THE PROTEIN PROFILING OF ASIAN GIANT TOAD SKIN SECRETIONS AND THEIR ANTIMICROBIAL ACTIVITY
Objective: The skin secretions of toads are a rich source of bioactive peptides and proteins, which offer a wide range of therapeutic application. The current study was designed to elucidate the antimicrobial activity of Bufo asper skin secretions.
Methods: Proteomic analysis of electrically stimulated skin secretions were mapped using SDS-PAGE followed by LC-MS/MS. In total,>50 proteins were identified with a molecular weight ranging from 20 to 250 KDa. The antimicrobial activity was performed by an agar-well diffusion method to measure the diameter of inhibition zone (DIZ) as well as microdilution technique to determine the minimum inhibitory concentration (MIC).
Results: Toad's skin secretion (TSS) exhibited broad spectrum growth inhibitory activity against both Gram-positive and Gram-negative bacteria; with more pronounce activity towards Staphylococcus aureus and Bacillus subtilis, with MIC 12.25Â±0.4 and 25Â±1.3 Î¼g/ml, respectively. Moreover, the proteomic profile of Bufo asper skin secretion has revealed the presence of interesting proteins such as, actin, histone H4 and heat shock proteins (HSP90, HSP70 and HSC70).
Conclusion: we anticipate that the collective functions of proteins and peptides with a wide range of diversity may contribute to the TSS antimicrobial activity.
Keywords: Bufo asper, Skin secretion, Antimicrobial, Proteomics.
2. Gomes A, Giri B, Kole L, Saha A, Debnath A, Gomes A. A crystalline compound (BM-ANF1) from the Indian toad (Bufo melanostictus, Schneider) skin extract, induced antiproliferation and apoptosis in leukemic and hepatoma cell line involving cell cycle proteins. Toxicon 2007;30:835-49.
3. Qi J, Tan CK, Hashimi SM, Zulfiker AH, Good D, Wei MQ. Toad glandular secretions and skin extractions as anti-inflammatory and anticancer agents. J Evidence-Based Complementary Altern Med 2014;6:1-9.
4. Clarke BT. The natural history of amphibian skin secretions, their normal functioning, and potential medical applications. Biol Rev Cambridge Philos Soc 1997;1:365-79.
5. Conlon JM, Leprince J. Identification and analysis of bioactive peptides in amphibian skin secretions. Peptidomics 2010;1:145-57.
6. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389-95.
7. Wang G, Li X, Wang Z. APD2: the updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Res 2009;37 Suppl 1:D933-7.
8. Tabana Y, Dahham SS, Al Akkad A, Al-Hindi B. Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) among medical staff in three syrian provinces: damascus, daraa and al-swayda. Midlle East J Sci Res 2015;81756-64.
9. CaÅ‚kosiÅ„ski I, Zasadowski A, Bronowicka-SzydeÅ‚ko A, Dzierzba K, Seweryn E, DobrzyÅ„ski M, et al. [Amphibian skin secretions as a new source of antibiotics and biologically active substances]. Postepy Hig Med Dosw 2009;63:537-48.
10. Chu HL, Yip BS, Chen KH, Yu HY, Chih YH, Cheng HT, et al. Novel antimicrobial peptides with high anticancer activity and selectivity. PLoS One 2015;1-14. Doi:10.1371/ journal. pone.0126390. [Article in Press]
11. Schweizer F. Cationic amphiphilic peptides with cancer-selective toxicity. Eur J Pharmacol 2009;625:190-4.
12. Cruz-Chamorro L, Puertollano MA, Puertollano E, de Cienfuegos GÃ, de Pablo MA. In vitro biological activities of magainin alone or in combination with nisin. Peptides 2006;27:1201-9.
13. Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci USA 1987;8:5449-53.
14. Cunha Filho GA, Schwartz CA, Resck IS, Murta MM, Lemos SS, Castro MS, et al. Antimicrobial activity of the bufadienolides marinobufagin and telo cinobufagin isolated as major components from skin secretion of the toad Bufo rubescens. Toxicon 2005;45:777-82.
15. Wang K, Zheng J. Signaling regulation of fetoplacental angiogenesis. J Endocrinol 2012;212:243-55.
16. Grismer LL, Youmans TM, Wood Jr PL, Ponce A, Johnson R, Wright SB, et al. Checklist of the herpetofauna of Pulau Langkawi with taxonomic comments. Hamadryad 2006;29:15-32.
17. Ibrahim J, Anuar S, Norhayati A, Shukor MN, Shahriza S, Nurul Ain E, et al. An annotated checklist of the herpetofauna of langkawi island, Kedah, Malaysia. Malay Nat J 2003;574:369-81.
18. Norsham Y, Bernard H, Chew KL, Yong HS, Yap MN, Lim BL. An annotated checklist of herpetofauna in the northern part of Belum forest reserve, Perak, Peninsular Malaysia. Malay Nat J 2000;54:245-53.
19. Laemmli UK. Most commonly used discontinuous buffer system for SDS electrophoresis. Nature 1970;227:680-5.
20. Gam LH, Aishah L. Identification and confirmation of urinary human chorionic gonadotropin using in-gel digestion and HPLC/MS/MS analysis. J Biosains 2002;13:27-34.
21. Dahham SS, Ali MN, Tabassum H, Khan M. Studies on antibacterial and antifungal activity of pomegranate (Punica granatum L.). Am Eur J Agric Environ Sci 2010;9:273-81.
22. Dahham SS, Tabana YM, Iqbal MA, Ahamed MB, Ezzat MO, Majid AS, et al. The Anticancer, Antioxidant and antimicrobial properties of the sesquiterpene Î²-caryophyllene from the essential oil of Aquilaria crassna. Molecules 2015;26:11808-29.
23. Maciel NM, Schwartz CA, Junior OR, Sebben A, Castro MS, Sousa MV, et al. Composition of indole alkylamines of Bufo rubescens cutaneous secretions compared to six other Brazilian bufonids with phylogenetic implications. Comp Biochem Physiol Part B: Biochem Mol Biol 2003;134:641-9.
24. Simmaco M, Mignogna G, Barra D. Antimicrobial peptides from amphibian skin: what do they tell us? Pept Sci 1998;47:435-50.
25. Hancock RE, Sahl HG. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 2006;24:1551-7.
26. Conlon JM, Mechkarska M, Prajeep M, Arafat K, Zaric M, Lukic ML, et al. Transformation of the naturally occurring frog skin peptide, alyteserin-2a into a potent, non-toxic anti-cancer agent. Amino Acids 2013;44:715-23.
27. Hancock RE. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001;1:156-64.
28. Smith VJ, Desbois AP, Dyrynda EA. Conventional and unconventional antimicrobials from fish, marine invertebrates and micro-algae. Mar Drugs 2010;8:1213-62.
29. Kawasaki H, Iwamuro S. Potential roles of histones in host defense as antimicrobial agents. Curr Drug Targets Infect Disord 2008;8:195-205.
30. Moraes TB, Ferreira JL, da Rosa CE, Sandrini JZ, Votto AP, Trindade GS, et al. Antioxidant properties of the mucus secreted by Laeonereis acuta (Polychaeta, Nereididae): a defense against environmental pro-oxidants? Comp Biochem Physiol Part C: Toxicol Pharmacol 2006; 142: 293-300.
31. Zhang A, Zhou X, Wang X, Zhou H. Characterization of two heat shock proteins (Hsp70/Hsc70) from grass carp (Ctenopharyngodon idella): evidence for their differential gene expression, protein synthesis and secretion in LPS-challenged peripheral blood lymphocytes. Comp Biochem Physiol Part B: Biochem Mol Biol 2011;159:109-14.
32. Blanchoin L, Boujemaa-Paterski R, Sykes C, Plastino J. Actin dynamics, architecture, and mechanics in cell motility. Physiol Rev 2014;94:235-63.