Probiotics Antimicrob Proteins. 2021 Oct 20; doi: 10.1007/s12602-021-09865-6. Epub 2021 Oct 20.

Inhibition of Serine Protease, α-Amylase and Growth of Phytopathogenic Fungi by Antimicrobial Peptides from Capsicum chinense Fruits.

Probiotics and antimicrobial proteins

Larissa Maximiano Resende, Érica de Oliveira Mello, Mariana Carvalho de Lima Aguieiras, Celso Shiniti Nagano, Renata Pinheiro Chaves, Gabriel Bonan Taveira, Marciele Souza da Silva, André de Oliveira Carvalho, Rosana Rodrigues, Valdirene Moreira Gomes

PMID: 34671924 DOI: 10.1007/s12602-021-09865-6

Abstract

Plant fungal diseases cause major problems for the global economy. Antimicrobial peptides have aroused great interest in the control of phytopathogens, as they are natural molecules and have a broad spectrum of inhibitory activity. Herein, we have tried to identify and characterize antimicrobial peptides present in fruits of Capsicum chinense and to evaluate their enzymatic and antifungal activities. The retained fraction obtained in the anion exchange chromatography with strong antifungal activity was subjected to molecular exclusion chromatography and obtained four fractions named G1, G2, G3, and G4. The 6.0-kDa protein band of G2 showed similarity with protease inhibitors type II, and it was able to inhibit 100% of trypsin and α-amylase activities. The protein band with approximately 6.5 kDa of G3 showed similarity with sequences of protease inhibitors from genus Capsicum and showed growth inhibition of 48% for Colletotrichum lindemuthianum, 49% for Fusarium lateritium, and 51% for F. solani and F. oxysporum. Additionally, G3 causes morphological changes, membrane permeabilization, and ROS increase in F. oxysporum cells. The 9-kDa protein band of G4 fraction was similar to a nsLTP type 1, and a protein band of 6.5 kDa was similar to a nsLTP type 2. The G4 fraction was able to inhibit 100% of the activities of glycosidases tested and showed growth inhibition of 35 and 50% of F. oxysporum and C. lindemuthianum, respectively. C. chinense fruits have peptides with antifungal activity and enzyme inhibition with biotechnological potential.

© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords: Antifungal; Antimicrobial peptide; Capsicum; Colletotrichum; Fusarium; Growth inhibition

References

1. Fones HN, Fisher MC, Gurr SJ (2017) Emerging fungal threats to plants and animals challenge agriculture and ecosystem resilience. Microbiol Spectr 5. https://doi.org/10.1128/microbiolspec.FUNK-0027-2016 - PubMed
2. Talamini V, Nunes MUC (2018) Estratégias de controle das principais doenças do tomateiro orgânico na região central de Sergipe. Embrapa Tabuleiros Costeiros-Comunicado Técnic, Aracajú, SE.1ª edição - PubMed
3. Revie NM, Iyer KR, Robbins N, Cowen LE (2018) Antifungal drug resistance: evolution, mechanisms and impact. Curr Opin Microbiol 45:70–76. https://doi.org/10.1016/j.mib.2018.02.005 - PubMed
4. Spader TB, Venturini TP, Rossato L et al (2013) Synergysm of voriconazole or itraconazole with other antifungal agents against species of Fusarium. Rev Iberoam Micol 30:200–204. https://doi.org/10.1016/j.riam.2013.01.002 - PubMed
5. Camiletti BX, Asensio CM, Gadban LC et al (2016) Essential oils and their combinations with iprodione fungicide as potential antifungal agents against withe rot (Sclerotium cepivorum Berk) in garlic (Allium sativum L.) crops. Ind Crops Prod 85:117–124. https://doi.org/10.1016/j.indcrop.2016.02.053 - PubMed
6. Ayoub F, Ben ON, Ayoub M et al (2018) In field control of Botrytis cinerea by synergistic action of a fungicide and organic sanitizer. J Integr Agric 17:1401–1408. https://doi.org/10.1016/S2095-3119(17)61875-6 - PubMed
7. Sharma C, Chowdhary A (2017) Molecular bases of antifungal resistance in filamentous fungi. Int J Antimicrob Agents 50:607–616. https://doi.org/10.1016/j.ijantimicag.2017.06.018 - PubMed
8. Zhu M, Liu P, Niu ZW (2017) A perspective on general direction and challenges facing antimicrobial peptides. Chinese Chem Lett 28:703–708. https://doi.org/10.1016/j.cclet.2016.10.001 - PubMed
9. Campos ML, Lião LM, Alves ESF et al (2018) A structural perspective of plant antimicrobial peptides. Biochem J 475:3359–3375. https://doi.org/10.1042/BCJ20180213 - PubMed
10. Mahlapuu M, Håkansson J, Ringstad L, Björn C (2016) Antimicrobial peptides: an emerging category of therapeutic agents. Front Cell Infect Microbiol 6:194. https://doi.org/10.3389/fcimb.2016.00194 - PubMed
11. Das K, Datta K, Karmakar S, Datta SK (2019) Antimicrobial peptides - small but mighty weapons for plants to fight phytopathogens. Protein Pept Lett 26:720–742. https://doi.org/10.2174/0929866526666190619112438 - PubMed
12. Koehbach J, Craik DJ (2019) The vast structural diversity of antimicrobial peptides. Trends Pharmacol Sci 40:517–528. https://doi.org/10.1016/j.tips.2019.04.012 - PubMed
13. Yeats TH, Rose JKC (2008) The biochemistry and biology of extracellular plant lipid-transfer proteins (LTPs). Protein Sci 17:191–198. https://doi.org/10.1110/ps.073300108 - PubMed
14. Odintsova TI, Slezina MP, Istomina EA et al (2019) Non-specific lipid transfer proteins in Triticum kiharae dorof. Et migush.: identification, characterization and expression profiling in response to pathogens and resistance inducers. Pathogens 8:221. https://doi.org/10.3390/pathogens8040221 - PubMed
15. Bard GCV, Zottich U, Souza TAM et al (2016) Purification, biochemical characterization, and antimicrobial activity of a new lipid transfer protein from Coffea canephora seeds. Genet Mol Res 15. https://doi.org/10.4238/gmr15048859 - PubMed
16. de Carvalho AO, Gomes VM (2007) Role of plant lipid transfer proteins in plant cell physiology - a concise review. Peptides 28:1144–1153. https://doi.org/10.1016/j.peptides.2007.03.004 - PubMed
17. Regente MC, de la Canal L (2000) Purification, characterization and antifungal properties of a lipid-transfer protein from sunflower (Helianthus annuus) seeds. Physiol Plant 110:158–163. https://doi.org/10.1034/j.1399-3054.2000.110203.x - PubMed
18. Nishimura S, Tatano S, Gomi K et al (2008) Chloroplast-localized nonspecific lipid transfer protein with anti-fungal activity from rough lemon. Physiol Mol Plant Pathol 72:134–140. https://doi.org/10.1016/j.pmpp.2008.07.003 - PubMed
19. Diz MS, Carvalho AO, Ribeiro SFFF et al (2011) Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel α-amylase inhibitory properties. Physiol Plant 142:233–246. https://doi.org/10.1111/j.1399-3054.2011.01464.x - PubMed
20. Zottich U, Da Cunha M, Carvalho AO et al (2011) Purification, biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties. Biochim Biophys Acta - Gen Subj 1810:375–383. https://doi.org/10.1016/j.bbagen.2010.12.002 - PubMed
21. Bogdanov IV, Shenkarev ZO, Finkina EI et al (2016) A novel lipid transfer protein from the pea Pisum sativum: isolation, recombinant expression, solution structure, antifungal activity, lipid binding, and allergenic properties. BMC Plant Biol 16:107. https://doi.org/10.1186/s12870-016-0792-6 - PubMed
22. Schmitt AJ, Sathoff AE, Holl C et al (2018) The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity. J Exp Bot 69(22):5587–5597. https://doi.org/10.1093/jxb/ery319 - PubMed
23. Clemente M, Corigliano M, Pariani S et al (2019) Plant serine protease inhibitors: biotechnology application in agriculture and molecular farming. Int J Mol Sci 20:1345. https://doi.org/10.3390/ijms20061345 - PubMed
24. Hellinger R, Gruber CW (2019) Peptide-based protease inhibitors from plants. Drug Discov Today 24:1877–1889. https://doi.org/10.1016/j.drudis.2019.05.026 - PubMed
25. Giudici AM, Regente MC, de la Canal L (2000) A potent antifungal protein from Helianthus annuus flowersis a trypsin inhibitor. Plant Physiol Biochem 38:881–888. https://doi.org/10.1016/S0981-9428(00)01191-8 - PubMed
26. Araújo NMS, Dias LP, Costa HPS et al (2019) ClTI, a Kunitz trypsin inhibitor purified from Cassia leiandra Benth. seeds, exerts a candidicidal effect on Candida albicans by inducing oxidative stress and necrosis. Biochim Biophys Acta - Biomembr 1861:183032. https://doi.org/10.1016/j.bbamem.2019.183032 - PubMed
27. Bleackley MR, Dawson CS, McKenna JA et al (2017) Synergistic activity between two antifungal proteins, the plant defensin NaD1 and the bovine pancreatic trypsin inhibitor. mSphere 2(5):e00390–17. https://doi.org/10.1128/mSphere.00390-17 - PubMed
28. Ribeiro SFF, Fernandes KVS, Santos IS et al (2013) New small proteinase inhibitors from Capsicum annuum seeds: characterization, stability, spectroscopic analysis and a cDNA cloning. Biopolymers 100:132–140. https://doi.org/10.1002/bip.22172 - PubMed
29. Silva MS, Ribeiro SFF, Taveira GB et al (2017) 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 97:3790–3801. https://doi.org/10.1002/jsfa.8243 - PubMed
30. Zhang X, Guo K, Dong Z et al (2020) Kunitz-type protease inhibitor BmSPI51 plays an antifungal role in the silkworm cocoon. Insect Biochem Mol Biol 116:103258. https://doi.org/10.1016/j.ibmb.2019.103258 - PubMed
31. Afroz M, Akter S, Ahmed A et al (2020) Ethnobotany and antimicrobial peptides from plants of the Solanaceae family: an update and future prospects. Front Pharmacol 11:565. https://doi.org/10.3389/fphar.2020.00565 - PubMed
32. Ribeiro SFF, Carvalho AO, Da Cunha M et al (2007) Isolation and characterization of novel peptides from chilli pepper seeds: antimicrobial activities against pathogenic yeasts. Toxicon 50:600–611. https://doi.org/10.1016/j.toxicon.2007.05.005 - PubMed
33. Taveira GB, Mathias LS, da Motta OV et al (2014) Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts. Biopolym - Pept Sci Sect 102:30–39. https://doi.org/10.1002/bip.22351 - PubMed
34. Maracahipes ÁC, Taveira GB, Mello EO et al (2019) Biochemical analysis of antimicrobial peptides in two different Capsicum genotypes after fruit infection by Colletotrichum gloeosporioides. Biosci Rep 39(4):BSR20181889. https://doi.org/10.1042/BSR20181889 - PubMed
35. Aguieiras MCL, Resende LM, Souza TAM et al (2021) Potent anti-Candida fraction isolated from Capsicum chinense fruits contains an antimicrobial peptide that is similar to plant defensin and is able to inhibit the activity of different α-amylase enzymes. Probiotics Antimicrob Proteins 13:862–872. https://doi.org/10.1007/s12602-020-09739-3 - PubMed
36. Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379. https://doi.org/10.1016/0003-2697(87)90587-2 - PubMed
37. Shevchenko A, Tomas H, Havli J et al (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860. https://doi.org/10.1038/nprot.2006.468 - PubMed
38. Carneiro RF, Viana JT, Torres RCF et al (2019) A new mucin-binding lectin from the marine sponge Aplysina fulva (AFL) exhibits antibiofilm effects. Arch Biochem Biophys 662:169–176. https://doi.org/10.1016/j.abb.2018.12.014 - PubMed
39. Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2 - PubMed
40. Larkin MA, Blackshields G, Brown NP et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. https://doi.org/10.1093/bioinformatics/btm404 - PubMed
41. Broekaert W, Terras FRG, Cammue BPA, Vanderleyden J (1990) An automated quantitative assay for fungal growth inhibition. FEMS Microbiol Lett 69:55–59. https://doi.org/10.1016/0378-1097(90)90412-J - PubMed
42. Thevissen K, Terras FRG, Broekaert WF (1999) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl Environ Microbiol 65:5451–5458. https://doi.org/10.1128/AEM.65.12.5451-5458.1999 - PubMed
43. Mello EO, Ribeiro SFF, Carvalho AO et al (2011) Antifungal activity of PvD - PubMed
44. da Silva FCV, do Nascimento VV, Fernandes KV, et al (2018) Recombinant production and α-amylase inhibitory activity of the lipid transfer protein from Vigna unguiculata (L. Walp.) seeds. Process Biochem 65:205–212. https://doi.org/10.1016/j.procbio.2017.10.018 - PubMed
45. Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85. https://doi.org/10.1016/0003-2697(85)90442-7 - PubMed
46. Franco OL, Rigden DJ, Melo FR et al (2000) Activity of wheat α-amylase inhibitors towards bruchid α-amylases and structural explanation of observed specificities. Eur J Biochem 267:2166–2173. https://doi.org/10.1046/j.1432-1327.2000.01199.x - PubMed
47. Meneguetti BT, dos Machado L, S, Oshiro KGN, et al (2017) Antimicrobial peptides from fruits and their potential use as biotechnological Tools-a review and outlook. Front Microbiol 7:2136 - PubMed
48. Moguel-Salazar F (2011) A review of a promising therapeutic and agronomical alternative: Antimicrobial peptides from Capsicum sp. African J Biotechnol 10:19918–19928. https://doi.org/10.5897/AJBX11.070 - PubMed
49. dos Santos L, de A, Taveira GB, Ribeiro S de FF, et al (2017) 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 132:97–107. https://doi.org/10.1016/j.pep.2017.01.013 - PubMed
50. Santos LA, Taveira GB, Silva MS et al (2020) Antimicrobial peptides from Capsicum chinense fruits: agronomic alternatives against phytopathogenic fungi. Biosci Rep 40(8):BSR20200950.  https://doi.org/10.1042/BSR20200950 - PubMed
51. Khani S, Seyedjavadi SS, Zare-Zardini H et al (2019) Isolation and functional characterization of an antifungal hydrophilic peptide, Skh-AMP1, derived from Satureja khuzistanica leaves. Phytochemistry 164:136–143. https://doi.org/10.1016/j.phytochem.2019.05.011 - PubMed
52. Kirubakaran SI, Begum SM, Ulaganathan K, Sakthivel N (2008) Characterization of a new antifungal lipid transfer protein from wheat. Plant Physiol Biochem 46:918–927. https://doi.org/10.1016/j.plaphy.2008.05.007 - PubMed
53. Moore J, Rajasekaran K, Cary JW, Chlan C (2019) Mode of action of the antimicrobial peptide D4E1 on Aspergillus flavus. Int J Pept Res Ther 25:1135–1145. https://doi.org/10.1007/s10989-018-9762-1 - PubMed
54. Pizzo E, Zanfardino A, Di Giuseppe AMA et al (2015) A new active antimicrobial peptide from PD-L4, a type 1 ribosome inactivating protein of Phytolacca dioica L.: a new function of RIPs for plant defence? FEBS Lett 589:2812–2818. https://doi.org/10.1016/j.febslet.2015.08.018 - PubMed
55. Maracahipes ÁC, Taveira GB, Sousa-Machado LY et al (2019) Characterization and antifungal activity of a plant peptide expressed in the interaction between Capsicum annuum fruits and the anthracnose fungus. Biosci Rep 39(12):BSR20192803. https://doi.org/10.1042/BSR20192803 - PubMed
56. Paege N, Warnecke D, Zäuner S et al (2019) Species-specific differences in the susceptibility of fungi to the antifungal protein AFP depend on C-3 saturation of glycosylceramides. mSphere 4(6):e00741–19. https://doi.org/10.1128/mSphere.00741-19 - PubMed
57. Corrêa JAF, Evangelista AG, de Nazareth T, M, Luciano FB, (2019) Fundamentals on the molecular mechanism of action of antimicrobial peptides. Materialia 8:100494. https://doi.org/10.1016/j.mtla.2019.100494 - PubMed
58. Soares JR, Tenório J, de Melo E, da Cunha M et al (2017) Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress. Biochim Biophys Acta - Gen Subj 1861:3429–3443. https://doi.org/10.1016/j.bbagen.2016.09.005 - PubMed
59. Osborn RW, De Samblanx GW, Thevissen K et al (1995) Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett 368:257–262. https://doi.org/10.1016/0014-5793(95)00666-W - PubMed
60. Zottich U, Da Cunha M, Carvalho AO et al (2013) An antifungal peptide from Coffea canephora seeds with sequence homology to glycine-rich proteins exerts membrane permeabilization and nuclear localization in fungi. Biochim Biophys Acta - Gen Subj 1830:3509–3516. https://doi.org/10.1016/j.bbagen.2013.03.007 - PubMed
61. Broekaert WF, Terras F, Cammue B, Osborn RW (1995) Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol 108:1353–1358. https://doi.org/10.1104/pp.108.4.1353 - PubMed
62. Boyce KJ, Chang H, D’Souza CA, Kronstad JW (2005) An Ustilago maydis septin is required for filamentous growth in culture and for full symptom development on maize. Eukaryot Cell 4:2044–2056. https://doi.org/10.1128/EC.4.12.2044-2056.2005 - PubMed
63. Pelegrini PB, Lay FT, Murad AM et al (2008) Novel insights on the mechanism of action of α-amylase inhibitors from the plant defensin family. Proteins Struct Funct Genet 73:719–729. https://doi.org/10.1002/prot.22086 - PubMed
64. Islamov RA, Fursov OV (2007) Bifunctional inhibitor of α-amylase/trypsin from wheat grain. Appl Biochem Microbiol 43:379–382. https://doi.org/10.1134/S0003683807040035 - PubMed
65. Gadge PP, Wagh SK, Shaikh FK et al (2015) A bifunctional α-amylase/trypsin inhibitor from pigeonpea seeds: Purification, biochemical characterization and its bio-efficacy against Helicoverpa armigera. Pestic Biochem Physiol 125:17–25. https://doi.org/10.1016/j.pestbp.2015.06.007 - PubMed
66. Pereira L da S, do Nascimento VV, Ribeiro S de FF et al (2018) Characterization of Capsicum annuum L. leaf and root antimicrobial peptides: antimicrobial activity against phytopathogenic microorganisms. Acta Physiol Plant 40:107. https://doi.org/10.1007/s11738-018-2685-9 - PubMed
67. Tamhane VA, Giri AP, Sainani MN, Gupta VS (2007) Diverse forms of Pin-II family proteinase inhibitors from Capsicum annuum adversely affect the growth and development of Helicoverpa armigera. Gene 403:29–38. https://doi.org/10.1016/j.gene.2007.07.024 - PubMed
68. da Silva FCV, Pessoa Costa E, Moreira Gomes V, de Oliveira CA (2020) Inhibition mechanism of human salivary α-amylase by lipid transfer protein from Vigna unguiculata. Comput Biol Chem 85:107193. https://doi.org/10.1016/j.compbiolchem.2019.107193 - PubMed
69. Tormo MA, Gil-Exojo I, de Tejada AR, Campillo JE (2004) Hypoglycaemic and anorexigenic activities of an α-amylase inhibitor from white kidney beans (Phaseolus vulgaris) in Wistar rats. Br J Nutr 92:785–790. https://doi.org/10.1079/bjn20041260 - PubMed

Publication Types

• Journal Article

Grant support

• 305495/ 2017-8/CNPq
• E-26/202.330/2019/FAPERJ
• E-26/202.616/2019/FAPERJ
• finance code 001/CAPES
• E-26/202.132/2015/Faperj