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Adjuvant role of Pseudomonas flagellin for Acinetobacter baumannii biofilm associated protein.

ABSTRACT: AIM:To study immunogenicity of Pseudomonas N terminal flagellin as an adjuvant for Acinetobacter baumannii (A. baumannii) biofilm associated protein (Bap). METHODS:The N terminal flagellin gene was amplified. The pET28a (+) and polymerase chain reaction products were digested with HindIII and EcoR I. The ligation of N terminal flagellin into pET28a (?+) was performed using T4 DNA ligase and was then transformed into Escherichia coli BL21 (DE3) as a suitable expression host. pET28a (?+) vector harboring a conserved region of Bap from our previous work was used. The recombinant proteins were expressed, analyzed by SDS-PAGE method and was purified by affinity chromatography with His-Tag residues followed by confirmation with western blotting. Mice were immunized with recombinant N terminal flagellin and Bap subunits. The immunized animals were intranasally (i.n) challenged with A. baumannii and Pseudomonas aeruginosa (P. aeruginosa). RESULTS:The flagellin enhanced the immunogenicity of Bap causing an increase in specific IgG titers in serum (P < 0.001). Internal organs, i.e., liver, lung and spleen of the Bap-Flagellin immunized group challenged with A. baumannii showed significantly lower bacterial load compared to the control group. The bacterial loads were studied in internal organs. A. baumannii infected immunized animals with Bap-Flagellin exhibited internal organs with minor bacterial load while P. aeruginosa PAO1 infected group showed heavy bacterial load of (4.3 ± 0.12) × 10(6), (1.1 ± 0.01) × 10(6) and (2.2 ± 0.22) × 10(6) per gram of lungs, liver and spleen respectively. Bacterial loads were detected per gram of lungs, liver and spleen of the mice group immunized with Bap were (1.2 ± 0.06) × 10(7), (11.1 ± 0.041) × 10(5) and (3.6 ± 0.42) × 10(6) respectively. In vivo neutralization assay indicated that all experimental mice groups, except for Flagellin administered group was significantly (P < 0.05) protected against A. baumannii. CONCLUSION:These results demonstrate that P. aeruginosa Flagellin as an adjuvant for Bap A. baumannii could be a useful model to evaluate new vaccine against A. baumannii.

SUBMITTER: Sefidi MD

PROVIDER: S-EPMC5031927 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Adjuvant role of Pseudomonas flagellin for Acinetobacter baumannii biofilm associated protein.

Sefidi Mozhgan Derakhshan MD Rasooli Iraj I Owlia Parviz P Talei Daryush D Astaneh Shakiba Darvish Alipour SD Nazarian Shahram S

World journal of methodology 20160926 3

<h4>Aim</h4>To study immunogenicity of Pseudomonas N terminal flagellin as an adjuvant for Acinetobacter baumannii (A. baumannii) biofilm associated protein (Bap).<h4>Methods</h4>The N terminal flagellin gene was amplified. The pET28a (+) and polymerase chain reaction products were digested with HindIII and EcoR I. The ligation of N terminal flagellin into pET28a (‏+) was performed using T4 DNA ligase and was then transformed into Escherichia coli BL21 (DE3) as a suitable expression host. pET28a ...[more]

PMID: 27679782

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Project description:Is there a universal genetically programmed defense providing tolerance to antibiotics when bacteria grow as biofilms? A comparison between biofilms of three different bacterial species by transcriptomic and metabolomic approaches uncovered no evidence of one. Single-species biofilms of three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii) were grown in vitro for three days then challenged with respective antibiotics (ciprofloxacin, daptomycin, tigecycline) for an additional 24 h. All three microorganisms displayed reduced susceptibility in biofilms compared to planktonic cultures. Global transcriptomic profiling of gene expression comparing biofilm to planktonic and antibiotic-treated biofilm to untreated biofilm was performed. Extracellular metabolites including 18 amino acids, glucose, lactate, acetate, formate, and ethanol were measured to characterize the utilization of carbon sources between biofilms, treated biofilms, and planktonic cells. While all three bacteria exhibited a species-specific signature of stationary phase, no conserved gene, gene set, or common functional pathway could be identified that changed consistently across the three microorganisms. Across the three species, glucose consumption was increased in biofilms compared to planktonic cells and alanine and aspartic acid utilization were decreased in biofilms compared to planktonic cells. The reasons for these changes were not readily apparent in the transcriptomes. No common shift in the utilization pattern of carbon sources was discerned when comparing untreated to antibiotic-exposed biofilms. Overall, our measurements do not support the existence of a common genetic or biochemical basis for biofilm tolerance against antibiotics. Rather, there are likely myriad genes, proteins, and metabolic pathways that influence the physiological state of microorganisms in biofilms contributing to antibiotic tolerance. The Acinetobacter baumannii microarray data from the study described above is deposited here.

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Adjuvant role of Pseudomonas flagellin for Acinetobacter baumannii biofilm associated protein.

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Adjuvant role of Pseudomonas flagellin for Acinetobacter baumannii biofilm associated protein.

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