Bridging the Gap to Non-toxic Fungal Control: Lupinus-Derived Blad-Containing Oligomer as a Novel Candidate to Combat Human Pathogenic Fungi

Abstract

The lack of antifungal drugs with novel modes of action reaching the clinic is a serious concern. Recently a novel antifungal protein referred to as Blad-containing oligomer (BCO) has received regulatory approval as an agricultural antifungal agent. Interestingly its spectrum of antifungal activity includes human pathogens such as Candida albicans, however, its mode of action has yet to be elucidated. Here we demonstrate that BCO exerts its antifungal activity through inhibition of metal ion homeostasis which results in apoptotic cell death in C. albicans. HIP HOP profiling in Saccharomyces cerevisiae using a panel of signature strains that are characteristic for common modes of action identified hypersensitivity in yeast lacking the iron-dependent transcription factor Aft1 suggesting restricted iron uptake as a mode of action. Furthermore, global transcriptome profiling in C. albicans also identified disruption of metal ion homeostasis as a potential mode of action. Experiments were carried out to assess the effect of divalent metal ions on the antifungal activity of BCO revealing that BCO activity is antagonized by metal ions such as Mn²⁺, Zn²⁺, and Fe²⁺. The transcriptome profile also implicated sterol synthesis as a possible secondary mode of action which was subsequently confirmed in sterol synthesis assays in C. albicans. Animal models for toxicity showed that BCO is generally well tolerated and presents a promising safety profile as a topical applied agent. Given its potent broad spectrum antifungal activity and novel multitarget mode of action, we propose BCO as a promising new antifungal agent for the topical treatment of fungal infections.

Keywords: Blad-containing oligomer; antifungal; metal chelation; metal homeostasis; multitarget mode of action; toxicology.

FIGURE 1

Assigning Blad-containing oligomer (BCO) responsive genes from Candida albicans to functional categories. The average logarithmic (log₂) fold change ratio from three independent experiments is shown. Genes in boldface have a FDR < 0.05. All other genes have a FDR < 0.1.

FIGURE 2

Blad-containing oligomer induced growth inhibition in a panel of yeast HIP HOP “signature strains” that are characteristic for common modes of action. Signature genes were identified previously through high-throughput genome-wide HIP HOP screening of a library of yeast inhibitory small molecules. Growth inhibition of the 27 strains over 24 h was determined in the presence of a sublethal inhibitory dose of BCO (0.024 μM). Cultures of each strain were diluted to OD₆₀₀ 0.00015 and grown in a 384 well plate with and without BCO. Readings were taken every 20 mins for 27 h to produce 48 separate growth curves. Increase in growth over 24 h was then used to determine growth inhibition of each strain relative to the untreated control of the same genetic background. The same procedure was carried out for the isogenic control strain BY4743. Error bars are SEM, n = 12.

FIGURE 3

Effect of sub-inhibitory concentrations of BCO, amphotericin B (AMB), FCL, and caspofungin on the inhibition of ergosterol synthesis in C. albicans. The results represent the mean ± standard deviation of three independent experiments. Statistical analysis was performed as described in the “Materials and Methods” section. Bars with a letter in common are not significantly different (P > 0.05).

FIGURE 4

Induction of C. albicans apoptosis by BCO. Annexin V and propidium iodide staining of C. albicans cells exposed to 2.4 μM BCO for 4 h. Cells labeled with annexin V (A), propidium iodide (B), or simply observed by bright field microscopy (C). Bar corresponds to 10 μm.

FIGURE 5

Detection of endogenous reactive oxygen species (ROS) production. DCFH-CA staining of C. albicans cells exposed for 4 h to 2.4 μM BCO. Cells labeled with DCFH-DA (A) or simply observed by bright field microscopy (B). Bar corresponds to 10 μm.