Genome-wide screening of genes associated with momilactone B sensitivity in the fission yeast Schizosaccharomyces pombe

Abstract

Momilactone B is a natural product with dual biological activities, including antimicrobial and allelopathic properties, and plays a major role in plant chemical defense against competitive plants and pathogens. The pharmacological effects of momilactone B on mammalian cells have also been reported. However, little is known about the molecular and cellular mechanisms underlying its broad bioactivity. In this study, the genetic determinants of momilactone B sensitivity in yeast were explored to gain insight into its mode of action. We screened fission yeast mutants resistant to momilactone B from a pooled culture containing genome-wide gene-overexpressing strains in a drug-hypersensitive genetic background. Overexpression of pmd1, bfr1, pap1, arp9, or SPAC9E9.06c conferred resistance to momilactone B. In addition, a drug-hypersensitive, barcoded deletion library was newly constructed and the genes that imparted altered sensitivity to momilactone B upon deletion were identified. Gene Ontology and fission yeast phenotype ontology enrichment analyses predicted the biological pathways related to the mode of action of momilactone B. The validation of predictions revealed that momilactone B induced abnormal phenotypes such as multiseptated cells and disrupted organization of the microtubule structure. This is the first investigation of the mechanism underlying the antifungal activity of momilactone B against yeast. The results and datasets obtained in this study narrow the possible targets of momilactone B and facilitate further studies regarding its mode of action.

Keywords: chromatin remodeling; microtubule; mode of action; phytoalexin; protein quality control; septum formation.

Figure 1

The chemical structures of momilactone A (left) and momilactone B (right).

Figure 2

Genome-wide screening for multicopy suppressors of momilactone B cytotoxicity. (A) Anti-proliferative activity of momilactone B against the drug-hypersensitive fission yeast. Log-phase cultures of JY265 (parental strain) and YA8 (drug-hypersensitive) were adjusted to the cell density of 10⁷ cells/mL and serially diluted by 10-fold. Aliquots of each dilution were spotted onto YES agar plates containing 1 µM of momilactone B and incubated for 4 days at 30°C. (B) Schematic representation of the screening procedure. A pooled culture containing fission yeast strains overexpressing a different ORF was spread onto EMM agar plates containing 1 µM momilactone B. After the incubation, growing colonies were picked up and the inserted ORFs were identified. (C) Validation of the results from the screening. Log-phase inoculum of the resistant strains in the drug-hypersensitive genetic background identified in the screen and the vector control YA9 were diluted to the cell density of 10⁷ cells/mL and serially diluted by 10-fold. Aliquots of each dilution were spotted onto EMM agar plates with or without 1 µM momilactone B and incubated for 4 days at 30°C.

Figure 3

Genome-wide screening for identifying deletion mutants hypersensitive or resistant to momilactone B. (A) Schematic representation of the screening process. A pooled culture containing 2,195 fission yeast deletion mutants in the drug-hypersensitive genetic background was incubated with or without momilactone B. Subsequently, the genomic DNA was extracted from the culture and the inserted barcode regions were PCR amplified and sequenced. Finally, to measure the relative fitness of each strain, Z-scores were calculated by comparing the read counts from the momilactone B treatment sample to the control. (B) Overall Z-scores from the genome-wide screen. Z-scores from the experiment (0.7 µM momilactone B, #2) were shown as a representative dataset. (C and D) Venn diagrams showing the overlap of deletion mutants identified as hypersensitive (C) or resistant (D) to momilactone B. The total number of genes that upon deletion altered the sensitivity to momilactone B at each dose is indicated in parentheses. (E) Validation of the results from the screening. Log-phase inoculums of the deletion strains of top-hit genes in the drug-hypersensitive genetic background were diluted to an OD₆₀₀ of 0.01 and exposed to 0.3 µM momilactone B in YES liquid medium for 18 h at 30°C. Bars indicate the growth of momilactone B-treated cells, which is normalized relative to the growth of untreated cells (n = 3, mean ± SD). Asterisks indicate significant differences relative to the wild type assessed by Welch’s t-test (*P < 0.01).

Figure 4

Abnormal septum and microtubule formation of S. pombe cells after treated with momilactone B. (A) Wild-type strain L972 was incubated for 24 h at 30°C with 10 µM momilactone B. Cells were fixed and stained with calcofluor white. (B) YMP384 strain expressing Hht1-mRFP and GFP-Atb2 was incubated for 24 h at 30°C with 10 µM momilactone B. Histone and microtubules were observed using fluorescent microscopy after incubation. Scale bar: 10 µm. Arrowheads indicate curved microtubules.