Analysis of antibacterial and antibiofilm activity of purified recombinant Azurin from Pseudomonas aeruginosa
-
Hajar Mohammadi-Barzelighi
,
Bahram Nasr-Esfahani
,
Bita Bakhshi
,
Bahram Daraei
,
Sharareh Moghim
,
Hossein Fazeli
Abstract
Background and Objectives: The aim of this study was to evaluate the antibacterial and antibiofilm activity of recombinant Azurin from Pseudomonas aeruginosa against different bacterial species.
Materials and Methods: The azurin gene was cloned in the pET21a vector. The pET21a-azurin construct was transformed into Escherichia coli BL21. The recombinant Azurin was expressed and purified using affinity chromatography and confirmed by Western blotting. The cytotoxicity of rAzurin was assessed on peripheral blood mononuclear cells. Antibacterial and antibiofilm activity of rAzurin with different concentrations were determined by micro-broth dilution and crystal violet methods, respectively. The effect of rAzurin on bacterial species was statistically analyzed by t- test and spearman correlation.
Results: The identity of purified protein was confirmed by blotting and distinguished as a 14 kDa band on 15% SDS-PAGE. The IC50 of rAzurin on Peripheral Blood Mononuclear Cell (PBMC) was determined as 377.91±0.5 µg/mL in 24 h. Vibrio cholerae and Campilobacter jejuni displayed the most sensitivity to rAzurin (27.5 and 55 μg/mL, respectively) and the highest resistance (220 μg/mL) was displayed by P. aeruginosa and E. coli. The MIC for other species was 110 μg/mL. The Minimum Biofilm Inhibition Concentration (MBIC) was determined as 220 μg/mL for Salmonella enterica and V. cholerae, 300 μg/mL for Shigella sonnei, Shigella flexneri and P. aeruginosa and 440 μg/mL for the other species. The antimicrobial effect of rAzurin on bacterial species were significant (p value<0.05) and correlation coefficient was negative.
Conclusion: The rAzurin appears to be an appropriate choice and a new strategy for prevention of bacterial infection. It inhibits bacterial growth and biofilm formation and candidates as antimicrobial peptides.
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20. Vila-Farrés X, Giralt E, Vila J. Update of peptides with antibacterial activity. Curr Med Chem 2012; 19:6188-6198.
21. Sancineto L, Piccioni M, De Marco S, Pagiotti R, Nascimento V, Braga AL, et al. Diphenyl diselenide derivatives inhibit microbial biofilm formation involved in wound infection. BMC Microbiol 2016; 16:220.
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24. Rios JL, Recio MC. Medicinal plants and antimicrobial activity. J Ethnopharmacol 2005; 100:80-84.
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27. Punj V, Bhattacharyya S, Saint-Dic D, Vasu C, Cunningham EA, Graves J. Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer. Oncogene 2004; 23:2367-2378.
28. Yamada T, Fialho AM, Punj V, Bratescu L, Gupta TKD, Chakrabarty AM. Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity. Cell Microbiol 2005; 7:1418-1431.
29. Scocchi M, Mardirossian M, Runti G, Benincasa M. Non-membrane permeabilizing modes of action of antimicrobial peptides on bacteria. Curr Top Med Chem 2016; 16:76-88.
30. Leuschner C, Hansel W. Membrane disrupting lytic peptides for cancer treatments. Curr Pharm Des 2004; 10:2299-2310.
31. Tommassen J. Assembly of outer-membrane proteins in bacteria and mitochondria. Microbiology 2010; 156:2587-2596.
32. Huang Y, Huang J, Chen Y. Alpha-helical cationic antimicrobial peptides: relationships of structure and function. Protein Cell 2010; 1:143-152.
33. Vaara M. Agents that increase the permeability of the outer membrane. Microbiol Rev 1992;56:395-411.
34. Seales EC, Jurado GA, Brunson BA, Wakefield JK, Frost AR, Bellis SL. Hypersialylation of β1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility. Cancer Res 2005; 65:4645-4652.
35. Rabin N, Zheng Y, Opoku-Temeng C, Du Y, Bonsu E, Sintim HO. Agents that inhibit bacterial biofilm formation. Future Med Chem 2015;7:647-671.
36. Bernardes N, Ribeiro AS, Seruca R, Paredes J, Fialho AM. Bacterial protein azurin as a new candidate drug to treat untreatable breast cancers. Bioengineering (ENBENG) 2011;1st Portuguese Meeting in: IEEE.
37. Boles BR, Horswill AR. Agr-mediated dispersal of Staphylococcus aureus biofilms. PLoS Pathog 2008; 4(4): e1000052.
38. Park J, Jagasia R, Kaufmann GF, Mathison JC, Ruiz DI, Moss JA, et al. Infection control by antibody disruption of bacterial quorum sensing signaling. Chem Biol 2007; 14:1119-1127.
39. Jennings JA, Courtney HS, Haggard WO. Cis-2-decenoic acid inhibits S. aureus growth and biofilm in vitro: a pilot study. Clin Orthop Relat Res 2012;470:2663-2670.
40. Davies DG, Marques CuNH. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol 2009; 191:1393-1403.
2. Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 2008; 3:163-175.
3. Coutinho HDM, Lobo KM, Bezerra DAC, Lobo I. Peptides and proteins with antimicrobial activity. Indian J Pharmacol 2008; 40:3-9.
4. Chung PY, Toh YS. Anti-biofilm agents: recent breakthrough against multi-drug resistant Staphylococcus aureus. Pathog Dis 2014; 70:231-239.
5. Roy R, Tiwari M, Donelli G, Tiwari V. Strategies for combating bacterial biofilms: A focus on anti-biofilm agents and their mechanisms of action. Virulence 2018; 9: 522-554.
6. Pletzer D, Hancock REW. Antibiofilm peptides: potential as broad-spectrum agents. J Bacteriol 2016; 198:2572-2578.
7. Conrady DG, Brescia CC, Horii K, Weiss AA, Hassett DJ, Herr AB. A zinc-dependent adhesion module is responsible for intercellular adhesion in Staphylococcal biofilms. Proc Natl Acad Sci U S A 2008; 105:19456-19461.
8. Rao SS, Mohan KVK, Atreya CD. A peptide derived from phage display library exhibits antibacterial activity against E. coli and Pseudomonas aeruginosa. PLoS One 2013; 8(2):e56081.
9. Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis 2002; 8:881-890.
10. Leon-Calvijo MaA, Leal-Castro AL, Almanzar-Reina GA, Rosas-Parez JE, Garcaa-Castaneda JE, Rivera-Monroy ZJ. Antibacterial activity of synthetic peptides derived from lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212. Biomed Res Int 2015;2015:453826.
11. Wu H-J, Seib KL, Edwards JL, Apicella MA, McEwan AG, Jennings MP. Azurin of pathogenic Neisseria spp. is involved in defense against hydrogen peroxide and survival within cervical epithelial cells. Infect Immun 2005; 73:8444-8448.
12. Chaudhari A, Fialho AM, Ratner D, Gupta P, Hong CS, Kahali S, et al. Azurin, Plasmodium falciparum malaria and HIV/AIDS: inhibition of parasitic and viral growth by Azurin. Cell Cycle 2006; 5:1642-1648.
13. Naguleswaran A, Fialho AM, Chaudhari A, Hong CS, Chakrabarty AM, Sullivan WJ. Azurin-like protein blocks invasion of Toxoplasma gondii through potential interactions with parasite surface antigen SAG1. Antimicrob Agents Chemother 2008; 52:402-408.
14. Arvidsson RHA, Nordling M, Lundberg LG. The azurin gene from Pseudomonas aeruginosa. Cloning and characterization. Eur J Biochem 1989;179:195-200.
15. Karlsson BGr, Pascher Tr, Nordling M, Arvidsson RHA, Lundberg LG. Expression of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli. FEBS Lett 1989;246:211-217.
16. Arai H, Sanbongi Y, Igarashi Y, Kodama T. Cloning and sequencing of the gene encoding cytochrome c-551 from Pseudomonas aeruginosa. FEBS Lett 1990;261:196-198.
17. Fang HW, Fang S, Chiang Chiau JS, Yeung CY, Chan WT, Jiang CB, et al. Inhibitory effects of Lactobacillus casei subsp. rhamnosus on Salmonella lipopolysaccharide-induced inflammation and epithelial barrier dysfunction in a co-culture model using Caco-2/peripheral blood mononuclear cells. J Med Microbiol 2010; 59, 573-579.
18. Seeligmüller N. Faster Isolation of PBMC Using Ficoll-Paque. Plus in the Eppendorf® Centrifuge 5920 R. Application Note 2016; 372:1-6.
19. Haller D, Bode C, Hammes WP, Pfeifer AMA, Schivrin EJ, Blum S. Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures. Gut 2000; 47:79-87.
20. Vila-Farrés X, Giralt E, Vila J. Update of peptides with antibacterial activity. Curr Med Chem 2012; 19:6188-6198.
21. Sancineto L, Piccioni M, De Marco S, Pagiotti R, Nascimento V, Braga AL, et al. Diphenyl diselenide derivatives inhibit microbial biofilm formation involved in wound infection. BMC Microbiol 2016; 16:220.
22. De Marco S, Piccioni M, Pagiotti R, Pietrella D. Antibiofilm and antioxidant activity of propolis and Bud Poplar Resins versus Pseudomonas aeruginosa. Evid Based Complement Alternat Med 2017;2017: 10.1155/2017/5163575.
23. Gould IM. The epidemiology of antibiotic resistance. Int J Antimicrob Agents 2008; 32:S2-S9.
24. Rios JL, Recio MC. Medicinal plants and antimicrobial activity. J Ethnopharmacol 2005; 100:80-84.
25. Dunne C, Murphy L, Flynn S, O'Mahony L, O'Halloran S, Feeney M, et al. Probiotics: from myth to reality. Demonstration of functionality in animal models of disease and in human clinical trials. Lactic acid bacteria: genetics, metabolism and applications. Antonie Van Leeuwenhoek 1999; 76: 279-292.
26. Flachbartova Z, Pulzova L, Bencurova E, Potocnakova L, Comor L, Bednarikova Z, et al. Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries. Microbiol Res 2016; 188:34-41.
27. Punj V, Bhattacharyya S, Saint-Dic D, Vasu C, Cunningham EA, Graves J. Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer. Oncogene 2004; 23:2367-2378.
28. Yamada T, Fialho AM, Punj V, Bratescu L, Gupta TKD, Chakrabarty AM. Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity. Cell Microbiol 2005; 7:1418-1431.
29. Scocchi M, Mardirossian M, Runti G, Benincasa M. Non-membrane permeabilizing modes of action of antimicrobial peptides on bacteria. Curr Top Med Chem 2016; 16:76-88.
30. Leuschner C, Hansel W. Membrane disrupting lytic peptides for cancer treatments. Curr Pharm Des 2004; 10:2299-2310.
31. Tommassen J. Assembly of outer-membrane proteins in bacteria and mitochondria. Microbiology 2010; 156:2587-2596.
32. Huang Y, Huang J, Chen Y. Alpha-helical cationic antimicrobial peptides: relationships of structure and function. Protein Cell 2010; 1:143-152.
33. Vaara M. Agents that increase the permeability of the outer membrane. Microbiol Rev 1992;56:395-411.
34. Seales EC, Jurado GA, Brunson BA, Wakefield JK, Frost AR, Bellis SL. Hypersialylation of β1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility. Cancer Res 2005; 65:4645-4652.
35. Rabin N, Zheng Y, Opoku-Temeng C, Du Y, Bonsu E, Sintim HO. Agents that inhibit bacterial biofilm formation. Future Med Chem 2015;7:647-671.
36. Bernardes N, Ribeiro AS, Seruca R, Paredes J, Fialho AM. Bacterial protein azurin as a new candidate drug to treat untreatable breast cancers. Bioengineering (ENBENG) 2011;1st Portuguese Meeting in: IEEE.
37. Boles BR, Horswill AR. Agr-mediated dispersal of Staphylococcus aureus biofilms. PLoS Pathog 2008; 4(4): e1000052.
38. Park J, Jagasia R, Kaufmann GF, Mathison JC, Ruiz DI, Moss JA, et al. Infection control by antibody disruption of bacterial quorum sensing signaling. Chem Biol 2007; 14:1119-1127.
39. Jennings JA, Courtney HS, Haggard WO. Cis-2-decenoic acid inhibits S. aureus growth and biofilm in vitro: a pilot study. Clin Orthop Relat Res 2012;470:2663-2670.
40. Davies DG, Marques CuNH. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol 2009; 191:1393-1403.
| Files | ||
| Issue | Vol 11 No 2 (2019) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v11i2.1083 | |
| Keywords | ||
| Antibacterial effect Antibiofilm activity Recombinant Azurin | ||
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How to Cite
1.
Mohammadi-Barzelighi H, Nasr-Esfahani B, Bakhshi B, Daraei B, Moghim S, Fazeli H. Analysis of antibacterial and antibiofilm activity of purified recombinant Azurin from Pseudomonas aeruginosa. Iran J Microbiol. 2019;11(2):166-176.
