An antifungal peptide from Coffea canephora seeds with sequence homology to glycine-rich proteins exerts membrane permeabilization and nuclear localization in fungi

• Published in: Biochimica et biophysica acta Vol. 1830; no. 6; pp. 3509 - 3516
• Main Authors: Zottich, Umberto, Da Cunha, Maura, Carvalho, André O., Dias, Germana B., Casarin, Nádia, Vasconcelos, Ilka M., Gomes, Valdirene M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2013
• Subjects: 4',6-diamidino-2-phenylindole; amino acids; AMPs; anti-infective properties; Antifungal Agents - chemistry; Antifungal Agents - isolation & purification; Antifungal Agents - pharmacology; Antimicrobial peptides; bacteria; biogenesis; cell walls; Coffea - chemistry; Coffea canephora; Coffee; Colletotrichum lindemuthianum; Defense plant; Fusarium - growth & development; Fusarium oxysporum; Glycine-rich peptide; in vitro studies; light microscopy; membrane permeability; molecular weight; pathogens; Peptides - chemistry; Peptides - isolation & purification; Peptides - pharmacology; plant proteins; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Plant Proteins - pharmacology; pollen; RNA; seeds; Seeds - chemistry; sequence homology; Sequence Homology, Amino Acid; stress tolerance; viruses; yeasts; AMPs; Glycine-rich peptide; Defense plant; Coffee; Antimicrobial peptides
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.03.007

The superfamily of glycine-rich proteins (GRPs) corresponds to a large and complex group of plant proteins that may be involved in many developmental and physiological processes such as RNA biogenesis, stress tolerance, pollen hydration and plant-pathogen interactions, showing defensive activity against fungi, bacteria and viruses. In this study, the peptides from Coffea canephora seeds were extracted according to the methods of Egorov et al. (2005). The purified peptide was submitted for amino acid sequencing and antimicrobial activity measurement. The purified peptide with a molecular weight of 7kDa, named Cc-GRP, was observed to display homology to GRPs. The Cc-GRP–fungi interaction led to morphological changes and membrane permeability, including the formation of pseudohyphae, which were visualized with the aid of SYTOX green dye. Additionally, Cc-GRP also prevented colony formation by yeasts. Antifungal assays of Fusarium oxysporum and Colletotrichum lindemuthianum, observed by light microscopy, showed that the two molds exhibited morphological changes after the growth assay. Cc-GRP coupled to FITC and its subsequent treatment with DAPI revealed the presence of the peptide in the cell wall, cell surface and nucleus of F. oxysporum. In this work we purified, characterized and evaluated the in vitro effect on fungi of a new peptide from coffee, named Cc-GRP, which is involved in the plant defense system against pathogens by acting through a membrane permeabilization mechanism and localized in the nuclei of fungal cells. We also showed, for the first time, the intracellular localization of Cc-GRP during antimicrobial assay. •Cc-GRP shows molecular weight of 7kDa and display homology to glycine rich proteins family.•Cc-GRP–fungi interaction led to yeast membrane permeability.•Cc-GRP exhibited strong antifungal activity against Candida albicans and C. tropicalis yeasts.•Cc-GRP promoted morphological changes in Fusarium oxysporum and Colletotrichum lindemuthianum fungi.•The intracellular localization of a GRP, in nucleus, was showed for the first time.