Cloning, Expression, and Purification of Antimicrobial Peptide LL-37 and Assessment of its Antimicrobial Effectiveness on Multiple-Drug-Resistant Acinetobacter Baumannii

Document Type : Original Article (s)

Authors

1 MSc Student, Department of Medical Biotechnology, School of Medicine, Arak University of Medical Sciences, Arak, Iran

2 Associate Professor, Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran

3 Department of Agricultural Biotechnology, School of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

Abstract

Background: Now a day, antibiotic resistance is a global problem. A way to solve this problem is production of alternative drugs. In this regard, today so many researches on antimicrobial peptides against pathogens are being done. LL-37 peptide is one of these peptides that is cationic and has antibacterial, antiviral, and anticancer activity. This project aimed to study the antimicrobial effectiveness of peptide LL-37 on multiple-drug-resistant (MDR) Acinetobacter baumannii.Methods: First, the LL-37 gene was linked to the pET-32a vector; then, recombinant DNA was transformed into the host bacteria and inducted to produce proteins. After the production and purification of recombinant proteins, to activate the protein, dialysis was performed in phosphate buffered saline (PBS). Then, the efficiency of peptide LL-37 on multiple-drug-resistant Acinetobacter baumannii was tested using common laboratory tests such as minimum inhibitory concentration (MIC).Findings: The minimum inhibitory concentration of LL-37 on Acinetobacter baumannii ATCC19606 was 1.5 µg/ml. In addition, the activity test showed that the recombinant proteins could inhibit the growth and decay the bacteria.Conclusion: The results of this study show that LL-37 protein, in comparison to other peptides and drugs in other studies, is more efficient and in low concentration can cause destruction of bacteria. This can herald a bright future for the treatment of infections caused by multiple-drug-resistant Acinetobacter baumannii.

Keywords

References

  1. Centers for Disease Control and Prevention (CDC). Antibiotic resistance threats in the United States. Atlanta, GA: CDC; 2013.
  2. Zeth K. Structure and mechanism of human antimicrobial peptide dermcidin and its antimicrobial potential. In: Mendez-Vilas A, editor. Microbial pathogens and strategies for combating them: Science, technology and education. Badajoz, Spain: Formatex Research Center; 2013. p. 1333-42.
  3. Rivera G, Bulnes J, Castillo C, Ajenjo MC, Garcia P, Labarca J. Extensively drug-resistant Acinetobacter baumannii isolated in a university hospital: Role of inter-hospital transmission. J Infect Dev Ctries 2016; 10(1): 96-9.
  4. Spence RP, Towner KJ, Henwood CJ, James D, Woodford N, Livermore DM. Population structure and antibiotic resistance of Acinetobacter DNA group 2 and 13TU isolates from hospitals in the UK. J Med Microbiol 2002; 51(12): 1107-12.
  5. Penchovsky R, Traykovska M. Designing drugs that overcome antibacterial resistance: where do we stand and what should we do? Expert Opin Drug Discov 2015; 10(6): 631-50.
  6. Nguyen LT, Haney EF, Vogel HJ. The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol 2011; 29(9): 464-72.
  7. Brogden KA. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 2005; 3(3): 238-50.
  8. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002; 415(6870): 389-95.
  9. Wang Z, Wang G. APD: The Antimicrobial Peptide Database. Nucleic Acids Res 2004; 32(Database issue): D590-D592.
  10. Wang G, Li X, Wang Z. APD2: the updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Res 2009; 37(Database issue): D933-D937.
  11. Zanetti M. Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 2004; 75(1): 39-48.
  12. Agerberth B, Charo J, Werr J, Olsson B, Idali F, Lindbom L, et al. The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Blood 2000; 96(9): 3086-93.
  13. Gudmundsson GH, Agerberth B, Odeberg J, Bergman T, Olsson B, Salcedo R. The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. Eur J Biochem 1996; 238(2): 325-32.
  14. Oren Z, Lerman JC, Gudmundsson GH, Agerberth B, Shai Y. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem J 1999; 341(Pt 3): 501-13.
  15. Wang G, Mishra B, Epand RF, Epand RM. High-quality 3D structures shine light on antibacterial, anti-biofilm and antiviral activities of human cathelicidin LL-37 and its fragments. Biochim Biophys Acta 2014; 1838(9): 2160-72.
  16. Mahmoudi S, Abtahi H, Bahador A, Mosayebi G, Salmanian AH, Teymuri M. Optimizing of Nutrients for High Level Expression of Recombinant Streptokinase Using pET32a Expression System. Maedica (Buchar) 2012; 7(3): 241-6.
  17. Farhangnia L, Ghaznavi-Rad E, Mollaee N, Abtahi H. Cloning, Expression, and Purification of Recombinant Lysostaphin From Staphylococcus simulans. Jundishapur J Microbiol 2014; 7(5): e10009.
  18. Krijgsveld J, Zaat SA, Meeldijk J, van Veelen PA, Fang G, Poolman B, et al. Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines. J Biol Chem 2000; 275(27): 20374-81.
  19. Jorgensen JH, Crawford SA. Assessment of two commercial susceptibility test methods for determination of daptomycin MICs. J Clin Microbiol 2006; 44(6): 2126-9.
  20. Prakash V, Lewis JS, Jorgensen JH. Vancomycin MICs for methicillin-resistant Staphylococcus aureus isolates differ based upon the susceptibility test method used. Antimicrob Agents Chemother 2008; 52(12): 4528.
  21. Koeth LM, DiFranco-Fisher JM, McCurdy S. A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically. J Vis Exp 2015; (103).
  22. Satishkumar R, Sankar S, Yurko Y, Lincourt A, Shipp J, Heniford BT, et al. Evaluation of the antimicrobial activity of lysostaphin-coated hernia repair meshes. Antimicrob Agents Chemother 2011; 55(9): 4379-85.
  23. Farhang Nia L, Ghaznavi Rad E, Molaee N, Abtahi H. Cloning, expression and purification of recombinant prolysostaphin protein and evaluating its in vitro antistaphylococcal activity. Koomesh 2014; 15 (4): 441-8. [In Persian].
  24. Hajikhani B, Najar Peerayeh S, Soleimanjahi H, Hassan ZM. Cloning, expression, purification and antigenicity of recombinant UreB332-HpaA fusion protein from Helicobacter pylori. Modares J Med Sci Pathol 2017; 13(2): 1-10. [In Persian].
  25. Demain AL, Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 2009; 27(3): 297-306.
  26. Baneyx F. Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 1999; 10(5): 411-21.
  27. Gordon YJ, Huang LC, Romanowski EG, Yates KA, Proske RJ, McDermott AM. Human cathelicidin (LL-37), a multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity. Curr Eye Res 2005; 30(5): 385-94.
  28. Moffatt JH, Harper M, Harrison P, Hale JD, Vinogradov E, Seemann T, et al. Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production. Antimicrob Agents Chemother 2010; 54(12): 4971-7.
  29. Vila-Farres X, Garcia dlM, Lopez-Rojas R, Pachon J, Giralt E, Vila J. In vitro activity of several antimicrobial peptides against colistin-susceptible and colistin-resistant Acinetobacter baumannii. Clin Microbiol Infect 2012; 18(4): 383-7.
Journal of Isfahan Medical School
Volume 35, Issue 441 - Serial Number 441
July and August 2017
Pages 954-960

Files

History

  • Receive Date: 13 June 2017
  • Revise Date: 08 May 2024
  • Accept Date: 06 April 2022

Share

How to cite

Statistics