Skip to main content

Advertisement

SpringerLink
Log in

Cloning and expression of nlpA gene as DNA vaccine candidate against Acinetobacter baumannii

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Acinetobacter baumannii is one of the highly antibiotic-resistant bacteria that cause infections with high rate of death. This bacterium is one the common causes of infection worldwide leading to endemic and epidemic nosocomial infections. Despite many efforts, there is no effective vaccine against A. baumannii. As NlpA is one of the important antigenic factors in biogenesis of outer membrane vesicles, and OMV-based reported vaccines in A. baumannii stimulated the immune responses, this study was aimed to clone and express nlpA gene in eukaryotic HDF cells and evaluate the induced immunization following the administration of resulting construct as DNA vaccine in BALB/c mice. The nlpA gene of A. baumannii was amplified using PCR. The PCR product was then cloned and subcloned into the pTZ57R/T and pEGFP-C2 vectors respectively. The cloning was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The pEGFP-C2-nlpA recombinant plasmid was transferred into the HDF cells using electroporation and the expression of target gene was validated by RT-PCR. The recombinant construct was injected to BALB/c mice through three IM injections and the levels of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 were determined using ELISA assay. The A. baumannii nlpA gene was amplified during PCR as 867 bp band which was successfully cloned in pEGFP-C2-nlpA vector. Obtained data from RT-PCR and presence of the 867 bp fragment in transformed HDF cells confirmed the nlpA gene expression. Following the injection of pEGFP-C2-nlpA showed the increased level of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 in serum of immunized mice. Overall, through this study recombinant pEGFP-C2-nlpA was generated and successfully expressed the A. baumannii nlpA gene in eukaryotic cells. Additionally, our in vivo study confirmed that the recombinant construct capable to induce the immune response in immunized mice. These findings suggest the pEGFP-C2-nlpA may be considered as DNA vaccine candidate against A. baumannii.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Ansari H, Doosti A, Kargar M, Bijanzadeh M, Jafarinya M (2017) Antimicrobial resistant determination and prokaryotic expression of smpA gene of Acinetobacter baumannii Isolated from admitted patients. Jundishapur J Microbiol 10:e59370. https://doi.org/10.5812/jjm.59370

    Article  Google Scholar 

  2. RazaviNikoo H, Ardebili A, Mardaneh J (2017) Systematic review of antimicrobial resistance of clinical Acinetobacter baumannii isolates in Iran: an update. Microb Drug Res 23:744–756. https://doi.org/10.1089/mdr.2016.0118

    Article  CAS  Google Scholar 

  3. Amorim AM, Nascimento JD (2017) Acinetobacter: an underrated foodborne pathogen? J Infect Dev Ctries 11:111–114

    Article  PubMed  CAS  Google Scholar 

  4. Ansari H, Kargar M, Bizhanzadeh M, Jafarinya M, Doosti A (2017) Cloning and sequencing of the ompA and smpA virulence genes of Acentobacter baumannii isolated in clinical samples. Armaghane-danesh 21:1207–1217

    Google Scholar 

  5. Odewale G, Adefioye OJ, Ojo J, Adewumi FA, Olowe OA (2016) Multidrug resistance of AcinetobacterBaumanniiin ladoke University Teaching Hospital, Osogbo, Nigeria. Eur J Microbiol Immunol (Bp) 6:238–243

    Article  CAS  Google Scholar 

  6. Pourhajibagher M, Hashemi FB, Pourakbari B, Aziemzadeh M, Bahador A (2016) Antimicrobial resistance of Acinetobacter baumannii to imipenem in Iran: a systematic review and meta-analysis. Open Microbiol J 10:32–42

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Ni Z, Chen Y, Ong E, He Y (2017) Antibiotic resistance determinant-focused Acinetobacter baumannii vaccine designed using reverse vaccinology. Int J Mol Sci 18:E458. https://doi.org/10.3390/ijms18020458

    Article  PubMed  CAS  Google Scholar 

  8. García Quintanilla M, Pulido MR, Pachón J, McConnell MJ (2014) Immunization with lipopolysaccharide-deficient whole cells provides protective immunity in an experimental mouse model of Acinetobacter baumannii infection. PLoS ONE 9:e114410. https://doi.org/10.1371/journal.pone.0114410

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Singh R, Garg N, Shukla G, Capalash N, Sharma P (2016) Immunoprotective efficacy of Acinetobacter baumannii outer membrane protein, FilF, predicted in silico as a potential vaccine candidate. Front Microbiol 7:158. https://doi.org/10.3389/fmicb.2016.00158

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Ahmad TA, Tawfik DM, Sheweita SA, Haroun M, El-Sayed LH (2016) Development of immunization trials against Acinetobacter baumannii. Trials Vaccinol 5:53–60

    Article  Google Scholar 

  11. Schwechheimer C, Kuehn MJ (2013) Synthetic effect between envelope stress and lack of outer membrane vesicle production in Escherichia coli. J Bacteriol 195(18):4161–4173

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Bodero MD, Pilonieta MC, Munson GP (2007) Repression of the inner membrane lipoprotein NlpA by Rns in enterotoxigenic Escherichia coli. J Bacteriol 189:1627–1632

    Article  PubMed  CAS  Google Scholar 

  13. Schwechheimer C, Kulp A, Kuehn MJ (2014) Modulation of bacterial outer membrane vesicle production by envelope structure and content. BMC Microbiol 14:324. https://doi.org/10.1186/s12866-014-0324-1

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Yamaguchi K, Inouye M (1988) Lipoprotein 28, an inner membrane protein of Escherichia coli encoded by nlpA, is not essential for growth. J Bacteriol 170:3747–3749

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Doosti A, Doosti E, Rahimi E, Ghasemi-Dehkordi P (2017) Frequency of antimicrobial-resistant genes in Salmonella enteritidis isolated from traditional and industrial iranian white cheeses. Proc Natl A Sci India B 87:73–80

    CAS  Google Scholar 

  16. McConnell MJ, Rumbo C, Bou G, Pachón J (2011) Outer membrane vesicles as an acellular vaccine against Acinetobacter baumannii. Vaccine 29:5705–5710

    Article  PubMed  CAS  Google Scholar 

  17. Wang Q, Cao W, Yang ZG, Zhao GF (2015) DC targeting DNA vaccines induce protective and therapeutic antitumor immunity in mice. Int J Clin Exp Med 8:17565–17577

    PubMed  PubMed Central  CAS  Google Scholar 

  18. Taneja N, Singh G, Singh M, Sharma M (2011) Emergence of tigecycline and colistin resistant Acinetobacter baumanii in patients with complicated urinary tract infections in north India. Indian J Med Res 133:681–684

    PubMed  PubMed Central  CAS  Google Scholar 

  19. Moradi J, Hashemi FB, Bahador A (2015) Antibiotic resistance of Acinetobacter baumannii in Iran: a systemic review of the published literature. Osong Public Health Res Perspect 6:79–86

    Article  PubMed  PubMed Central  Google Scholar 

  20. Huang W, Wang S, Yao Y, Xia Y, Yang X et al (2015) OmpW is a potential target for eliciting protective immunity against Acinetobacter baumannii infections. Vaccine 33:4479–4485

    Article  PubMed  CAS  Google Scholar 

  21. Huang W, Yao Y, Wang S, Xia Y, Yang X et al (2016) Immunization with a 22-kDa outer membrane protein elicits protective immunity to multidrug-resistant Acinetobacter baumannii. Sci Rep 6:20724. https://doi.org/10.1038/srep20724

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Lin L, Tan B, Pantapalangkoor P, Ho T, Hujer AM, Taracila MA et al (2013) Acinetobacter baumannii OmpA vaccine dose alters immune polarization and immunodominant epitopes. Vaccine 31:313–318

    Article  PubMed  CAS  Google Scholar 

  23. Huang W, Yufeng Y, Qiong L, Xu Y, Wenjia S, Cunbao Let al (2014) Immunization against multidrug-resistant Acinetobacter baumannii effectively protects mice in both pneumonia and sepsis models. PLoS ONE 9:e100727

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Esmaeilkhani H, Rasooli I, Nazarian S, Sefid F (2016) In vivo validation of the immunogenicity of recombinant Baumannii Acinetobactin utilization a protein (rBauA). Microb Pathog 98:77–81

    Article  PubMed  CAS  Google Scholar 

  25. Modarresi F, Azizi O, Shakibaie MR, Motamedifar M, Mansouri S (2016) Cloning and expression of quorum sensing N-acyl-homoserine synthase (LuxI) gene detected in Acinetobacter baumannii. Iran J Microbiol 8:139–143

    PubMed  PubMed Central  Google Scholar 

  26. Alzubaidi AN, Alkozai ZM (2015) Immunogenic properties of outer membrane protein of Acinetobacter baumannii that loaded on chitosan nanoparticles. Am J BioMed 3:59–74

    Google Scholar 

Download references

Acknowledgements

The authors wish to thank the staff of Biotechnology Research Center of Shahrekord Branch, Islamic Azad University and Ahvaz Jundishapur University of Medical Sciences for their help.

Author information

Authors and Affiliations

  1. Department of Molecular Genetics, Fars Science and Research Branch, Islamic Azad University, Shiraz, Islamic Republic of Iran

    Rassoul Hashemzehi

  2. Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Islamic Republic of Iran

    Abbas Doosti

  3. Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Islamic Republic of Iran

    Mohammad Kargar

  4. Department of Molecular Genetics, Marvdasht Branch, Islamic Azad University, Marvdasht, Islamic Republic of Iran

    Rassoul Hashemzehi & Mojtaba Jaafarinia

Authors
  1. Rassoul Hashemzehi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abbas Doosti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohammad Kargar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mojtaba Jaafarinia
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RH; literature review, manuscript writing and data collection. AD; literature review and manuscript revision. MK; manuscript revision and MJ; literature review.

Corresponding author

Correspondence to Abbas Doosti.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hashemzehi, R., Doosti, A., Kargar, M. et al. Cloning and expression of nlpA gene as DNA vaccine candidate against Acinetobacter baumannii. Mol Biol Rep 45, 395–401 (2018). https://doi.org/10.1007/s11033-018-4167-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-018-4167-y

Keywords

Search

Navigation