Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Methodol. Sep 26, 2016; 6(3): 190-199
Published online Sep 26, 2016. doi: 10.5662/wjm.v6.i3.190
Adjuvant role of Pseudomonas flagellin for Acinetobacter baumannii biofilm associated protein
Mozhgan Derakhshan Sefidi, Iraj Rasooli, Parviz Owlia, Daryush Talei, Shakiba Darvish Alipour Astaneh, Shahram Nazarian
Mozhgan Derakhshan Sefidi, Iraj Rasooli, Department of Biology, Shahed University, Tehran 3319118651, Iran
Iraj Rasooli, Parviz Owlia, Molecular Microbiology Research Center, Shahed University, Tehran 3319118651, Iran
Daryush Talei, Shahed University, Tehran 3319118651, Iran
Shakiba Darvish Alipour Astaneh, Department of Biotechnology, Semnan University, Semnan 3513119111, Iran
Shahram Nazarian, Department of Biology, Faculty of Science, Imam Hussein University, Tehran 3319118651, Iran
Author contributions: Rasooli I contributed to the conception and design of the study, or acquisition of data, or analysis and interpretation of data; Sefidi MD carried out the experimental part of the project; Owlia P revised it critically for important intellectual content; Talei D carried out the statistical analyses; Astaneh SDA supervised the experiments; Nazarian S drafted the article; and final approval of the version to be submitted was made by Rasooli I.
Institutional review board statement: This work was carried out as a part of MSc thesis of Ms. Mozhgan Derakhshan Sefidi at Shahed University. The thesis proposal was approved by Post Graduate Committee in Biology Department of the Aforementioned University.
Institutional animal care and use committee statement: The animal care protocol was approved by Shahed University. 4-6 wk old (16-22 g) BALB/c mice procured from the Razi Institute, Tehran, Iran were housed in clean standard animal care facility of Shahed University. The research was carried out in compliance with the Animal Welfare Act and regulations related to experiments involving animals. The animal protocol was designed to minimize pain or discomfort to the animals. The animals were acclimatized to laboratory conditions (23 °C, 12 h/12 h light/dark, 50% humidity, ad libitum access to food and water) for two weeks prior to experimentation. All animals were euthanized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium) for tissue collection.
Conflict-of-interest statement: The authors declare no conflict of interests.
Data sharing statement: Technical details and dataset available from the corresponding author at rasooli@shahed.ac.ir. No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Iraj Rasooli, Professor, Department of Biology, Shahed University, Tehran-Qom Express way, Tehran 3319118651, Iran. rasooli@shahed.ac.ir
Telephone: +98-21-51212200 Fax: +98-21-51212201
Received: April 12, 2016
Peer-review started: April 15, 2016
First decision: May 19, 2016
Revised: June 19, 2016
Accepted: July 14, 2016
Article in press: July 16, 2016
Published online: September 26, 2016
Processing time: 160 Days and 1.3 Hours
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) × 106, (1.1 ± 0.01) × 106 and (2.2 ± 0.22) × 106 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) × 107, (11.1 ± 0.041) × 105 and (3.6 ± 0.42) × 106 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 BapA. baumannii could be a useful model to evaluate new vaccine against A. baumannii.

Keywords: Acinetobacter baumannii; Pseudomonas aeruginosa; Vaccine; Immunogen; Biofilm associated protein

Core tip: The increasing frequency of Acinetobacter baumannii (A. baumannii) infections and its drug resistance challenge health authorities. Flagellin is an effective immune activator stimulating various biologic functions identified by Toll like receptor 5. Conserved regions of biofilm associated protein (Bap) have already been identified and their immunoprotectivity against A. baumannii have been established. In order to enhance their immunogenic activities, we designed a study on adjuvant role of flagellin from Pseudomonas aeruginosa for Bap.