A sunflower lectin with antifungal properties and putative medical mycology applications.

Lectins are carbohydrate-binding proteins with a high specificity for a variety of glycoconjugate sugar motifs. The jacalin-related lectins (JRL) are considered to be a small sub-family composed of galactose- and mannose-specific members. Using a proteomics approach, we have detected a 16 kDa protein (Helja) in sunflower seedlings that were further purified by mannose-agarose affinity chromatography. The aim of this work was to characterize the biological activity of Helja and to explore potential applications for the antifungal activity of this plant lectin against medically important yeasts. To initially assess the agglutination properties of the lectin, Saccharomyces cerevisiae cells were incubated with increasing concentrations of the purified lectin. At a concentration of 120 µg/ml, Helja clearly agglutinated these cells. The ability of different sugars to inhibit S. cerevisiae cell agglutination determined its carbohydrate-specificity. Among the monosaccharides tested, D-mannose had the greatest inhibitory effect, with a minimal concentration of 1.5 mM required to prevent cell agglutination. The antifungal activity was evaluated using pathogenic fungi belonging to the Candida and Pichia genera. We demonstrate that 200 µg/ml of Helja inhibited the growth of all yeasts, and it induced morphological changes, particularly through pseudohyphae formation on Candida tropicalis. Helja alters the membrane permeability of the tested fungi and is also able to induce the production of reactive oxygen species in C. tropicalis cells. We concluded that Helja is a mannose-binding JRL with cell agglutination capabilities and antifungal activity against yeasts. The biological properties of Helja may have practical applications in the control of human pathogens.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel α-amylase inhibitory properties.

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1) . Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein's biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1) , showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of α-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian α-amylase activity in vitro.

Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds.

The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.

Antimicrobial peptides from chili pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells.

During the last few years, a growing number of cysteine-rich antimicrobial peptides has been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides play an important role in the protection of plants against microbial infection. In this work, proteins from chili pepper (Capsicum annuum L.) seeds were extracted in phosphate buffer, pH 5.4 and peptides purification were performed by employing ion-exchange chromatographies on DEAE, CM-Sepharose, Sephacryl S-100 and reverse phase in HPLC. Three peptide enriched fractions, namely F1, F2 and F3, were obtained after the CM-Sepharose chromatography. The F1 fraction, mainly composed of three peptides ranging from 6 to 10 kDa, was submitted to N-terminal amino acid sequencing. The closer to 10 kDa peptide showed high sequence homology to lipid transfer proteins (LTPs) previously isolated from others seeds. F1 fraction exhibited strong fungicidal activity against Candida albicans, Saccharomyces cerevisiae and Schizosaccharomyces pombe and also promoted several morphological changes to C. albicans, including the formation of pseudohyphae, as revealed by scanning electron micrography. F1 fraction also reduced the glucose stimulated acidification of the medium mediated by H(+)-ATPase of S. cerevisiae cells in a dose-dependent manner and caused the permeabilization of yeast plasma membrane to the dye SYTOX Green, as verified by confocal laser microscopy.