Thionin-like peptide from Capsicum annuum fruits: mechanism of action and synergism with fluconazole against Candida species

Abstract

Background: Thionins are a family of plant antimicrobial peptides (AMPs), which participate in plant defense system against pathogens. Here we describe some aspects of the CaThi thionin-like action mechanism, previously isolated from Capsicum annuum fruits. Thionin-like peptide was submitted to antimicrobial activity assays against Candida species for IC50 determination and synergism with fluconazole evaluation. Viability and plasma membrane permeabilization assays, induction of intracellular ROS production analysis and CaThi localization in yeast cells were also investigated.

Results: CaThi had strong antimicrobial activity against six tested pathogenic Candida species, with IC50 ranging from 10 to 40 μg.mL(-1). CaThi antimicrobial activity on Candida species was candidacidal. Moreover, CaThi caused plasma membrane permeabilization in all yeasts tested and induces oxidative stresses only in Candida tropicalis. CaThi was intracellularly localized in C. albicans and C. tropicalis, however localized in nuclei in C. tropicalis, suggesting a possible nuclear target. CaThi performed synergistically with fluconazole inhibiting all tested yeasts, reaching 100% inhibition in C. parapsilosis. The inhibiting concentrations for the synergic pair ranged from 1.3 to 4.0 times below CaThi IC50 and from zero to 2.0 times below fluconazole IC50.

Conclusion: The results reported herein may ultimately contribute to future efforts aiming to employ this plant-derived AMP as a new therapeutic substance against yeasts.

Fig. 1

Cell viability loss. a Photographs of the Petri dishes showing the viability of yeasts cells after the treatment with IC₅₀ CaThi for 24 h. b The table shows the percentage of viability loss of yeasts cells after the treatment with IC₅₀ CaThi for 24 h. CFU = Colony forming unit. (*) Indicates significance by the T test (P < 0.05) among the experiments and their respective controls. The experiments were carried out in triplicate

Fig. 2

Membrane permeabilization assay. Photomicrography of different yeast cells after membrane permeabilization assay by fluorescence microscopy using the fluorescent probe Sytox green. Cells were treated with CaThi for 24 h and then assayed for membrane permeabilization. Control cells were treated only with probe Sytox green. Bars 5 μm

Fig. 3

Oxidative stress assay. Photomicrography of different yeast cells after reactive oxygen species assay detection by fluorescence microscopy using the fluorescent probe 2′,7′ dichlorofluorescein diacetate (H₂DCFDA). Cells were treated with CaThi for 24 h and then assayed for ROS detection. Control cells were treated only with probe (H₂DCFDA). Bars 5 μm

Fig. 4

Localization of CaThi in yeast cells. Photomicrography of Candida albicans and Candida tropicalis cells incubated for 24 h with 10 μg.mL⁻¹ CaThi-FITC (green fluorescence, open arrows) by fluorescence microscopy. Nuclei were visualized by 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) after the CaThi-FITC incubation period (blue fluorescence, filled arrows). Overlap of the DAPI and FITC images (dotted arrows). Bars 20 μm. (*) Indicates the position of digital enlargement

Fig. 5

Effect of CaThi, FLC, and CaThi plus FLC on yeast growth. a Photomicrography of Candida albicans, Candida tropicalis, Candida parapsilosis, Candida pelliculosa, Candida buinensis, and Candida mogii cells by light microscopy after the growth inhibition assay. Bars 5 μm. b Scanning electron microscopy of Candida pelliculosa and Candida buinensis. Filled arrow (formation of pseudohyphae); open arrow (cell agglomeration); asterisk (amorphous material). Bars 10 μm. Cells grown in the presence of Fluconazole (FLC), CaThi, and FLC plus CaThi