,we predict and analyze the structure and characteristic of Stenotrophomonas maltophilia Omp A,and investigate the protective effects of recombinant Omp A(r Omp A)immunization against S.maltophilia infection in mice.Part ?: Bioinformatics analysis of Stenotrophomonas maltophilia outer membrane protein AObjective: The purpose of this part is to predict and analyze the structure and characteristic of Stenotrophomonas maltophilia Omp A.Methods: Bioinformatic software and biological databases were used to analyze the physicochemical properties,signal peptides,and transmembrane structures of the Omp A protein and to predict the subcellular localization,secondary and tertiary structures,sequence homology and conserved domains,and epitopes of Omp A.Results:Omp A protein had strong hydrophilicity but without transmembrane helices in mature protein,and position 1-22 of the sequence predicted as signal peptide.In the second structure random coil helix,?lpha-helix,beta-turn and extended strand made up 50.27%,24.59%,9.29%,15.85%,respectively.The three-dimensional structure of Omp A was ?-barrel.Omp A is highly conserved among S.maltophilia strains but shares minimal homology with human and mouse proteins.The N-terminal domain of Omp A was OM-channels superfamily and the C-terminal domain of Omp A was Omp A_C-like superfamily;Omp A protein contains 11 dominant antigen epitopes.Part ?: Mucosal immune protection study of Stenotrophomonas maltophilia outer membrane protein AObjective: We aimed to further investigate the immune responses induced by intranasally immunizing mice with S.maltophilia r Omp A,and the protective effects of r Omp A immunization against S.maltophilia infection.Methods: Recombinant Omp A were prepared and used to intranasally immune mice.To study antigen-specific responses generated by intranasal immunization with recombinant antigens,the antibody leveals and isotypes and splenocytes-secreted cytokines induced by immunization were evaluated by Enzyme-linked immuno sorbent assay(ELISA).To further evaluate the protection against S.maltophilia elicited by intranasally immunization with r Omp A,the bacterial loads in lungs and pro-inflammatory cytokines(TNF-? and IL-6)level in bronchoalveolar lavage fluid(BALF)of the mice were detected after intransally challenge with S.maltophilia.Results: In mice,intranasal immunization with S.maltophilia rOmpA without additional adjuvant induced sustained mucosal and systemic r Omp A-specific antibody responses.Treatment with r Omp A stimulated significantly higher levels of secretion of IFN-?,IL-2,and IL-17A(All P<0.05)from the primary splenocytes isolated from r Omp A-immunized mice than from the primary splenocytes isolated from PBS-immunized mice.Furthermore,mice immunized with r Omp A showed significantly reduced bacterial burden in the lung and reduced levels of pro-inflammatory cytokines(TNF-? and IL-6)in BALF 24 hours after intranasal S.maltophilia infection,indicating that immunization with r Omp A may have protective effects against S.maltophilia challenge in mice.Conclusions:(1)The properties of Omp A were successfully obtained by bioinformatic analysis.These bioinformatic predictions not only provide reference for the study of structure and novel characteristic of Omp A,but also provide theoretical basis for the research on related new subunit vaccines.(2)Our findings suggest that intranasal immunization with r Omp A may induce mucosal and systemic immune responses in mice,trigger Th1-and Th17-mediated cellular immune responses,and thus protect mice from S.maltophilia challenge.Therefore,r Omp A can be a promising candidate for the development of a subunit vaccine to prevent S.maltophilia infection.