Rice Defensin OsAFP1 is a New Drug Candidate against Human Pathogenic Fungi

Abstract

Fungal infections, such as candidiasis and aspergillosis, are some of the most frequent infections in humans. Although antifungal drugs are available for the treatment of these infections, antifungal agents with new mechanisms of action should be developed because of the increasing incidence of drug-resistant pathogens in recent years. In this study, a basic functional analysis of rice defensin OsAFP1, a novel antifungal drug candidate, was conducted. OsAFP1 exerted fungicidal activity against Candida albicans, the most common pathogenic fungus in humans, at 4 μM concentration, but it did not inhibit the growth of human pathogenic bacteria. In addition, OsAFP1 retained structural stability after heat treatment at 100 °C for 10 min and after serum treatment at 37 °C for 24 h. A propidium iodide (PI) uptake assay and mutational analysis revealed that amino acid residues within the C-terminal γ-core motif of OsAFP1, particularly Leu-39 and Lys-41, play an important role in its antifungal activity. Further, PI uptake and apoptosis assays suggested that OsAFP1 exerts its antifungal activity by inducing apoptosis of target cells. Immunohistochemistry showed that the OsAFP1 target molecule was located in the cell wall. These findings indicate that OsAFP1 may be developed into a potent antifungal drug.

Figure 1

Antifungal activity and stability of OsAFP1. Antifungal activity of OsAFP1 against C. albicans CAI4 (a) and S. cerevisiae BY4742 (b) cells. Cells were incubated with different OsAFP1 concentrations, as indicated. After incubation, OD₆₅₀ was measured. (c) Fungicidal effect of OsAFP1. C. albicans CAI4 cells were incubated with OsAFP1 at 16 μM (4 × MIC) for the time indicated. After incubation, CFUs at each time point were calculated. (d) Antifungal activity of OsAFP1 after heat treatment at 100 °C for 10 min. C. albicans CAI4 cells were incubated with different concentrations of heat-treated OsAFP1 (0, 0.25, 0.5, 1, 2, 4, and 8 μM). Data for panel (a–d) are presented as the means ± standard deviations (n = 3). (e) Biodegradation of OsAFP1 after serum treatment. OsAFP1 was incubated with human serum at 37 °C for 0, 6, 12, and 24 h. OsAFP1 degradation at each time point was analysed by Tricine-SDS-PAGE. Data are presented as means ± standard deviation (n = 2). The culture conditions and full experimental details are described in the Materials and Methods section.

Figure 2

PI uptake assay. C. albicans CAI4 cells were treated with PBS (a), melittin (b), OsAFP1 (c), peptide 1 (d), or peptide 7 (e) at 4 × IC₅₀ (6 μM melittin, 8 μM OsAFP1, 20 μM peptide 1, and 40 μM peptide 7) and incubated at 30 °C for 3 h. The PI fluorescence of the cells was analysed by flow cytometry; the cell counts are enumerated in the right field of each histogram. The experimental details have been provided in the Materials and Methods section.

Figure 3

Primary structure of OsAFP1 and design of its partial peptides. (a) Alignment of amino acid sequences of plant defensins: OsAFP1 from rice (UniProtKB accession no.: Q6K209); MtDef4 from M. truncatula (UniProtKB accession no.: G7L736); MsDef1 from M. sativa (UniProtKB accession no.: Q9FPM3); RsAFP2 from R. sativus (UniProtKB accession no.: P30230); Psd1 from P. sativum (UniProtKB accession no.: P81929); and NaD1 from N. alata (UniProtKB accession no.: Q8GTM0). Amino acid sequences were aligned using the Clustal Omega program. Identical, strongly conserved, and weakly conserved amino acid residues are denoted by asterisks, colons, and dots, respectively. Putative secondary structural elements of OsAFP1 are displayed above the sequences. Cysteine residues that are presumed to form disulphide bonds are highlighted in black. The C-terminal γ-core motif is enclosed within a solid box. (b) Designed peptides corresponding to partial amino acid sequences of OsAFP1. The peptides correspond to eight overlapping OsAFP1 sequence fragments.

Figure 4

Microscopic observations of C. albicans CAI4 apoptotic cells. C. albicans CAI4 cells were treated with OsAFP1 at 16 μM (4 × MIC) and incubated at 30 °C for 0, 0.5, 1, 2, and 4 h. At each time point, apoptotic cells were observed under an epifluorescence microscope. C. albicans CAI4 cells treated with water and incubated at 30 °C for 4 h was used as a negative control. The top row corresponds to bright field, and the middle and bottom row correspond to dark field microscopic images. The white bars correspond to 10 μm, and the scale is the same in all images. The method is described in detail in the Materials and Methods section.

Figure 5

ICC staining of C. albicans CAI4 treated with OsAFP1. C. albicans cells treated without (a) or with (b) OsAFP1 were analysed by ICC staining. C. albicans CAI4 cells were treated with OsAFP1 at 16 μM (4 × MIC), incubated at 30 °C for 0.5 h, and used for ICC staining. The method is described in detail in the Materials and Methods section.

Figure 6

Homology model of OsAFP1. (a) The homology model of OsAFP1 generated using the MODELLER program. Detailed parameters are specified in the Materials and Methods section. The surface regions corresponding to peptides 1 and 7 are coloured orange and red, respectively. (b) Detailed loop structure corresponding to peptide 7. The atoms of the amino acid residues are designated by different colours, as follows: oxygen, red; nitrogen, deep blue; carbon, magenta; sulphur, yellow.