ABSTRACT
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.
Subject(s)
Antifungal Agents , Candida , Protease Inhibitors , Antifungal Agents/pharmacology , Candida/drug effects , Candida/growth & development , Protease Inhibitors/pharmacology , Microbial Sensitivity Tests , Animals , Capsicum/microbiology , Reactive Oxygen Species/metabolism , Seeds/growth & development , Plant Extracts/pharmacology , Plant Extracts/chemistry , Erythrocytes/drug effects , Larva/microbiology , Larva/growth & development , Larva/drug effectsABSTRACT
In the present study, we identified and characterized two defensin-like peptides in an antifungal fraction obtained from Capsicum chinense pepper fruits and inhibited the growth of Colletotrichum scovillei, which causes anthracnose. AMPs were extracted from the pericarp of C. chinense peppers and subjected to ion exchange, molecular exclusion, and reversed-phase in a high-performance liquid chromatography system. We investigated the endogenous increase in reactive oxygen species (ROS), the loss of mitochondrial functioning, and the ultrastructure of hyphae. The peptides obtained from the G3 fraction through molecular exclusion chromatography were subsequently fractionated using reverse-phase chromatography, resulting in the isolation of fractions F1, F2, F3, F4, and F5. The F1-Fraction suppressed C. scovillei growth by 90, 70.4, and 44% at 100, 50, and 25 µg mL-1, respectively. At 24 h, the IC50 and minimum inhibitory concentration were 21.5 µg mL-1 and 200 µg mL-1, respectively. We found an increase in ROS, which may have resulted in an oxidative burst, loss of mitochondrial functioning, and cytoplasm retraction, as well as an increase in autophagic vacuoles. MS/MS analysis of the F1-Fraction indicated the presence of two defensin-like proteins, and we were able to identify the expression of three defensin sequences in our C. chinense fruit extract. The F1-Fraction was also found to inhibit the activity of insect α-amylases. In summary, the F1-Fraction of C. chinense exhibits antifungal activity against a major pepper pathogen that causes anthracnose. These defensin-like compounds are promising prospects for further research into antifungal and insecticide biotechnology applications. © 2024 Society of Chemical Industry.
Subject(s)
Capsicum , Colletotrichum , Defensins , Mitochondria , Reactive Oxygen Species , Colletotrichum/drug effects , Colletotrichum/growth & development , Capsicum/microbiology , Reactive Oxygen Species/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Defensins/pharmacology , Defensins/chemistry , Antifungal Agents/pharmacology , Antifungal Agents/chemistry , Plant Proteins/pharmacology , Plant Proteins/chemistry , Plant Proteins/metabolism , Fruit/microbiologyABSTRACT
Chagas disease is a neglected tropical disease caused by the protozoan pathogen Trypanosoma cruzi. The disease is a major public health problem affecting about 6 to 7 million people worldwide, mostly in Latin America. The available therapy for this disease is based on two drugs, nifurtimox and benznidazole, which exhibit severe side effects, including resistance, severe cytotoxicity, variable efficacy and inefficiency in the chronic phase. Therefore, new drugs are urgently needed. Coordination compounds may be an interesting alternative for antiparasite therapy against Leishmania spp., Toxoplasma gondii and T. cruzi. Herein, we tested the in vitro effect on T. cruzi epimastigotes (Y strain) of two new µ-oxo Fe(III) dinuclear complexes: [(HL1)(Cl)Fe(µ-O)Fe(Cl)(HL2)](Cl)2·(CH3CH2OH)2·H2O (1) and [(HL2)(Cl)Fe(µ-O)Fe(Cl)(HL2)](Cl)2·H2O (2) where HL1 and HL2 are ligands which contain two pyridines, amine and alcohol moieties with a naphthyl pendant unit yielding a N3O coordination environment. Complexes (1) and (2), which are isomers, were completely characterized, including X-ray diffraction studies for complex (1). Parasites were treated with the complexes and the outcome was analyzed. Complex (1) exhibited the lowest IC50 values, which were 99 ± 3, 97 ± 2 and 110 ± 39 nM, after 48, 72 and 120 h of treatment, respectively. Complex (2) showed IC50 values of 118 ± 5, 122 ± 6 and 104 ± 29 nM for the same treatment times. Low cytotoxicity to the host cell LLC-MK2 was found for both complexes, resulting in impressive selectivity indexes of 106 for complex (1) and 178 for (2), after 120 h of treatment. Treatment with both complexes reduced the mitochondrial membrane potential of the parasite. Ultrastructural analysis of the parasite after treatment with complexes showed that the mitochondria outer membrane presented swelling and abnormal disposition around the kinetoplast; in addition, reservosomes presented anomalous spicules and rupture. The complexes showed low nanomolar IC50 values affecting mitochondria and reservosomes, essential organelles for the survival of the parasite. The low IC50 and the high selectivity index show that both complexes act as a new prototype of drugs against T. cruzi and may be used for further development in drug discovery to treat Chagas disease.