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Identification of Novel Bioactive Hexapeptides Against Phytopathogenic Bacteria Through Rapid Screening of a Synthetic Combinatorial Library

  • Choi, Jae-Hyuk (Department of Molecular Science and Technology Ajou University) ;
  • Moon, Eun-Pyo (Department of Biological Sciences, Ajou University)
  • Published : 2009.08.31

Abstract

Antimicrobial peptides (AMPs) are considered to be a promising alternative to conventional antibiotics for future generations. We identified four novel hexapeptides with antimicrobial activity: KCM11 (TWWRWW-$NH_2$), KCM12 (KWRWlW-$NH_2$), KCM21 (KWWWRW-$NH_2$), and KRS22 (WRWFIH-$NH_2$), through positional scanning of a synthetic peptide combinatorial library (PS-SCL). The ability of these peptides to inhibit the growth of a variety of bacteria and unicellular fungi was evaluated. KCM11 and KRS22 preferentially inhibited the normal growth of fungal strains, whereas KCM12 and KCM21 were more active against bacterial strains. Bactericidal activity was addressed in a clear zone assay against phytopathogenic bacteria, including Pectobacterium spp., Xanthomonas spp., Pseudomonas spp., etc. KCM21 showed the highest activity and was effective against a wide range of target organisms. Application of KCM21 with inoculation of Pectobacterium carotovorum subsp. carotovorum on detached cabbage leaves resulted in an immune phenotype or a significant reduction in symptom development, depending on the peptide concentration. Cytotoxicity of the four hexapeptides was evaluated in mouse and human epithelial cell lines using an MTT test. The results revealed a lack of cytotoxic effects.

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References

  1. Agrios, G. N., 2005. Plant Pathology, 4th Ed. Academic Press, London
  2. Bader, M. W., S. Sanowar, M. E. Daley, A. R. Schneider, U. Cho, W. Xu, R. E. Klevit, H. Le Moual, and S. I. Miller. 2005. Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122: 461-472 https://doi.org/10.1016/j.cell.2005.05.030
  3. Blondelle, S. E., E. Crooks, R. Aligue, N. Agell, O. Bachs, V. Esteve, R. Tejero, B. Celda, M. T. Pastor, and E. Perez-Paya. 2000. Novel, potent calmodulin antagonists derived from an all- D hexapeptide combinatorial library that inhibits in vivo cell proliferation: Activity and structural characterization. J. Pept. Res. 55: 148-162 https://doi.org/10.1034/j.1399-3011.2000.00162.x
  4. Blondelle, S. E. and R. A. Houghten. 1996. Novel antimicrobial compounds identified using synthetic combinatorial library technology. Trends Biotechnol. 14: 60-65 https://doi.org/10.1016/0167-7799(96)80922-X
  5. Blondelle, S. E. and K. Lohner. 2000. Combinatorial libraries: A tool to design antimicrobial and antifungal peptide analogues having lytic specificities for structure-activity relationship studies. Biopolymers 55: 74-87 https://doi.org/10.1002/1097-0282(2000)55:1<74::AID-BIP70>3.0.CO;2-S
  6. Blondelle, S. E., E. Perez-Paya, and R. A. Houghten. 1996. Synthetic combinatorial libraries: Novel discovery strategy for identification of antimicrobial agents. Antimicrob. Agents Chemother. 40: 1067-1071
  7. Blondelle, S. E., C. Pinilla, and C. Boggiano. 2003. Synthetic combinatorial libraries as an alternative strategy for the development of novel treatments for infectious diseases. Methods Enzymol. 369: 322-344 https://doi.org/10.1016/S0076-6879(03)69018-X
  8. Brogden, K. A. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3: 238-250 https://doi.org/10.1038/nrmicro1098
  9. Cabrefiga, J. and E. Montesinos. 2005. Analysis of Aggressiveness of Erwinia mylovora Using Disease-Dose and Time Relationships. p. 1430-1437 https://doi.org/10.1094/PHYTO-95-1430
  10. Chen, Y., M. T. Guarnieri, A. I. Vasil, M. L. Vasil, C. T. Mant, and R. S. Hodges. 2007. Role of peptide hydrophobicity in the mechanism of action of alpha-helical antimicrobial peptides. Antimicrob. Agents Chemother. 51: 1398-1406 https://doi.org/10.1128/AAC.00925-06
  11. Feng, Y., N. Huang, Q. Wu, L. Bao, and B. Y. Wang. 2005. Alpha-helical domain is essential for antimicrobial activity of high mobility group nucleosomal binding domain 2 (HMGN2). Acta Pharmacol. Sin. 26: 1087-1092 https://doi.org/10.1111/j.1745-7254.2005.00132.x
  12. Fields, G. B. and R. L. Noble. 1990. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35: 161-214 https://doi.org/10.1111/j.1399-3011.1990.tb00939.x
  13. Hancock, R. E. and H. G. Sahl. 2006. Antimicrobial and hostdefense peptides as new anti-infective therapeutic strategies. Nat. Biotechnol. 24: 1551-1557 https://doi.org/10.1038/nbt1267
  14. Heu, S., J. Oh, Y. Kang, S. Ryu, S. K. Cho, Y. Cho, and M. Cho. 2001. gly Gene cloning and expression and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines 8ra. Appl. Environ. Microbiol. 67: 4105-4110 https://doi.org/10.1128/AEM.67.9.4105-4110.2001
  15. Hong, S. Y., J. E. Oh, M. Kwon, M. J. Choi, J. H. Lee, B. L. Lee, H. M. Moon, and K. H. Lee. 1998. Identification and characterization of novel antimicrobial decapeptides generated by combinatorial chemistry. Antimicrob. Agents Chemother. 42: 2534-2541
  16. Houghten, R. A. 2000. Parallel array and mixture-based synthetic combinatorial chemistry: Tools for the next millennium. Annu. Rev. Pharmacol. Toxicol. 40: 273-282 https://doi.org/10.1146/annurev.pharmtox.40.1.273
  17. Hughes, S. R., P. F. Dowd, R. E. Hector, T. Panavas, D. E. Sterner, N. Qureshi, et al. 2008. Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain. J. Pept. Sci. 14: 1039-1050 https://doi.org/10.1002/psc.1040
  18. Kyte, J. and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132 https://doi.org/10.1016/0022-2836(82)90515-0
  19. Lopez-Garcia, B., L. Gonzalez-Candelas, E. Perez-Paya, and J. F. Marcos. 2000. Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits. Mol. Plant Microbe Interact. 13: 837-846 https://doi.org/10.1094/MPMI.2000.13.8.837
  20. Lopez-Garcia, B., J. F. Marcos, C. Abad, and E. Perez-Paya. 2004. Stabilisation of mixed peptide/lipid complexes in selective antifungal hexapeptides. Biochim. Biophys. Acta 1660: 131- 137 https://doi.org/10.1016/j.bbamem.2003.11.006
  21. Lopez-Garcia, B., E. Perez-Paya, and J. F. Marcos. 2002. Identification of novel hexapeptides bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library. Appl. Environ. Microbiol. 68: 2453-2460 https://doi.org/10.1128/AEM.68.5.2453-2460.2002
  22. Lopez-Garcia, B., W. Ubhayasekera, R. L. Gallo, and J. F. Marcos. 2007. Parallel evaluation of antimicrobial peptides derived from the synthetic PAF26 and the human LL37. Biochem. Biophys. Res. Commun. 356: 107-113 https://doi.org/10.1016/j.bbrc.2007.02.093
  23. Marcos, J. F., R. N. Beachy, R. A. Houghten, S. E. Blondelle, and E. Perez-Paya. 1995. Inhibition of a plant virus infection by analogs of melittin. Proc. Natl. Acad. Sci. U.S.A. 92: 12466- 12469 https://doi.org/10.1073/pnas.92.26.12466
  24. Matsuzaki, K. 1999. Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim. Biophys. Acta 1462: 1-10 https://doi.org/10.1016/S0005-2736(99)00197-2
  25. Montesinos, E. 2007. Antimicrobial peptides and plant disease control. FEMS Microbiol. Lett. 270: 1-11 https://doi.org/10.1111/j.1574-6968.2007.00683.x
  26. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55-63 https://doi.org/10.1016/0022-1759(83)90303-4
  27. Munoz, A., B. Lopez-Garcia, E. Perez-Paya, and J. F. Marcos. 2007. Antimicrobial properties of derivatives of the cationic tryptophan-rich hexapeptide PAF26. Biochem. Biophys. Res. Commun. 354: 172-177 https://doi.org/10.1016/j.bbrc.2006.12.173
  28. Ostresh, J. M., G. M. Husar, S. E. Blondelle, B. Dorner, P. A. Weber, and R. A. Houghten. 1994. 'Libraries from libraries': Chemical transformation of ombinatorial libraries to extend the range and repertoire of chemical diversity. Proc. Natl. Acad. Sci. U.S.A. 91: 11138-11142 https://doi.org/10.1073/pnas.91.23.11138
  29. Rajasekaran, K., J. W. Cary, J. M. Jaynes, and T. E. Cleveland. 2005. Disease resistance conferred by the expression of a gene encoding a synthetic peptide in transgenic cotton (Gossypium hirsutum L.) plants. Plant Biotechnol. J. 3: 545-554 https://doi.org/10.1111/j.1467-7652.2005.00145.x
  30. Reddy, K. V., R. D. Yedery, and C. Aranha. 2004. Antimicrobial peptides: Premises and promises. Int. J. Antimicrob. Agents 24: 536-547 https://doi.org/10.1016/j.ijantimicag.2004.09.005
  31. Sundin, G. W. and C. L. Bender. 1993. Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae. Appl. Environ. Microbiol. 59: 1018- 1024
  32. Vidaver, A. K. 2002. Uses of antimicrobials in plant agriculture. Clin. Infect. Dis. 34 Suppl 3: S107-S110 https://doi.org/10.1086/340247
  33. Wan, Y. K., S. P. Tian, and G. Z. Qin. 2003. Enhancement of biocontrol activity of yeasts by adding sodium bicarbonate or ammonium molybdate to control postharvest disease of jujube fruits. Lett. Appl. Microbiol. 37: 249-253 https://doi.org/10.1046/j.1472-765X.2003.01385.x
  34. Yeaman, M. R. and N. Y. Yount. 2003. Mechanisms of antimicrobial peptide action and resistance. Pharmacol. Rev. 55: 27-55 https://doi.org/10.1124/pr.55.1.2
  35. Yedery, R. D. and K. V. Reddy. 2005. Antimicrobial peptides as microbicidal contraceptives: Prophecies for prophylactics - a mini review. Eur. J. Contracept. Reprod. Health Care 10: 32-42 https://doi.org/10.1080/13625180500035124
  36. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415: 389-395 https://doi.org/10.1038/415389a
  37. Zhao, C., T. Nguyen, L. M. Boo, T. Hong, C. Espiritu, D. Orlov, W. Wang, A. Waring, and R. I. Lehrer. 2001. RL-37, an alpha-helical antimicrobial peptide of the rhesus monkey. Antimicrob. Agents Chemother. 45: 2695-2702 https://doi.org/10.1128/AAC.45.10.2695-2702.2001

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