Crystal structure of rice defensin OsAFP1 and molecular insight into lipid-binding.

Defensins are antibacterial peptides that function in the innate immune system. OsAFP1, a defensin identified from Oryza sativa (rice), exhibits antimicrobial activity against rice pathogens. Intriguingly, OsAFP1 was also shown to demonstrate potent antifungal activity against the human pathogenic fungus Candida albicans by inducing apoptosis in target cells, suggesting that OsAFP1 represents a potential new antibiotic candidate; however, further analyses, particularly at the structural level, are required to elucidate the mechanistic underpinnings of OsAFP1 antifungal activity. Here, we determined the three-dimensional structure of OsAFP1 using X-ray crystallography. OsAFP1 features the cysteine-stabilized αß structure highly conserved in plant defensins and presents a dimeric structure that appears necessary for antifungal activity. Superimposition of the OsAFP1 structure with that of Nicotiana alata NaD1 complexed with phosphatidic acid indicated that the target molecule is likely trapped between the S2-S3 loops of each OsAFP1 dimer. In lipid-binding analyses performed using nitrocellulose membranes immobilized with various membrane lipid components, OsAFP1 was found to bind to phosphatidylinositols (PIPs) harboring phosphate groups, particularly PI(3)P. These results indicate that OsAFP1 exerts antifungal activity by binding to PI(3)P contained in the C. albicans cell membrane, thereby applying cellular stress and inducing apoptosis. Furthermore, the OsAFP1 structure and site-specific-mutation analyses revealed that Arg1, His2, Leu4, Arg9, and Phe10 play critical roles in OsAFP1 dimer formation. Thus, our study provides novel insights into the antifungal mechanism of OsAFP1.

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

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.

Partial peptides from rice defensin OsAFP1 exhibited antifungal activity against the rice blast pathogen Pyricularia oryzae.

Rice blast caused by Pyricularia oryzae is one of the most devastating diseases worldwide. This study aimed to investigate the antifungal activity of rice defensin OsAFP1 and its partial peptides against P. oryzae. The partial peptides near the N- and C-terminal regions of OsAFP1 exhibited approximately the same antifungal activity as the entire protein against P. oryzae. These partial peptides have the potential to be used as fungicides.

Identification and characterization of a gene encoding an ABC transporter expressed in the dicarboxylic acid-producing yeast Candida maltosa.

A gene, CmCDR1, encoding an ABC transporter of the dicarboxylic acid (DCA)-producing yeast Candida maltosa was cloned. Transcription of CmCDR1 was upregulated in a DCA-hyper-producing mutant of C. maltosa in a later phase of culture on n-dodecane, but not in its parental strain. CmCDR1 expression was significantly induced by the longer-chain DCA in this mutant.

A Gcn4p homolog is essential for the induction of a ribosomal protein L41 variant responsible for cycloheximide resistance in the yeast Candida maltosa.

Cycloheximide (CYH) resistance in the yeast Candida maltosa is based on the inducible expression of genes encoding a variant of ribosomal protein L41-Q, with glutamine at position 56 instead of the proline found in normal L41. The promoter of L41-Q2a, one of the L41-Q gene alleles encoding L41-Q, has an element similar to the Gcn4p-responsive element of Saccharomyces cerevisiae. In a previous study, this element was shown to be essential for the induction of L41-Q by CYH. In the present study, a C. maltosa GCN4 homolog, C-GCN4, was cloned. It had a long 5'-leader region with three upstream open reading frames. Enhanced expression of the C-GCN4 reporter fusion gene upon the addition of 3-aminotriazole or by mutations in start codons of all three upstream open reading frames indicates that C-GCN4 expression is under translation repression as was seen with GCN4. The C-GCN4-depleted mutant was unable to grow in a nutrient medium containing CYH and did not express L41-Q genes. Recombinant C-Gcn4p bound to the consensus DNA element for Gcn4p, 5'-(G/A)TGACTCAT-3', located upstream of L41-Q2a. Thus, C-Gcn4p, which likely functions in the general control of amino acid biosynthesis, is essential for the expression of L41-Q genes.