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

Masayuki Taniguchi,Kodai Ogawa,Takumi Kanaoka,Yoshiyuki Sagehashi,Masahiro Ohori,Takaaki Tanaka,Akihito Ochiai,Minami Fukuda

doi:10.1038/s41598-018-29715-w

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.

Highlights

Plants produce antimicrobial peptides and proteins, e.g. thionin, defensin, lipid transfer protein, cyclotide, hevein-like protein, and knottin-type peptide, as part of their innate immune response to protect themselves against pathogen-associated infections and stress caused by unfavourable environmental conditions[7,8]
The defensin, at concentrations up to 32 μM, did not show any growth inhibition against five bacterial strains, namely, E. coli K-12, Porphyromonas gingivalis ATCC 33277, Streptococcus mutans JCM 5705, Staphylococcus aureus NBRC 12732, and Propionibacterium acnes JCM 6473 (Table 1). This suggested that OsAFP1 is a fungi-specific antibiotic
The activities of L39A and K41A mutants were so low that the IC50 values could not be calculated, in contrast with the remaining mutants (Table 3). These results clearly indicated that amino acid residues within the γ-core motif, in particular Leu-39 and Lys-41, play critical roles in the antifungal activity of OsAFP1

Summary

Introduction

Plants produce antimicrobial peptides and proteins, e.g. thionin, defensin, lipid transfer protein, cyclotide, hevein-like protein, and knottin-type peptide, as part of their innate immune response to protect themselves against pathogen-associated infections and stress caused by unfavourable environmental conditions[7,8] Many of these substances are rich in cysteine residues, which contribute to the stabilization of their structures by disulphide bond formation. DEF7 (designated OsAFP1 in subsequent papers) was reported to exhibit antimicrobial activity against rice pathogenic microbes, such as Pyricularia oryzae[21] and Helminthosporium oryzae[22] Based on this latest knowledge, basic functional analyses of O. sativa OsAFP1 were conducted to assess its potency as a novel antifungal drug. The antimicrobial spectrum of OsAFP1 against various human pathogenic microorganisms, stability upon heat and serum treatments, mechanism of action, and structural factors responsible for its antifungal activity were analysed

Methods

Results

Discussion

Conclusion