Using a combination of genomic and genetic approaches, we have shown that heme plays an important role in the antifungal activity of the plant alkaloid sampangine. This conclusion is based on several lines of evidence obtained from the present work. (i) The transcriptome response of S. cerevisiae to sampangine is indicative of a hypoxia-like response, which is known to be mediated by heme through the transcription factors Hap1p, Hap2/3/4/5p, Rox1p, and Upc2p. (ii) Ergosterol reduced the susceptibility of S. cerevisiae to sampangine, suggesting that exposure to sampangine mimics heme deficiency, a condition which allows the uptake of exogenous sterol. (iii) A hem1Δ mutant of both S. cerevisiae and C. albicans exhibited increased sensitivity to sampangine, and exogenous heme provided as hemin reduced the sensitivity to sampangine in both organisms. (iv) Upon prolonged exposure to high concentrations of sampangine, both organisms produced red pigmentation which can be attributed to increased levels of heme biosynthetic precursors (free porphyrins) in the cells, based on spectrophotometric analysis of alkaline pyridine extracts. (v) S. cerevisiae mutants with heterozygous deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased sensitivity to sampangine.
While our results strongly suggest that the antifungal activity of sampangine most likely involves a disruption in heme metabolism or function, the exact mechanism behind this effect is unclear at this time. One possibility is that sampangine directly inhibits the activity of one of the enzymes in the heme biosynthetic pathway. However, it is unlikely that sampangine inhibits the enzyme ALA synthase (the first committed step in heme biosynthesis), given that exogenously supplied ALA had no apparent effect on sensitivity to sampangine (data not shown). In addition, the production of red pigmentation in the presence of sampangine, indicating the accumulation of porphyrin intermediates, would be consistent with pathway inhibition occurring at a later step.
Sampangine could also interfere with heme metabolism through indirect mechanisms, such as causing a reduction in available iron or the misdirection of biosynthetic intermediates, since heme biosynthesis in S. cerevisiae
occurs as a multistep pathway spatially separated between the cytosol and the mitochondria (37
). Heme biosynthesis is tightly coupled to iron availability due to the requirement of iron by the enzyme ferrochelatase, which catalyzes the final step in the heme biosynthetic pathway (38
). Two mitochondrial iron transporters, Mrs3p and Mrs4p, have been shown to play an important role in the rapid transport of iron into the mitochondria for heme biosynthesis in S. cerevisiae
). Interestingly, MRS4
is induced by sampangine treatment in S. cerevisiae
cells (see Table S1 in the supplemental material), which could indicate a response to decreased mitochondrial iron levels. As mentioned above, another possibility is that sampangine could be involved in the misdirection of heme biosynthetic intermediates. In S. cerevisiae
, most of the porphyrin intermediates are synthesized in the cytosol, and the final two steps leading to the synthesis of heme occur in the mitochondria. Thus, if sampangine were to, for example, interfere with the transport of porphyrin intermediates into the mitochondria, it would cause a reduction in the synthesis of heme. ATP-binding cassette-type transporters involved in mitochondrial porphyrin transport have been identified for mammalian systems (reviewed in reference 24
), although the corresponding transporters in S. cerevisiae
have yet to be identified.
From the results obtained for S. cerevisiae
, it is evident that sampangine induces oxidative stress. This conclusion is supported by the following observations: (i) the induction of oxidative stress-responsive genes, (ii) the increased sensitivity of deletion mutants of YAP1
to sampangine, and (iii) the increased levels of protein carbonylation in the presence of sampangine. These results are consistent with previous reports indicating that exposure to sampangine, as well as to the structurally related marine alkaloid ascididemin, induces the formation of reactive oxygen species in cancer cell lines (33
). While the present data do not point to a specific mechanism for how sampangine could cause oxidative stress, one possibility would be directly related to the iminoquinone moiety within its structure (Table ), which could participate in redox cycling reactions leading to the generation of reactive oxygen species within cells (48
). Alternatively, oxidative stress could occur as a by-product of heme deficiency through the resultant accumulation of porphyrins, which are known to be potent generators of singlet oxygen in the presence of light (26
). Heme depletion could also result in the generation of superoxide radicals through a mechanism involving a decrease in the activity of complex IV, the heme A-containing terminal complex in the electron transport chain, resulting in the leakage of electrons to molecular oxygen. Such a mechanism has been demonstrated with human fibroblasts (reviewed in reference 3
). Further experimentation will be required to determine whether oxidative stress and perturbations in heme metabolism represent distinct or interrelated mechanisms in the inhibitory activity of sampangine.
As previously discussed, significant parallels were observed between the transcriptional responses in S. cerevisiae
and C. albicans
to sampangine treatment, although in S. cerevisiae
, the global response was highly reminiscent of hypoxia-induced gene expression changes, which was not apparent in the case of C. albicans
. This disparity is perhaps not unexpected given the very different strategies employed by these two organisms in responding to alterations in cellular oxygen levels. While S. cerevisiae
responds to hypoxia by altering respiration, sterol transport, and cell wall biogenesis (reviewed in reference 35
), C. albicans
responds to hypoxia primarily by inducing glycolysis and the expression of hypha-specific genes (61
). Setiadi et al. (61
) have suggested that transcription factors required for regulating the hypoxic response in S. cerevisiae
are either missing or have acquired divergent functions in C. albicans
. For example, no apparent homolog of HAP1
exists in C. albicans
, and the RFG1
gene of C. albicans
(homolog of ROX1
) does not participate in the regulation of hypoxic genes but instead functions in the regulation of filamentous growth (29
). Based on these considerations, the differences seen in the genomic profiling of sampangine exposure between these species are not surprising, given the possibility that heme-related pathways could be altered by this compound. A clearer answer could likely be obtained from transcription profiling studies using comparable heme-deficient mutants from both organisms.
Given its essential role in numerous cellular processes, heme biosynthesis or heme signaling could serve as a highly effective target for inhibiting fungal growth and could contribute to a chemical defense strategy against fungal pathogens in aporphinoid-producing plant species of the Annonaceae
). However, due to the importance of heme in all eukaryotes, a therapeutic drug that targets heme metabolism could most likely lack specificity for the fungal pathogen. Yet it is possible that specificity could be achieved if heme metabolism is under different regulatory controls between fungal and mammalian cells. For example, heme deficiency leads to mitochondrial iron accumulation in mammalian erythroid cells (59
), whereas this is not the case in yeast (13
). Further studies will be required to determine the utility of heme-inhibiting compounds like sampangine as therapeutic antifungal agents. Irrespective of these potential limitations, such compounds could serve as important pharmacological tools in investigating the consequences of heme deficiency and the relationships between heme, oxygen, sterol, and iron regulation in fungal cells.
This study lays the groundwork for future studies to determine the precise mechanism of action of sampangine. While our data strongly suggest an important role for heme in its antifungal activity, further analysis will be required to confirm that sampangine causes heme depletion and to determine the mechanism involved in this effect.