ISOLATION OF A NOVEL ANTIMICROBIAL POLYPEPTIDE FROM AN Aspergillus niger ISOLATE

Year 2023, , 41 - 48, 15.10.2023

Ayşe Üstün , Ayşenur Yazıcı , Serkan Örtucu

https://doi.org/10.23902/trkjnat.1247186

Abstract

In this study the extracellular proteins from the isolate LC3 belonging to Aspergillus were purified for new antimicrobial polypeptide (AMP) discovery and then tested for antimicrobial activity against Staphylococcus aureus (ATCC 25923) and Methicillin-resistant S. aureus (MRSA). Antimicrobial activity was determined by the trypsin/proteinase K assay, which was polypeptide-based, and it was observed that this protein was a protein of about 11 kDa by gel overlay assay. The minimum inhibitory concentration of purified AMP molecule against S. aureus ATCC 25923 and MRSA was 8 µg/ml and 32 µg/ml, respectively and the AMP molecule was confirmed. ITS sequence analysis showed that isolate LC3 was identified as Aspergillus niger, using the Bioedit sequence assembly program. The sequence was deposited with the GenBank database with accession number MK332597. The results indicate that the purified AMP molecule has the potential to be used in infections caused by S. aureus.

Keywords

Antimicrobial polypeptide, Aspergillus niger, Gel overlay assay, Staphylococcus aureus.

Ethical Statement

Since the article does not contain any studies with human or animal subject, its approval to the ethics committee was not required.

Supporting Institution

Scientific Research Projects of Erzurum Technical University

Project Number

2019/04

Thanks

We thank the High Technology Research and Application Center (YUTAM) for providing research facilities to carry out this research work.

References

• 1. Alanis, A.J. 2005. Resistance to Antibiotics: Are We in the Post-Antibiotic Era? Archives of Medical Research 36: 697-705. https://doi.org/10.1016/j.arcmed.2005.06.009
• 2. Al-Fakih, A.A. & Almaqtri, W.Q.A. 2019. Overview on antibacterial metabolites from terrestrial Aspergillus spp. Mycology 10: 191-209. https://doi.org/10.1080/21501203.2019.1604576 3. Al-Shaibani, A.B.A., Al-Shakarchi, F.I. & Ameen, R.S. 2013. Extraction and Characterization of Antibacterial Compound from Aspergillus niger. Al-Nahrain Journal of Science 16: 167-174.
• 4. Apan, T.Z. 2004. Effects Of Magainin And Pexiganan As New Peptide Antibiotics. Turkish Bulletin of Hygiene and Experimental Biology, 61: 37-40. 5. Bachère, E., Gueguen, Y. & Gonzalez, M. 2004. Insights into the anti-microbial defense of marine invertebrates: the penaeid shrimps and the oyster Crassostrea gigas. Immunological Reviews 198: 149-168. https://doi.org/10.1111/j.0105-2896.2004.00115.x
• 6. Chen, Y-Y., Lin, S-Y. & Yeh, Y-Y. 2005. A modified protein precipitation procedure for efficient removal of albumin from serum. ELECTROPHORESIS 26: 2117-2127. https://doi.org/10.1002/elps.200410381
• 7. Ebbensgaard, A., Mordhorst, H. & Overgaard, M.T. 2015. Comparative Evaluation of the Antimicrobial Activity of Different Antimicrobial Peptides against a Range of Pathogenic Bacteria. PLOS ONE, 10: e0144611. https://doi.org/10.1371/journal.pone.0144611
• 8. Frieri M, Kumar K. & Boutin A. 2017. Antibiotic resistance. Journal of Infection and Public Health 10: 369-378. https://doi.org/10.1016/j.jiph.2016.08.007
• 9. Galatenko, OA. & Terekhova, LP. 1990. Isolation of antibiotic-producing Actinomycetes from soil samples exposed to UV light. Antibiot Khimioter, 35:6-8
• 10. Gun Lee, D., Yub Shin, S. & Maeng, C-Y. 1999. Isolation and Characterization of a Novel Antifungal Peptide from Aspergillus niger. Biochemical and Biophysical Research Communications, 263: 646-651. https://doi.org/10.1006/bbrc.1999.1428
• 11. Hancock, R.E.W. & Diamond, G. 2000. The role of cationic antimicrobial peptides in innate host defences. Trends in Microbiology 8: 402-410. https://doi.org/10.1016/S0966-842X(00)01823-0
• 12. Huan, Y., Kong, Q., Mou, H. & Yi, H. 2020. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Frontiers in Microbiology, 11: 582779. https://doi.org/10.3389/fmicb.2020.582779
• 13. Jenssen, H., Hamill, P. & Hancock, R.E.W. 2006. Peptide Antimicrobial Agents. Clinical Microbiology Reviews 19: 491-511. https://doi.org/10.1128/CMR.00056-05
• 14. Kalyani, P. & Hemalatha, K. 2017. In vitro Antimicrobial Potential of Aspergillus niger (MTCC-961). International Journal of ChemTech Research, 10(4): 430-435.
• 15. Kowalska-Krochmal, B. & Dudek-Wicher, R. 2021. The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance. Pathogens 10: 165. https://doi.org/10.3390/pathogens10020165
• 16. Kruger, N.J. 2009. The Bradford Method For Protein Quantitation, pp. 17-24. In: Walker, J.M. (ed) The Protein Protocols Handbook. Humana Press, Totowa, NJ, LXX+1984 pp.
• 17. Lertcanawanichakul, M. & Sawangnop, S. 2011. A Comparison of Two Methods Used for Measuring the Antagonistic Activity of Bacillus Species. Walailak Journal of Science and Technology (WJST) 5: 161-171. https://doi.org/10.2004/WJST.V5I2.86
• 18. Liu, S., Wilkinson, B.J. & Bischoff, K.M. 2012. Novel antibacterial polypeptide laparaxin produced by Lactobacillus paracasei strain NRRL B-50314 via fermentation. Journal of petroleum & environmental biotechnology, 3: 121. https://doi.org/10.4172/2157-7463.1000121
• 19. Luong, H.X., Thanh, T.T. & Tran, T.H. 2020. Antimicrobial peptides–Advances in development of therapeutic applications. Life Sciences. 260: 118407.
• 20. Mataraci, E. & Dosler, S. 2012. In Vitro Activities of Antibiotics and Antimicrobial Cationic Peptides Alone and in Combination against Methicillin-Resistant Staphylococcus aureus Biofilms. Antimicrobial Agents and Chemotherapy, 56: 6366-6371. https://doi.org/10.1128/AAC.01180-12
• 21. Mygind, P.H., Fischer, R.L. & Schnorr, K.M. 2005. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature 437: 975-980. https://doi.org/10.1038/nature04051
• 22. Nizet, V., Ohtake, T. & Lauth, X. 2001. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414: 454-457. https://doi.org/10.1038/35106587
• 23. Omeike, S.O., Kareem, S.O. & Lasisi, A.A. 2019. Potential antibiotic-producing fungal strains isolated from pharmaceutical waste sludge. Beni-Suef University Journal of Basic and Applied Sciences, 8: 1-7. https://doi.org/10.1186/s43088-019-0026-8
• 24. Omeike, S.O., Kareem, S.O. & Nandanwar, H. 2021. Purification, De Novo Characterization and Antibacterial Properties of a Novel, Narrow-Spectrum Bacteriostatic Tripeptide from Geotrichum candidum OMON-1. Arabian Journal for Science and Engineering, 46: 5275-5283. https://doi.org/10.1007/s13369-020-05024-1
• 25. Park, C.B., Kim, H.S. & Kim, S.C. 1998. Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochem Biophys Res Commun, 244: 253-257. https://doi.org/10.1006/bbrc.1998.8159
• 26. Sambrook, J. & Russell, D.W. 2006. Purification of Nucleic Acids by Extraction with Phenol: Chloroform. Cold Spring Harb Protoc, 2006: 4455. https://doi.org/10.1101/pdb.prot4455
• 27. Schägger, H. 2006. Tricine–SDS-PAGE. Nature protocols, 1: 16-22. https://doi.org/10.1038/nprot.2006.4
• 28. Smith, J.E., Sullivan, R. & Rowan, N. 2003. The Role of Polysaccharides Derived from Medicinal Mushrooms in Cancer Treatment Programs: Current Perspectives. International Journal of Medicinal Mushrooms, 5: 217-234. https://doi.org/10.1615/Interjmedicmush.V5.I3.10
• 29. Subhash, S., Babu, P. & Vijayakumar, A. 2022. Aspergillus niger Culture Filtrate (ACF) Mediated Biocontrol of Enteric Pathogens in Wastewater. Water, 14: 119. https://doi.org/10.3390/w14010119
• 30. Sultana, A., Luo, H. & Ramakrishna, S. 2021. Antimicrobial Peptides and Their Applications in Biomedical Sector. Antibiotics, 10:1094. https://doi.org/10.3390/antibiotics10091094
• 31. Tong, S.Y.C., Davis, J.S. & Eichenberger, E. 2015. Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management. Clinical Microbiology Reviews, 28: 603-661. https://doi.org/10.1128/CMR.00134-14
• 32. Valore, E.V., Martin, E., Harwig, S.S. & Ganz, T. 1996. Intramolecular inhibition of human defensin HNP-1 by its propiece. The Journal of Clinical Investigation, 97(7): 1624-1629. https://doi.org/10.1172/JCI118588
• 33. Wasser, S.P. 2011. Current findings, future trends, and unsolved problems in studies of medicinal mushrooms. Applied Microbiology and Biotechnology, 89: 1323-1332. https://doi.org/10.1007/s00253-010-3067-4
• 34. Wiegand, I., Hilpert, K. & Hancock, R.E.W. 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols, 3: 163-175. https://doi.org/10.1038/nprot.2007.521
• 35. Yazici, A., Örtücü, S. & Taşkin, M. 2021. Screening and characterization of a novel Antibiofilm polypeptide derived from filamentous Fungi. Journal of Proteomics, 233: 104075. https://doi.org/10.1016/j.jprot.2020.104075 36. Yazici, A., Ortucu, S., Taskin, M. & Marinelli L. 2018. Natural-based Antibiofilm and Antimicrobial Peptides from Micro-organisms. Current Topics in Medicinal Chemistry, 18: 2102-2107. https://doi.org/10.2174/1568026618666181112143351
• 37. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature, 415: 389-395. https://doi.org/10.1038/415389a
• 38. Zhang, Z., Schwartz, S., Wagner, L. & Miller, W. 2000. A Greedy Algorithm for Aligning DNA Sequences. Journal of Computational Biology, 7: 203-214. https://doi.org/10.1089/10665270050081478
• 39. Zjawiony, J.K. 2004. Biologically Active Compounds from Aphyllophorales (Polypore) Fungi. Journal of Natural Products, 67(2): 300-310. https://doi.org/10.1021/np030372w