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


CONCLUSIONS
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.
Mozhgan Derakhshan Sefidi, Iraj Rasooli, Department of Biology, Shahed University, Tehran 3319118651, Iran.
Mozhgan Derakhshan
MD
Sefidi
Mozhgan Derakhshan Sefidi, Iraj Rasooli, Department of Biology, Shahed University, Tehran 3319118651, Iran.
Iraj
I
Rasooli
Mozhgan Derakhshan Sefidi, Iraj Rasooli, Department of Biology, Shahed University, Tehran 3319118651, Iran.
Parviz
P
Owlia
Mozhgan Derakhshan Sefidi, Iraj Rasooli, Department of Biology, Shahed University, Tehran 3319118651, Iran.
Daryush
D
Talei
Mozhgan Derakhshan Sefidi, Iraj Rasooli, Department of Biology, Shahed University, Tehran 3319118651, Iran.
Shakiba Darvish Alipour
SD
Astaneh
Mozhgan Derakhshan Sefidi, Iraj Rasooli, Department of Biology, Shahed University, Tehran 3319118651, Iran.
Shahram
S
Nazarian
Acinetobacter baumannii; Biofilm associated protein; Immunogen; Pseudomonas aeruginosa; Vaccine
Vaccine. 2007 Jan 8;25(4):763-75
16968658
Vaccine. 2010 Feb 3;28(5):1373-82
19932669
Int J Antimicrob Agents. 2010 Mar;35(3):219-26
20047818
Infect Immun. 2005 Mar;73(3):1706-13
15731071
Annu Rev Immunol. 2003;21:335-76
12524386
J Theor Biol. 2010 Sep 21;266(2):275-90
20600143
J Bacteriol. 2008 Feb;190(3):1036-44
18024522
J Bacteriol. 2009 Oct;191(19):5953-63
19633088
J Bacteriol. 2001 May;183(9):2888-96
11292810
Microb Pathog. 2011 Dec;51(6):402-6
21946278
J Clin Microbiol. 1997 Jun;35(6):1394-7
9163451
Science. 2005 Jun 10;308(5728):1626-9
15860593
Indian J Med Microbiol. 2008 Oct-Dec;26(4):333-7
18974485
Vaccine. 2002 May 31;20 Suppl 3:S56-64
12184368
Microbiology. 2003 Dec;149(Pt 12):3473-84
14663080
Vaccine. 2010 Apr 1;28(16):2818-26
20170765
Infect Immun. 2012 Feb;80(2):651-6
22104104
J Immunol. 2001 Jan 15;166(2):1248-60
11145708
Enferm Infecc Microbiol Clin. 2008 Apr;26(4):185-6
18381036
J Bacteriol. 2003 Jul;185(14):4243-7
12837800
Infect Immun. 2009 Aug;77(8):3150-60
19470746
Vaccine. 2013 Feb 6;31(8):1210-6
23306365
Vaccine. 2008 Dec 9;26(52):6777-83
18835576
Clin Microbiol Rev. 1996 Apr;9(2):148-65
8964033
J Biotechnol. 1999 Jul 30;73(1):1-33
10483112
Trends Microbiol. 2009 Feb;17(2):73-87
19162483
Biochem Biophys Res Commun. 2013 Oct 25;440(3):437-42
24076159
Nucleic Acids Res. 2003 Jul 1;31(13):3784-8
12824418
Nat Immunol. 2003 Dec;4(12):1247-53
14625549
Nat Immunol. 2004 Oct;5(10):987-95
15454922
Nat Rev Drug Discov. 2003 Sep;2(9):727-35
12951579
Vaccine. 2012 Aug 31;30(40):5856-63
22819990
J Clin Microbiol. 1998 Jul;36(7):1938-41
9650940
J Hosp Infect. 2009 Dec;73(4):355-63
19700220
J Biol Chem. 2002 Oct 25;277(43):40456-61
12185085
Clin Vaccine Immunol. 2008 Feb;15(2):367-75
18094113