A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production

Abstract

<i>Candida</i> species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. <i>M. oleifera</i> Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from <i>M. oleifera</i> seeds and to evaluate its antifungal properties against <i>Candida</i> species. The purified protein, named <i>Mo</i>-CBP₂, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, <i>Mo</i>-CBP₂ presented 13,309 Da. However, by SDS-PAGE, <i>Mo</i>-CBP₂ migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of <i>Mo</i>-CBP₂ under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that <i>Mo</i>-CBP₂ exists in different oligomeric forms. Moreover, <i>Mo</i>-CBP₂ is a basic glycoprotein (pI 10.9) with 4.1% (m/m) sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from <i>Mo</i>-CBP₂ in solution, after LC-ESI-MS/MS, revealed similarity with other <i>M. oleifera</i> proteins, as the 2S albumin <i>Mo</i>-CBP₃ and flocculating proteins, and 2S albumins from different species. <i>Mo</i>-CBP₂ possesses <i>in vitro</i> antifungal activity against <i>Candida albicans</i>, <i>C. parapsilosis</i>, <i>C. krusei</i>, and <i>C. tropicalis</i>, with MIC₅₀ and MIC₉₀ values ranging between 9.45-37.90 and 155.84-260.29 μM, respectively. In addition, <i>Mo</i>-CBP₂ (18.90 μM) increased the cell membrane permeabilization and reactive oxygen species production in <i>C. albicans</i> and promoted degradation of circular plasmid DNA (pUC18) from <i>Escherichia coli</i>. The data presented in this study highlight the potential use of <i>Mo</i>-CBP₂ as an anticandidal agent, based on its ability to inhibit <i>Candida</i> spp. growth with apparently low toxicity on mammalian cells.