Role of heme in the antifungal activity of the azaoxoaporphine alkaloid sampangine

Abstract

Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In the present study, transcriptional profiling experiments coupled with analyses of mutants were performed in an effort to elucidate its mechanism of action. Using Saccharomyces cerevisiae as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low-oxygen conditions. Several additional lines of evidence obtained suggest that these responses could involve effects on heme. First, the hem1Delta mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells. In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine. Furthermore, spectral analyses of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells. Transcriptional profiling experiments were also performed with C. albicans to investigate the response of a pathogenic fungal species to sampangine. Taking into account the known differences in the physiological responses of C. albicans and S. cerevisiae to low oxygen, significant correlations were observed between the two transcription profiles, suggestive of heme-related defects. Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme.

FIG. 1.

Distribution of genes responding to sampangine in S. cerevisiae. Data are shown for a subset of genes that were significantly induced or repressed (P value, ≤0.001) in three biological replicate samples. Data were organized into various biological processes using GO Term Finder and GO Slim Mapper tools in SGD. GO terms shown are those that were considered significantly overrepresented by the analyses. The terms “cell wall organization” and “other processes” were included separately to highlight additional genes that are relevant to the overall biological response to sampangine. For simplicity, not all “child terms” within a “parent term” are shown. Numbers in parentheses represent average change in gene expression. Positive values indicate induction, and negative values indicate repression. A complete list of all significant genes can be found in Table S1 in the supplemental material.

FIG. 2.

Role of heme in the activity of sampangine in S. cerevisiae. (A) Dilutions (fivefold) of wild-type (BY4741) and hem1Δ (yMH339) strains grown as described in Materials and Methods were inoculated onto YPD + ALA (8 μg/ml) agar medium and incubated for 2 days at 30°C. −SMP, medium containing solvent (DMSO); +SMP, medium containing sampangine at 1.25 μg/ml. (B) Results shown are from broth microdilution assays performed in triplicate using S. cerevisiae strain S288C in the presence of various concentrations (Conc.) of sampangine with 32.5 μg/ml hemin (+hemin) or without hemin (−hemin). Percent growth is shown as the mean ± standard error of the mean (SEM). (C) S. cerevisiae S288C colonies grown on YPD agar were streaked on SD agar medium containing various concentrations of sampangine and incubated for 3 days at 30°C. The 0 represents medium containing solvent (DMSO). (D) Absorption spectra of alkaline pyridine extracts prepared from S. cerevisiae S288C cells treated with sampangine (solid line) or with DMSO solvent (dashed line). (E) Results are shown from broth microdilution assays performed in triplicate with S. cerevisiae strain S288C in the presence of various concentrations of sampangine with 10 μg/ml ergosterol (+Ergo) or without ergosterol (−Ergo). Percent growth is shown as the mean ± SEM.

FIG. 3.

Effect of sampangine on the growth of heterozygous mutants harboring deletions in heme biosynthetic genes. Wild-type (BY4743), HEM1/hem1Δ, HEM2/hem2Δ, HEM3/hem3Δ, HEM4/hem4Δ, HEM12/hem12Δ, HEM13/hem13Δ, HEM14/hem14Δ, and HEM15/hem15Δ strains were grown as described in Materials and Methods. Dilutions (fivefold) were prepared from each culture, inoculated onto SC agar medium, and incubated for 3 days at 30°C. −SMP, medium containing solvent (DMSO); +SMP, medium containing sampangine at 2.0 μg/ml.

FIG. 4.

Role of oxidative stress in the activity of sampangine in S. cerevisiae. (A) S. cerevisiae S288C cells in early exponential phase were grown in the presence or absence of sampangine (SMP) for the indicated amount of time. Cells were harvested, protein extracts were prepared, and 20 μg of protein from each extract was derivatized with DNPH. The derivatized proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted to polyvinylidene difluoride membrane, and detected with anti-DNP antibody. A stained protein gel on which aliquots of the same samples were separated is shown on the right. (B) Dilutions (fivefold) of wild-type (BY4743), yap1Δ/Δ, and sod2Δ/Δ strains grown as described in Materials and Methods were inoculated onto SC agar medium and incubated for 2 days at 30°C. −SMP, medium containing solvent (DMSO); +SMP, medium containing sampangine at 1.25 μg/ml.

FIG. 5.

Effect of CIN5 overexpression and deletion on susceptibility to sampangine. (A) JK93dα cells containing vector pRS426 (TXSc001) and a CIN5-overexpressing plasmid (TXSc025) were inoculated onto SC-Ura agar medium and incubated for 5 days at 30°C. (B) Dilutions (fivefold) of wild-type (BY4743) and cin5Δ/Δ strains were inoculated onto SC agar medium and incubated for 2 days at 30°C. −SMP, medium containing solvent (DMSO); +SMP, medium containing sampangine at 4 μg/ml (A) and 1.25 μg/ml (B).

FIG. 6.

Distribution of genes responding to sampangine in C. albicans. Data are shown for a subset of genes that were significantly induced (P value, ≤0.001) in three biological replicate samples. Data were organized into various biological processes using the GO Term Finder tool in CGD. GO terms shown are those that were considered significantly overrepresented by the analysis. For simplicity, not all “child terms” within a “parent term” are shown. Numbers in parentheses represent average changes in gene expression. Gene names shown represent S. cerevisiae homologs. The systematic (orf19) name from CGD is used for genes with no known S. cerevisiae homologs. A complete list of all significant genes can be found in Table S2 in the supplemental material.

FIG. 7.

Role of heme in the activity of sampangine in C. albicans. (A) Dilutions (fivefold) of wild-type (BWP17) and hem1Δ/Δ (KRC1) strains grown as described in Materials and Methods were inoculated onto YPD + Uri + ALA (8 μg/ml) agar medium and incubated for 2 days at 30°C. −SMP, medium containing solvent (DMSO); +SMP, medium containing sampangine at 5 μg/ml. (B) Results shown are from broth microdilution assays performed in triplicate on C. albicans strain SC5314 in the presence of various concentrations (Conc.) of sampangine with 32.5 μg/ml hemin (+hemin) or without hemin (−hemin). Percent growth is shown as the mean ± SEM. (C) C. albicans strain SC5314 colonies grown on YPD agar were streaked on SD agar medium containing various concentrations (Conc.) of sampangine and incubated for 3 days at 30°C. The 0 represents medium containing solvent (DMSO). (D) Absorption spectra of alkaline pyridine extracts prepared from C. albicans strain SC5314 cells treated with sampangine (solid line) or with DMSO solvent (dashed line). (E) Results are shown from broth microdilution assays performed in triplicate on C. albicans strain SC5314 in the presence of various concentrations (Conc.) of sampangine with 10 μg/ml ergosterol (+Ergo) or without ergosterol (−Ergo). Percent growth is shown as the mean ± SEM.