Login Register Cart 0
Generic placeholder image

Protein & Peptide Letters

Editor-in-Chief

ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Bifunctional Inhibitors from Capsicum chinense Seeds with Antimicrobial Activity and Specific Mechanism of Action Against Phytopathogenic Fungi

Author(s): Marciele Souza da Silva, Valdirene Moreira Gomes*, Gabriel Bonan Taveira, Layrana de Azevedo dos Santos, Álan C. Maracahipes, Rosana Rodrigues, André de Oliveira Carvalho, Katia Valevski S. Fernandes and Antonia Elenir A. Oliveira

Volume 28, Issue 2, 2021

Published on: 17 June, 2020

Page: [149 - 163] Pages: 15

DOI: 10.2174/0929866527666200617124221

Price: $65

Abstract

Background: Antimicrobial peptides (AMPs) are found in the defense system in virtually all life forms, being present in many, if not all, plant species.

Objective: The present work evaluated the antimicrobial, enzymatic activity and mechanism of action of the PEF2 fraction from Capsicum chinense Jack. seeds against phytopathogenic fungi.

Methods: Peptides were extracted from C. chinense seeds and subjected to reverse-phase chromatography on an HPLC system using a C18 column coupled to a C8 guard column, then the obtained PEF2 fraction was rechromatographed using a C2/C18 column. Two fractions, named PEF2A and PEF2B, were obtained. The fractions were tested for antimicrobial activity on Colletotrichum gloeosporioides, Colletotrichum lindemuthianum, Fusarium oxysporum and Fusarium solani. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and α-amylase activity assays were also performed. The mechanism of action by which PEF2 acts on filamentous fungi was studied through analysis of membrane permeability and production of reactive oxygen species (ROS). Additionally, we investigated mitochondrial functionality and caspase activation in fungal cells.

Results: It is possible to observe that PEF2 significantly inhibited trypsin activity and T. molitor larval α-amylase activity. The PEF2 fraction was able to inhibit the growth of C. gloeosporioides, C. lindemuthianum and F. oxysporum. PEF2A inhibited the growth of C. lindemuthianum (75%) and F. solani (43%). PEF2B inhibited C. lindemuthianum growth (66%) and F. solani (94%). PEF2 permeabilized F. solani cell membranes and induced ROS in F. oxysporum and F. solani. PEF2 could dissipate mitochondrial membrane potential but did not cause the activation of caspases in all studied fungi.

Conclusion: The results may contribute to the biotechnological application of these AMPs in the control of pathogenic microorganisms in plants of agronomic importance.

Keywords: Plant defense, pepper, antimicrobial peptides, Fusarium, protease inhibitor, α-amylase inhibitor.

« Previous Next »
Graphical Abstract

[1]
Perry, L.; Dickau, R.; Zarrillo, S.; Holst, I.; Pearsall, D.M.; Piperno, D.R.; Berman, M.J.; Cooke, R.G.; Rademaker, K.; Ranere, A.J.; Raymond, J.S.; Sandweiss, D.H.; Scaramelli, F.; Tarble, K.; Zeidler, J.A. Starch fossils and the domestication and dispersal of chili peppers (Capsicum spp. L.) in the Americas. Science, 2007, 315(5814), 986-988.
[http://dx.doi.org/10.1126/science.1136914] [PMID: 17303753]
[2]
Carrizo, G.C.; Sterpetti, M.; Volpi, P.; Ummarino, M.; Saccardo, F. Wild Capsicums: identification and in situ analysis of Brazilian species XVth EUCARPIA meeting on genetics and breeding of Capsicum and Eggplant, 2013, 205-213.
[3]
Nascimento, K.O.; Vicente, J.; Saldanha, T.; Barbosa, J.J.L.; Barbosa, M.I.M.J. Caracterização química e informação nutricional de geleia de pimenta Cambuci orgânica (Capsicum baccatum L.). Rev. Verde, 2012, 7(2), 283-288.
[4]
Hill, T.A.; Ashrafi, H.; Wo, R.C.S.; Yao, J.; Stoffel, K.; Truco, J.M.; Kozik, A.; Michelmore, R.W.; Deynze, A.V. Characterization of Capsicum annuum genetic diversity and population structure based on parallel polymorphism discovery with a 30K unigene pepper gene chip. PLoS One, 2013, 8(2), 1-16.
[http://dx.doi.org/10.1371/journal.pone.0056200]
[5]
Maracahipes, A.C.; Viscovini, K.K.C.G.; Annunciatto, E.S.; Neves, L.G.; Da Luz, P.B.; Araujo, K.L. Genetic diversity of the germplasm active bank of’ Capsicum of UNEMAT based on components resistant to the fungus Colletotrichum gloeosporioides. Aust. J. Crop Sci., 2016, 10(7), 940-948.
[http://dx.doi.org/10.21475/ajcs.2016.10.07.p7437]
[6]
Silva, M.S.; Ribeiro, S.F.F.; Taveira, G.B.; Rodrigues, R.; Fernandes, K.V.S.; Carvalho, A.O.; Vasconcelos, I.M.; Mello, E.O.; Gomes, V.M. Application and bioactive properties of CaTI, a trypsin inhibitor from Capsicum annuum seeds: membrane permeabilization, oxidative stress and intracellular target in phytopathogenic fungi cells. J. Sci. Food Agric., 2017, 97(11), 3790-3801.
[http://dx.doi.org/10.1002/jsfa.8243] [PMID: 28139827]
[7]
Dos Santos, L.A.; Taveira, G.B.; Ribeiro, S.F.F.; Pereira, L.D.S.; Carvalho, A.O.; Rodrigues, R.; Oliveira, A.E.A.; Machado, O.L.T.; Araújo, J.D.S.; Vasconcelos, I.M.; Gomes, V.M. Purification and characterization of peptides from Capsicum annuum fruits which are α-amylase inhibitors and exhibit high antimicrobial activity against fungi of agronomic importance. Protein Expr. Purif., 2017, 132, 97-107.
[http://dx.doi.org/10.1016/j.pep.2017.01.013] [PMID: 28161544]
[8]
Nordström, R.; Malmsten, M. Delivery systems for antimicrobial peptides. Adv. Colloid Interface Sci., 2017, 242, 17-34.
[http://dx.doi.org/10.1016/j.cis.2017.01.005] [PMID: 28159168]
[9]
Giuliani, A.; Pirri, G.; Nicoletto, S.F. Antimicrobial peptides: an overview of a promising class of therapeutics. Electron. J. Biotechnol., 2007, 2(1), 1-33.
[10]
Baumann, T.; Kämpfer, U.; Schürch, S.; Schaller, J.; Largiadèr, C.; Nentwig, W.; Kuhn-Nentwig, L. Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei. Cell. Mol. Life Sci., 2010, 67(16), 2787-2798.
[http://dx.doi.org/10.1007/s00018-010-0364-0] [PMID: 20369272]
[11]
Cammue, B.P.; De Bolle, M.F.; Terras, F.R.; Proost, P.; Van Damme, J.; Rees, S.B.; Vanderleyden, J.; Broekaert, W.F. Isolation and characterization of a novel class of plant antimicrobial peptides form Mirabilis jalapa L. seeds. J. Biol. Chem., 1992, 267(4), 2228-2233.
[PMID: 1733929]
[12]
Benko-Iseppon, A.M.; Galdino, S.L.; Calsa, T., Jr; Kido, E.A.; Tossi, A.; Belarmino, L.C.; Crovella, S. Overview on plant antimicrobial peptides. Curr. Protein Pept. Sci., 2010, 11(3), 181-188.
[http://dx.doi.org/10.2174/138920310791112075] [PMID: 20088772]
[13]
Brogden, K.A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol., 2005, 3(3), 238-250.
[http://dx.doi.org/10.1038/nrmicro1098] [PMID: 15703760]
[14]
Andrés, M.T.; Acosta-Zaldívar, M.; Fierro, J.F. Antifungal mechanism of action of lactoferrin: identification of H+-ATPase (P3A-Type) as a new apoptotic-cell membrane receptor. Antimicrob. Agents Chemother., 2016, 60(7), 4206-4216.
[http://dx.doi.org/10.1128/AAC.03130-15] [PMID: 27139463]
[15]
Peters, B.M.; Shirtliff, M.E.; Jabra-Rizk, M.A. Antimicrobial peptides: primeval molecules or future drugs? PLoS Pathog., 2010, 6(10), e1001067.
[http://dx.doi.org/10.1371/journal.ppat.1001067] [PMID: 21060861]
[16]
Schneider, T.; Kruse, T.; Wimmer, R.; Wiedemann, I.; Sass, V.; Pag, U.; Jansen, A.; Nielsen, A.K.; Mygind, P.H.; Raventós, D.S.; Neve, S.; Ravn, B.; Bonvin, A.M.; De Maria, L.; Andersen, A.S.; Gammelgaard, L.K.; Sahl, H.G.; Kristensen, H.H. Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II. Science, 2010, 328(5982), 1168-1172.
[http://dx.doi.org/10.1126/science.1185723] [PMID: 20508130]
[17]
Tang, S.S.; Prodhan, Z.H.; Biswas, S.K.; Le, C.F.; Sekaran, S.D. Antimicrobial peptides from different plant sources: isolation, characterisation, and purification. Phytochemistry, 2018, 154, 94-105.
[http://dx.doi.org/10.1016/j.phytochem.2018.07.002] [PMID: 30031244]
[18]
Franco, O.L.; Rigden, D.J.; Melo, F.R.; Grossi-De-Sá, M.F. Plant alpha-amylase inhibitors and their interaction with insect alpha-amylases. Eur. J. Biochem., 2002, 269(2), 397-412.
[http://dx.doi.org/10.1046/j.0014-2956.2001.02656.x] [PMID: 11856298]
[19]
Valueva, T.A.; Mosolov, V.V. Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms. Biochemistry (Mosc.), 2004, 69(11), 1305-1309.
[http://dx.doi.org/10.1007/s10541-005-0015-5] [PMID: 15627384]
[20]
Dayler, C.S.A.; Mendes, P.A.M.; Prates, M.V.; Bloch, C., Jr; Franco, O.L.; Grossi-de-Sá, M.F. Identification of a novel bean α-amylase inhibitor with chitinolytic activity. FEBS Lett., 2005, 579(25), 5616-5620.
[http://dx.doi.org/10.1016/j.febslet.2005.09.030] [PMID: 16213488]
[21]
Diz, M.S.S.; Carvalho, A.O.; Rodrigues, R.; Neves-Ferreira, A.G.; Da Cunha, M.; Alves, E.W.; Okorokova-Façanha, A.L.; Oliveira, M.A.; Perales, J.; Machado, O.L.; Gomes, V.M. Antimicrobial peptides from chili pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells. Biochim. Biophys. Acta, 2006, 1760(9), 1323-1332.
[http://dx.doi.org/10.1016/j.bbagen.2006.04.010] [PMID: 16784815]
[22]
Taveira, G.B.; Mathias, L.S.; da Motta, O.V.; Machado, O.L.T.; Rodrigues, R.; Carvalho, A.O.; Teixeira-Ferreira, A.; Perales, J.; Vasconcelos, I.M.; Gomes, V.M. Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts. Biopolymers, 2014, 102(1), 30-39.
[http://dx.doi.org/10.1002/bip.22351] [PMID: 23896704]
[23]
Ribeiro, S.F.F.; Silva, M.S.; Da Cunha, M.; Carvalho, A.O.; Dias, G.B.; Rabelo, G.; Mello, É.O.; Santa-Catarina, C.; Rodrigues, R.; Gomes, V.M. Capsicum annuum L. trypsin inhibitor as a template scaffold for new drug development against pathogenic yeast A Van Leeuw., 2012, 101(3), 657-670.
[24]
Dias, G.B.; Gomes, V.M.; Pereira, U.Z.; Ribeiro, S.F.; Carvalho, A.O.; Rodrigues, R.; Machado, O.L.; Fernandes, K.V.; Ferreira, A.T.; Perales, J.; Da Cunha, M.; Cunha, M.D. Isolation, characterization and antifungal activity of proteinase inhibitors from Capsicum chinense Jacq. Seeds. Protein J., 2013, 32(1), 15-26.
[http://dx.doi.org/10.1007/s10930-012-9456-z] [PMID: 23117889]
[25]
Schägger, H.; von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem., 1987, 166(2), 368-379.
[http://dx.doi.org/10.1016/0003-2697(87)90587-2] [PMID: 2449095]
[26]
Macedo, M.L.R.; Garcia, V.A.; Freire, Md.; Richardson, M. Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd. Phytochemistry, 2007, 68(8), 1104-1111.
[http://dx.doi.org/10.1016/j.phytochem.2007.01.024] [PMID: 17363015]
[27]
Felicioli, R.; Garzelli, B.; Vaccari, L.; Melfi, D.; Balestreri, E. Activity staining of protein inhibitors of proteases on gelatin-containing polyacrylamide gel electrophoresis. Anal. Biochem., 1997, 244(1), 176-179.
[http://dx.doi.org/10.1006/abio.1996.9917] [PMID: 9025927]
[28]
da Silva, F.C.V.; do Nascimento, V.V.; Machado, O.L.T.; Pereira, L.D.S.; Gomes, V.M.; de Oliveira Carvalho, A. Insight into the α-Amylase inhibitory activity of plant lipid transfer proteins. J. Chem. Inf. Model., 2018, 58(11), 2294-2304.
[http://dx.doi.org/10.1021/acs.jcim.8b00540] [PMID: 30388003]
[29]
Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano, M.D.; Fujimoto, E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C. Measurement of protein using bicinchoninic acid. Anal. Biochem., 1985, 150(1), 76-85.
[http://dx.doi.org/10.1016/0003-2697(85)90442-7] [PMID: 3843705]
[30]
Thevissen, K.; Terras, F.R.G.; Broekaert, W.F. Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl. Environ. Microbiol., 1999, 65(12), 5451-5458.
[http://dx.doi.org/10.1128/AEM.65.12.5451-5458.1999] [PMID: 10584003]
[31]
Mello, E.O.; Ribeiro, S.F.F.; Carvalho, A.O.; Santos, I.S.; Da Cunha, M.; Santa-Catarina, C.; Gomes, V.M. Antifungal activity of PvD1 defensin involves plasma membrane permeabilization, inhibition of medium acidification, and induction of ROS in fungi cells. Curr. Microbiol., 2011, 62(4), 1209-1217.
[http://dx.doi.org/10.1007/s00284-010-9847-3] [PMID: 21170711]
[32]
Taveira, G.B.; Mello, E.O.; Souza, S.B.; Monteiro, R.M.; Ramos, A.C.; Carvalho, A.O.; Rodrigues, R.; Okorokov, L.A.; Gomes, V.M. Programmed cell death in yeast by thionin-like peptide from Capsicum annuum fruits involving activation of caspases and extracellular H+ flux. Biosci. Rep., 2018, 38(2), 38.
[http://dx.doi.org/10.1042/BSR20180119] [PMID: 29599127]
[33]
Mishra, M.; Mahajan, N.; Tamhane, V.A.; Kulkarni, M.J.; Baldwin, I.T.; Gupta, V.S.; Giri, A.P. Stress inducible proteinase inhibitor diversity in Capsicum annuum. BMC Plant Biol., 2012, 12(12), 217.
[http://dx.doi.org/10.1186/1471-2229-12-217] [PMID: 23153298]
[34]
Strange, R.N.; Scott, P.R. Plant disease: a threat to global food security. Annu. Rev. Phytopathol., 2005, 43(1), 83-116.
[http://dx.doi.org/10.1146/annurev.phyto.43.113004.133839] [PMID: 16078878]
[35]
Vriens, K.; Cammue, B.P.A.; Thevissen, K. Antifungal plant defensins: mechanisms of action and production. Molecules, 2014, 19(8), 12280-12303.
[http://dx.doi.org/10.3390/molecules190812280] [PMID: 25153857]
[36]
Jamal, F.; Pandey, P.K.; Singh, D.; Khan, M.Y. Serine protease inhibitors in plants: nature’s arsenal crafted for insect predators. Phytochem. Rev., 2013, 12(1), 1-34.
[http://dx.doi.org/10.1007/s11101-012-9231-y]
[37]
Svensson, B.; Fukuda, K.; Nielsen, P.K.; Bønsager, B.C. Proteinaceous alpha-amylase inhibitors. Biochim. Biophys. Acta, 2004, 1696(2), 145-156.
[http://dx.doi.org/10.1016/j.bbapap.2003.07.004] [PMID: 14871655]
[38]
Silva, S.M.; Koehnlein, E.A.; Bracht, A.; Castoldi, R.; Morais, R.G.; Baesso, M.L.; Peralta, R.A.; Souza, C.G.M.; Sá-Nakanishi, B.A.; Sá-Nakanishi, A.B.; Peralta, R.M. Inhibition of salivary and pancreatic α-amylases by a pinhão coat (Araucaria angustifolia) extract rich in condensed tannin. Food Res. Int., 2014, 56, 1-8.
[http://dx.doi.org/10.1016/j.foodres.2013.12.004]
[39]
Islamov, R.A.; Furusov, O.V. Bifunctional inhibitor of alpha-amylase/trypsin from wheat grain. Prikl. Biokhim. Mikrobiol., 2007, 43(4), 419-423.
[PMID: 17929568]
[40]
Gadge, P.P.; Wagh, S.K.; Shaikh, F.K.; Tak, R.D.; Padul, M.V.; Kachole, M.S. A bifunctional α-amylase/trypsin inhibitor from pigeonpea seeds: purification, biochemical characterization and its bio-efficacy against Helicoverpa armigera. Pestic. Biochem. Physiol., 2015, 125, 17-25.
[http://dx.doi.org/10.1016/j.pestbp.2015.06.007] [PMID: 26615146]
[41]
Revina, T.A.; Gerasimova, N.G.; Kladnitskaia, G.V.; Chalenko, G.I.; Valueva, T.A. Effect of proteinaceous proteinase inhibitors from potato tubers on the growth and development of phytopathogenic microorganisms. Prikl. Biokhim. Mikrobiol., 2008, 44(1), 101-105.
[PMID: 18491605]
[42]
Antcheva, N.; Patthy, A.; Athanasiadis, A.; Tchorbanov, B.; Zakhariev, S.; Pongor, S. Primary structure and specificity of a serine proteinase inhibitor from paprika (Capsicum annuum) seeds. Biochim. Biophys. Acta, 1996, 1298(1), 95-101.
[http://dx.doi.org/10.1016/S0167-4838(96)00121-5] [PMID: 8948493]
[43]
Silva, R.G.G.; Vasconcelos, I.M.; Filho, A.J.U.B.; Carvalho, A.F.U.; Souza, T.M.; Gondima, D.M.F.; Varela, A.L.N.; Oliveira, J.T.A. Castor bean cake contains a trypsin inhibitor that displays antifungal activity against Colletotrichum gloeosporioides and inhibits the midgut proteases of the dengue mosquito larvae. Ind. Crops Prod., 2015, 70, 48-55.
[http://dx.doi.org/10.1016/j.indcrop.2015.02.058]
[44]
Jenssen, H.; Hamill, P.; Hancock, R.E.W. Peptide antimicrobial agents. Clin. Microbiol. Rev., 2006, 19(3), 491-511.
[http://dx.doi.org/10.1128/CMR.00056-05] [PMID: 16847082]
[45]
Dib, H.X.; de Oliveira, D.G.L.; de Oliveira, C.F.R.; Taveira, G.B.; de Oliveira Mello, E.; Verbisk, N.V.; Chang, M.R.; Corrêa Junior, D.; Gomes, V.M.; Macedo, M.L.R. Biochemical characterization of a Kunitz inhibitor from Inga edulis seeds with antifungal activity against Candida spp. Arch. Microbiol., 2019, 201(2), 223-233.
[http://dx.doi.org/10.1007/s00203-018-1598-8] [PMID: 30483842]
[46]
Teixeira, V.; Feio, M.J.; Bastos, M. Role of lipids in the interaction of antimicrobial peptides with membranes. Prog. Lipid Res., 2012, 51(2), 149-177.
[http://dx.doi.org/10.1016/j.plipres.2011.12.005] [PMID: 22245454]
[47]
Addabbo, F.; Montagnani, M.; Goligorsky, M.S. Mitochondria and reactive oxygen species. Hypertension, 2009, 53(6), 885-892.
[http://dx.doi.org/10.1161/HYPERTENSIONAHA.109.130054] [PMID: 19398655]
[48]
Wang, K.; Dang, W.; Xie, J.; Zhu, R.; Sun, M.; Jia, F.; Zhao, Y.; An, X.; Qiu, S.; Li, X.; Ma, Z.; Yan, W.; Wang, R. Antimicrobial peptide protonectin disturbs the membrane integrity and induces ROS production in yeast cells. Biochim. Biophys. Acta, 2015, 1848(10 Pt A), 2365-2373.
[http://dx.doi.org/10.1016/j.bbamem.2015.07.008] [PMID: 26209560]
[49]
Scandalios, J.G. Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz. J. Med. Biol. Res., 2005, 38(7), 995-1014.
[http://dx.doi.org/10.1590/S0100-879X2005000700003] [PMID: 16007271]
[50]
Taveira, G.B.; Carvalho, A.O.; Rodrigues, R.; Trindade, F.G.; Da Cunha, M.; Gomes, V.M. Thionin-like peptide from Capsicum annuum fruits: mechanism of action and synergism with fluconazole against Candida species. BMC Microbiol., 2016, 16, 12.
[http://dx.doi.org/10.1186/s12866-016-0626-6] [PMID: 26819228]
[51]
Rocha, G.L.; Fernandez, J.H.; Oliveira, A.E.A.; Fernandes, K.V.S. Programmed cell death-related proteases in plants Enzyme Inhibitors and Activators., Senturk, M.; Ed., IntechOpen. Available from: . https://www.intechopen.com/books/enzyme -inhibitors-and-activators/programmed-cell-death-related-proteases-in-plants
[52]
Hwang, B.; Hwang, J.S.; Lee, J.; Lee, D.G. The antimicrobial peptide, psacotheasin induces reactive oxygen species and triggers apoptosis in Candida albicans. Biochem. Biophys. Res. Commun., 2011, 405(2), 267-271.
[http://dx.doi.org/10.1016/j.bbrc.2011.01.026] [PMID: 21219857]
[53]
Moguel-Salazar, F.; Brito-Argáez, L.; Díaz-Brito, M.; Islas-Flores, I. A review of a promising therapeutic and agronomical alternative: Antimicrobial peptides from Capsicum sp. Afr. J. Biotechnol., 2011, 10(86), 19918-19928.
[http://dx.doi.org/10.5897/AJBX11.070]

Rights & Permissions Print Cite
Article Metrics
31
© 2024 Bentham Science Publishers | Privacy Policy