Diverse pharmacological properties have been attributed to marine-derived cyclic peroxides, yet at present a paucity of information exists concerning their mechanism of action. Using a combination of genomic, genetic, and biochemical approaches, we have shown that the marine cyclic peroxide PFA mediates its antifungal activity by interfering with Ca2+ homeostasis. This conclusion is based on several lines of evidence obtained from the present work: (i) the transcriptome response of S. cerevisiae to PFA was indicative of Ca2+ stress, (ii) four different Ca2+ transporter mutants showed increased sensitivity to PFA, (iii) loss or inhibition of calcineurin function significantly enhanced the antifungal activity of PFA, and PFA was shown to activate the calcineurin-dependent transcription of a reporter gene, and (iv) intracellular calcium levels were significantly increased in PFA-treated cells relative to those in controls.
While the present results provide significant insights concerning PFA's mode of action, the specific mechanism underlying the disruption in Ca2+
homeostasis caused by PFA is unclear at this time. One possibility is that PFA interferes with the activity of Ca2+
ATPases, given that yeast mutants lacking Ca2+
ATPases, such as Pmr1 (Golgi-related pump) and Pmc1 (vacuole-related pump) accumulate excess calcium in the cytosol (7
). Interestingly, the antimalarial drug artemisinin, which, like PFA, contains an endoperoxide moiety, has been shown to inhibit the SERCA-type Ca2+
transporter of the malaria parasite Plasmodium falciparum
). Although yeast cells lack homologs of SERCA-type pumps, the Golgi-related pump Pmr1 is considered to be the functional equivalent of SERCA Ca2+
transporters (e.g., see reference 13
). Another Ca2+
ATPase transporter, Spf1, which plays a role in endoplasmic reticulum (ER) function and in maintaining calcium homeostasis in yeast cells, has been identified (6
). Given the presence of the endoperoxide moiety in the structures of both PFA and artemisinin, it is possible that PFA similarly inhibits the activity of a Ca2+
ATPase transporter in fungal cells.
PFA could also interfere with Ca2+
homeostasis by other mechanisms, such as by causing an influx of Ca2+
through interaction with the plasma membrane. It has been recently shown that the anti-arrhythmia drug AMD, due to its amphipathic nature, associates with the plasma membrane of yeast cells, causing membrane hyperpolarization that leads to the influx of Ca2+
into the cytoplasm (23
). It has been proposed that yeast cells may possess hyperpolarization-activated Ca2+
channels, similar to those described for plant root hairs and pollen tubes that are required for cell elongation and growth. Given the presence of the long hydrocarbon chain and the carboxylic group in the structure of PFA (Table ), it is possible that PFA might cause Ca2+
influx in fungal cells by hyperpolarization of the plasma membrane.
The work presented here indicates that PFA targets molecular pathways that are distinct from the pathways targeted by clinically used antifungal drugs. There appears to be no substantial overlap between the transcription profile of PFA and those of the four antifungal drugs, amphotericin B, ketoconazole, caspofungin, and 5-fluorocytosine. On the other hand, the transcriptional response to PFA resembles the response exhibited by the Ca2+
homeostasis-inhibiting drug AMD. PFA and AMD appear to have similarities with each other not only in their transcription profiles but also in their ability to increase cellular calcium levels and in their increased activities against Ca2+
transporter mutants (20
). Recent studies have shown that the fungicidal activity of AMD is tightly coupled to its ability to induce Ca2+
influx in yeast cells (26
); thus, a similar mechanism of Ca2+
-mediated cell death could be involved in PFA's antifungal activity.
Given the central role Ca2+
plays in various physiological processes, such as cell wall synthesis, cell cycle progression, and vesicular transport (24
), it would be expected that a severe Ca2+
imbalance would have devastating effects on fungal cell viability and growth. For example, yeast mutants deficient in both Pmr1 and Pmc1 Ca2+
ATPases are nonviable, thus demonstrating the inability of yeast to tolerate the resultant overaccumulation of cytosolic Ca2+
). Moreover, the observation that mutants of C
and Cryptococcus neoformans
lacking either specific Ca2+
transporters or the phosphatase calcineurin are avirulent in animal models of fungal infection demonstrates the requirement for normal Ca2+
homeostasis during fungal pathogenesis (3
). Thus, the disruption of Ca2+
homeostasis represents a promising new target pathway for the development of antifungal drug therapies.
Taken together, our findings strongly suggest that calcium homeostasis represents a key cellular target for the antifungal marine endoperoxide PFA. PFA could therefore serve as a useful tool for the further characterization of this cellular process as an antifungal drug target and as a novel pharmacological probe for dissecting the molecular mechanisms underlying calcium homeostasis in fungal and potentially other eukaryotic cell types.