Understanding the mechanism of action of protease inhibitors in controlling the growth of the Candida Genus: potential candidates for development of new antifungal molecules.

There is a growing imperative for research into alternative compounds for the treatment of the fungal infections. Thus, many studies have focused on the analysis of antifungal proteins and peptides from different plant sources. Among these molecules are protease inhibitors (PIs). Previously, PIs present in the peptide-rich fractions called PEF1, PEF2 and PEF3 were identified from Capsicum chinense seeds, which have strong activity against phytopathogenic fungi. The aim of this study was to evaluate the mechanism of action and antimicrobial activity of PIs from PEF2 and PEF3 on the growth of yeasts of the genus Candida. In this work, analyses of their antimicrobial activity and cell viability were carried out. Subsequently, the mechanism of action by which the PIs cause the death of the yeasts was evaluated. Cytotoxicity was assessed in vitro by erythrocytes lysis and in vivo in Galleria mellonella larvae. PEF2 and PEF3 caused 100% of the growth inhibition of C. tropicalis and C. buinensis. For C. albicans inhibition was approximately 60% for both fractions. The PEF2 and PEF3 caused a reduction in mitochondrial functionality of 54% and 46% for C. albicans, 26% and 30% for C. tropicalis, and 71% and 68% for C. buinensis, respectively. These fractions induced morphological alterations, led to membrane permeabilization, elevated ROS levels, and resulted in necrotic cell death in C. tropicalis, whilst demonstrating low toxicity toward host cells. From the results obtained here, we intend to contribute to the understanding of the action of PIs in the control of fungal diseases of medical importance.

Anti-Candida Potential of Peptides from Immature and Ripe Fruits of Capsicum chinense Jacq.

The objective of this work was to purify and evaluate the antifungal potential of peptides present in immature and ripe fruits of Capsicum chinense Jacq. (accession UENF 1706) on the medical importance yeasts. Initially the proteins of these seedless fruits were extracted, precipitated with ammonium sulfate at 70% saturation, followed by heating at 80 °C. Subsequently, the peptide-rich extract was fractionated by DEAE-Sepharose anion exchange. The whole process was monitored by tricine-SDS-PAGE. The results revealed that the fraction retained in anion exchange column, called D2, of immature and ripe fruits significantly inhibit the growth of Candida albicans and C. tropicalis yeasts. Due to the higher yield, the D2 fraction of immature fruits was selected for further purification by reverse phase chromatography on HPLC, where sixteen different fractions (H1-H16) were obtained and these were subjected to antifungal assay at 50 µg mL-1. Although almost all fractions tested had significant growth inhibition, the HI9 fraction inhibit 99% of the two yeasts tested. The effect of treatment with HI3, HI8, HI9, and HI14 fractions on the viability of yeast cells was analyzed due to their strong growth inhibition. We observed that only 50 µg mL-1 of the HI9 fraction is the lethal dose for 100% of the cells of C. albicans and C. tropicalis in the original assay. Although the HI9 fraction had a fungicidal effect on both tested yeasts, we only observed membrane permeabilization for C. tropicalis cells treated with 50 µg mL-1 of this fraction. Through mass spectrometry, we identified that the 6 kDa peptide band of HI9 fraction showed similarity with antimicrobial peptides belonging to the plant defensin family.

Biotechnological applications of versatile plant lipid transfer proteins (LTPs).

Plant AMPs are usually cysteine-rich, and can be classified in several classes, including lipid transfer proteins (LTPs). LTPs are small plant cationic peptides, and can be classified in two subclasses, LTP1 (9-10 kDa) and LTP2 (7 kDa). They have been identified and isolated from various plant species and can be involved in a number of processes, including responses against several phytopathogens. LTP1 presents 4 parallel α- helices and a 310-helix fragment. These structures form a tunnel with large and small entrances. LTP2 presents 3 parallel α- helices, which form a cavity with triangular structure. Both LTP subclasses present a hydrophobic cavity, which makes interaction with different lipids and general hydrophobic molecules possible. Several studies report a broad spectrum of activity of plant LTPs, including antibacterial, antifungal, antiviral, antitumoral, and insecticidal activity. Thus, these molecules can be employed in human and animal health as an alternative to the conventional treatment of disease, well as providing the source of novel drugs. However, employing peptides in human health can present challenges, such as the toxicity of peptides, the difference between the results found in in vitro assays and in pre-clinical or clinical tests and their low efficiency against Gram-negative bacteria. In this context, plant LTPs can be an interesting alternative means by which to bypass such challenges. This review addresses the versatility of plant LTPs, their broad spectrum of activities and their potential applications in human and animal health and in agricultural production, and examines challenges in their biotechnological application.

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.

Antimicrobial peptides (AMPs) are molecules present in several life forms, possess broad-spectrum of inhibitory activity against pathogenic microorganisms, and are a promising alternative to combat the multidrug resistant pathogens. The aim of this work was to identify and characterize AMPs from Capsicum chinense fruits and to evaluate their inhibitory activities against yeasts of the genus Candida and α-amylases. Initially, after protein extraction from fruits, the extract was submitted to anion exchange chromatography resulting two fractions. Fraction D1 was further fractionated by molecular exclusion chromatography, and three fractions were obtained. These fractions showed low molecular mass peptides, and in fraction F3, only two protein bands of approximately 6.5 kDa were observed. Through mass spectrometry, we identified that the lowest molecular mass protein band of fraction F3 showed similarity with AMPs from plant defensin family. We named this peptide CcDef3 (Capsicum chinense defensin 3). The antifungal activity of these fractions was analyzed against yeasts of the genus Candida. At 200 µg/mL, fraction F1 inhibited the growth of C. tropicalis by 26%, fraction F2 inhibited 35% of the growth of C. buinensis, and fraction F3 inhibited all tested yeasts, exhibiting greater inhibition activity on the growth of the yeast C. albicans (86%) followed by C. buinensis (69%) and C. tropicalis (21%). Fractions F1 and F2 promoted membrane permeabilization of all tested yeasts and increased the endogenous induction of reactive oxygen species (ROS) in C. buinensis and C. tropicalis, respectively. We also observed that fraction F3 at a concentration of 50 µg/mL inhibited the α-amylase activities of Tenebrio molitor larvae by 96% and human salivary by 100%. Thus, our results show that fraction F3, which contains CcDef3, is a very promising protein fraction because it has antifungal potential and is able to inhibit the activity of different α-amylase enzymes.