ABSTRACT
Invasive fungal diseases are a leading cause of mortality among immunocompromised populations. Treatment is notoriously difficult due to the limited number of antifungal drugs as well as the emergence of drug resistance. Tamoxifen (TAM), a selective estrogen receptor modulator frequently used for the treatment of breast cancer, has been found to have antifungal activities and may be a useful addition to the agents used to treat fungal infectious diseases. However, the molecular mechanisms underlying its antifungal actions remain obscure. Here, we screened for mutations that confer sensitivity to azole antifungal drugs by using the fission yeast Schizosaccharomyces pombe as a model and isolated a mutant with a mutation in cls1 (ccr1), an allele of the gene encoding the NADPH-cytochrome P450 reductase Ccr1. We found that strains with a deletion of the ccr1+ gene exhibited hypersensitivities to various drugs, including antifungal drugs (azoles, terbinafine, micafungin), the immunosuppressor FK506, and the anticancer drugs TAM and 5-fluorouracil (5-FU). Unexpectedly, the overexpression of Ccr1 caused yeast cell resistance to TAM but not the other drugs tested here. Additionally, strains with a deletion of Ccr1 displayed pleiotropic phenotypes, including defects in cell wall integrity and vacuole fusion, enhanced calcineurin activity, as well as increased intracellular Ca2+ levels. Overexpression of the constitutively active calcineurin suppressed the drug-sensitive phenotypes of the Δccr1 cells. Notably, TAM treatment of wild-type cells resulted in pleiotropic phenotypes, similar to those of cells lacking Ccr1. Furthermore, TAM inhibited Ccr1 NADPH-cytochrome P450 reductase activities in a dose-dependent manner. Moreover, TAM treatment also inhibited the NADPH-cytochrome P450 reductase activities of Candida albicans and resulted in defective cell wall integrity. Collectively, our findings suggest that Ccr1 is a novel target of TAM and is involved in the antifungal activity of TAM by regulating cell wall integrity in fission yeast.
INTRODUCTION
Invasive fungal infections are a global threat to human health, and their incidence has increased over the past few decades. This is mostly due to the growing number of people who are living with a compromised immune function, such as people receiving aggressive anticancer chemotherapy and people with immunodeficiency syndrome (AIDS), and who are, consequently, susceptible to infections from opportunistic pathogenic fungi. Fungal diseases are notoriously difficult to treat, in large part because of the close evolutionary relationships that fungi share with their human hosts (1, 2). While a limited number of distinct antifungal drug classes are available, the problem of antifungal drug resistance, both intrinsic and acquired, is common among all of the fungal pathogens across multiple antifungal drug classes, which leads to a serious threat to continued effective chemotherapy (3). There is a pressing need to discover novel therapeutic strategies to treat fungal infectious diseases.
Nowadays, the repurposing of existing drugs is a promising strategy for product development, as it is quite time-consuming to introduce completely new antifungal drugs onto the market. The aim of repurposing is to accelerate the drug development process by identifying novel biological activities for existing drugs and then applying those drugs to treat a new disease. The pressing need for new antifungal drugs has dovetailed with a growing focus on drug repurposing (4). Tamoxifen (TAM), a selective estrogen receptor modulator (SERM) most widely used as an anticancer drug to manage estrogen receptor-positive breast cancer, has been reported to have antifungal activities (5–7). Beggs et al. proposed that TAM could have an antifungal effect even more lethal than that of imidazole against Candida albicans (8). Further studies deepened and expanded the in vitro fungicidal effects and in vivo activities of TAM against laboratory fungal strains as well as in animal models of fungal infections (9). We and other researchers found that TAM potentiates the activity of existing antifungal drugs, such as azoles, terbinafine, polyenes, and echinocandins, in diverse fungal species (7, 10). More recently, it has been reported that TAM inhibits the growth of clinical C. albicans strains of the oral cavity as well as well-characterized azole-resistant C. albicans strains isolated from an immunocompromised patient (11). Furthermore, a randomized clinical trial showed initial efficacy and safety data for TAM combined with antifungals, including amphotericin B and fluconazole, in HIV-infected or uninfected adults with cryptococcal meningitis (12). These growing evidences indicate that TAM may be a novel antifungal drug or an antifungal drug sensitizer. While multiple TAM targets different from estrogen receptors have been revealed and its aggressiveness against disease and the sensitivity to TAM chemotherapy have been determined, there is a great deal of evidence suggesting that the mechanism of TAM action is far more complex. In particular, the molecular mechanism underlying the antifungal action of TAM remains largely unknown.
We have been studying the molecular mechanisms underlying the actions of antifungal drugs by using the fission yeast Schizosaccharomyces pombe as a model organism, since it shares many features with some pathogenic fungi and is amenable to genetic analysis. The fission yeast is also an excellent organism for the identification of the molecular targets of various drugs, since the major signaling pathways and processes involved in the cellular response to cytotoxic agents are conserved between yeast and mammalian cells (13). We previously performed several genome-wide screens for altered sensitivity to antifungal drugs in fission yeast and identified a host of genes associated with sensitivity and resistance to the existing antifungal drugs (14, 15). In this study, we performed in fission yeast a genetic screen for mutants that showed hypersensitivity to azoles, the most commonly used antifungal drugs in the clinic, and isolated a mutant with a mutation in cls1/ccr1-1, an allele of the gene encoding the NADPH-cytochrome P450 reductase Ccr1. It has been reported that the budding yeast homolog of Ccr1 plays a role in cell wall assembly (16). Consistently, our data indicated that strains with a deletion of Ccr1 displayed defects in cell wall integrity. Surprisingly, overexpression of Ccr1 caused yeast cell resistance to TAM but not the other drugs tested here. Notably, TAM inhibited the Ccr1 function, which in turn caused defects in yeast cell wall integrity. The current study provides a strong foundation for future efforts to optimize TAM for medicinal drug repurposing in the development of antifungal agents.
RESULTS
Isolation of the ccr1-1 mutant.Since therapeutic strategies can be improved by enhancing the efficacy of existing antifungal drugs, such as azoles, it is important to identify the genes and cellular pathways involved in susceptibility to these drugs. In order to identify the regulatory processes as well as key molecules involved in susceptibility to azole antifungal drugs, we performed a genetic screen for mutants that were hypersensitive to clotrimazole and isolated a mutant (the cls1 [for clotrimazole-sensitive 1] mutant). As shown in Fig. 1A, cls1/ccr1-1 mutant cells grew as well as wild-type (wt) cells did on yeast extract-peptone-dextrose (YPD) plates at 27°C. However, the cls1/ccr1-1 mutant could not grow on YPD plates containing 0.01 μg/ml clotrimazole, whereas wild-type cells grew normally (Fig. 1A).
A mutation in the cls1+/ccr1+ gene causes a clotrimazole-sensitive phenotype. (A) Wild-type (wt), ccr1 mutant, or Δccr1 cells transformed with the multicopy vector pDB248 or the vector containing the ccr1+ gene were streaked on the plates, as indicated, and then incubated for 4 days at 27°C. (B) Comparison of the amino acid sequences among fission yeast Ccr1, budding yeast Ncp1, and human POR. The amino acid sequences were aligned using the Clustal W program. Filled boxes indicate the mutated amino acids.
cls1 is a mutant allele of the ccr1+ gene encoding NADPH-cytochrome P450 reductase.As described in Materials and Methods, the ccr1+ gene was cloned by complementation of the clotrimazole-sensitive growth defect of cls1/ccr1-1 mutant cells (Fig. 1A, cls1+ccr1+). Nucleotide sequencing of the cloned DNA fragment revealed that the cls1+ gene is identical to the ccr1+ gene (SPBC29A10.01), which encodes NADPH-cytochrome P450 reductase, a protein of 678 amino acids that is highly similar to human POR (37% identity) and Saccharomyces cerevisiae Ncp1p (38% identify) (Fig. 1B). Linkage analysis was performed (see Materials and Methods), and the results indicated the allelism between the ccr1+ gene and the cls1 mutation. We therefore renamed cls1 as ccr1-1. The ccr1 deletion cells were viable, and the Δccr1 cells also showed clotrimazole sensitivity, similar to that of the ccr1-1 mutants (Fig. 1A, Δccr1 + vector).
To characterize the mutation site in the ccr1-1 mutant, genomic DNA was isolated from the ccr1-1 mutant, and DNA sequencing of the full-length coding region of the ccr1-1 mutant revealed that the G-to-A nucleotide substitution caused a highly conserved glycine to be altered to an aspartate codon at amino acid position 115, which lies in the flavodoxin domain (Fig. 1B, solid arrow).
The ccr1 deletion cells showed defects in cell wall integrity as well as vacuole fusion.Since the ccr1 mutant cells displayed hypersensitivity to the antifungal drug clotrimazole, which functions by disrupting ergosterol biosynthesis through inhibition of the cytochrome P450-dependent enzyme lanosterol 14-α-demethylase, encoded by the erg11+ gene, we then examined whether the ccr1 mutants showed hypersensitivity to the other azoles, such as fluconazole and miconazole, as well as other ergosterol biosynthesis inhibitors, including terbinafine, which targets the erg1+ gene, and fenpropimorph, which targets the erg2+ and erg24+ genes. Meanwhile, the immunosuppressant FK506, the antifungal drug micafungin, the anticancer drug TAM, and 5-fluorouracil (5-FU) were also examined. As shown in Fig. 2A, the results showed that the Δccr1 cells exhibited hypersensitivity to all these drugs, although the ccr1-1 mutant cells were mostly less sensitive to these drugs (Fig. 2A). Thus, the ccr1-1 mutant and Δccr1 cells displayed similar but distinct phenotypes, suggesting that NADPH-cytochrome P450 reductase is partially deficient in the ccr1-1 mutant.
Phenotypes of the ccr1 mutant and Δccr1 cells. (A) The Δccr1 cells displayed sensitivities to various drugs, including fluconazole, miconazole, terbinafine, fenpropimorph, micafungin, TAM, FK506, and 5-FU. Cells were spotted onto each plate, as indicated, and then incubated for 4 days at 27°C. (B) The ccr1 deletion markedly enhanced the sensitivity to β-glucanase. Wild-type and Δccr1 cells exponentially growing in YPD medium were harvested and incubated with β-glucanase (Zymolyase 20T) at 27°C with vigorous shaking. Cell lysis was monitored by measurement of the optical density at 660 nm (the value before adding the enzyme was taken as 100%). The data represent the mean ± SD from three determinations. (C) The Δccr1 cells were defective in vacuole fusion. Wild-type (wt) cells and Δccr1 cells were grown to log phase in YPD medium at 27°C. Then, the cells were harvested, resuspended in water, and examined by DIC microscopy. Photographs were taken after resuspension in water for 0 min and 90 min. (D) The drug-sensitive phenotypes of Δccr1 cells were suppressed by the expression of constitutively active calcineurin. Wild-type cells or Δccr1 cells transformed with a control vector, the vector containing full-length ppb1+ (Δccr1+ppb1+), or the vector containing the constitutively active truncated calcineurin gene (Δccr1+ppb1ΔC) were spotted onto each YPD plate, as indicated, and incubated for 4 days at 27°C.
The drug sensitivity phenotypes of Δccr1 cells were further evaluated by determining the MIC values. As shown in Table 1, deletion of Ccr1 resulted in MIC values of clotrimazole, fluconazole, miconazole, terbinafine, micafungin, fenpropimorph, FK506, TAM, and 5-FU lower for the deletion mutant than for wild-type cells (Table 1). These results were in good agreement with the findings from the spot assay described above and further demonstrated the sensitivity phenotypes caused by the deletion of Ccr1.
MICs of multiple drugs for S. pombe ccr1 deletion strains
The results presented above that the Δccr1 cells were sensitive to the 1,3-β-d-glucan synthase inhibitor micafungin prompted us to examine the effects of β-glucanase treatments on the Δccr1 cells. As shown in Fig. 2B, the Δccr1 cells lysed faster than the wild-type cells, although the sensitivity of the Δccr1 cells to β-glucanase was not as severe as that of the Δpmk1 cells (Fig. 2B), which lack a mitogen-activated protein kinase regulating the cell wall integrity of fission yeast (17), indicating that the Δccr1 cells are defective in cell wall integrity.
Several mutants that we have studied were defective in cell wall integrity and also showed defects in vacuole fusion (18–20). Therefore, we then examined the vacuolar fusion induced by osmotic stress in wild-type cells and Δccr1 cells, since hypotonic stress can cause a dramatic fusion of vacuoles in S. pombe (21). When the cells were collected, washed, and resuspended in water for 90 min, the wild-type cells had significantly large vacuoles that resulted from vacuole fusion (Fig. 2C, wt). In clear contrast, vacuoles remained small and numerous in the Δccr1 cells suspended in water, indicating that Ccr1 is essential for vacuole fusion in fission yeast (Fig. 2C, Δccr1).
Overexpression of constitutively active calcineurin suppressed the phenotypes of the ccr1 deletion cells.Since the Δccr1 cells were sensitive to FK506, a specific inhibitor of calcineurin phosphatase in both mammals and fission yeast, it was of interest to examine the effect of overexpression of calcineurin on the Δccr1 cells. Then, the Δccr1 cells were transformed with a vector containing the full-length calcineurin gene (ppb1+), the constitutively active truncated calcineurin gene (ppb1ΔC), or a control multicopy vector. The results showed that the overexpression of the constitutively active calcineurin, but not the full-length calcineurin, suppressed the drug-sensitive phenotypes of the Δccr1 cells (Fig. 2D). These results further suggest that Ccr1 is implicated in cell wall integrity.
Altered calcineurin activity in ccr1 deletion cells.To further characterize the calcineurin-related phenotypes associated with Δccr1 cells, we used the 3xCDRE::luc(R2.2) reporter system, which was developed to monitor the real-time activity of the Ca2+/calcineurin signaling pathway (22). As shown in Fig. 3A, the Δccr1 cells showed an enhanced 3xCDRE::luc(R2.2) reporter response in the presence of various concentrations of extracellular CaCl2 (0 to 200 mM) compared with that of the wild-type cells (Fig. 3A). Notably, compared with the wild-type cells, the Δccr1 cells displayed a continuous increase in the response of the calcineurin-dependent response element (CDRE) (Fig. 3A).
Altered calcineurin activity in ccr1 deletion cells. (A) Enhanced CDRE response in Δccr1 cells. Wild-type and Δccr1 cells harboring the multicopy plasmid 3хCDRE::luc(R2.2) reporter vector were incubated with d-luciferin, treated with various concentrations of CaCl2, and then analyzed as described in Materials and Methods. Data from three independent experiments are expressed as the mean ± SD. RLU, relative light units. (B) Increased intracellular Ca2+ concentration in Δccr1 cells. Wild-type and Δccr1 cells transformed with plasmid GFP-19-AEQ were incubated with coelenterazine and treated with various concentrations of CaCl2, and then the aequorin assay was performed as described in Materials and Methods. The data from the area under the curve (AUC) of three independent experiments are expressed as the mean ± SD. (C) The Δccr1 cells exhibited Ca2+ hypersensitivity. Wild-type cells and Δccr1 cells were spotted onto the plates, as indicated, and incubated for 4 days at 27°C. (D) Translocation of GFP-Prz1 to the nucleus when ccr1 was deleted. Wild-type and Δccr1 cells transformed with the vector containing GFP-Prz1 were grown in EMM containing thiamine at 27°C and then observed under a fluorescence microscope. ***, P < 0.001.
To provide additional information regarding enhanced calcineurin activity in the Δccr1 cells, we monitored intracellular Ca2+ levels in wild-type and Δccr1 cells. As shown in Fig. 3B, the Δccr1 cells exhibited a Ca2+ concentration higher than that observed in the wild-type cells (Fig. 3B). In agreement with this result, we found that the Δccr1 cells were hypersensitive to CaCl2 (Fig. 3C).
Given that calcineurin dephosphorylates and activates Prz1, a C2H2-type zinc finger transcriptional factor, which in turn translocates from the cytoplasm to the nucleus in fission yeast (23), we then tested the effect of Ccr1 deletion on the subcellular localization of Prz1. The results showed that, in wild-type cells, green fluorescent protein (GFP)-Prz1 largely localized to the cytosol. However, in the Δccr1 cells, GFP-Prz1 was observed mostly in the nucleus (Fig. 3D), further suggesting enhanced calcineurin activity in the Δccr1 cells.
Ccr1 is a molecular target of TAM.The results presented above indicating that the Δccr1 cells were sensitive to various drugs, including azoles, terbinafine, fenpropimorph, micafungin, FK506, TAM, and 5-FU, prompted us to examine whether overexpression of Ccr1 altered the sensitivities to these drugs. Strikingly, the results clearly showed that the overexpression of Ccr1 caused yeast cell resistance to TAM but not the other drugs tested (Fig. 4A), indicating that Ccr1 may be a new target of TAM.
Ccr1 is a molecular target of TAM. (A) Overexpression of Ccr1 caused yeast cell resistance to TAM. Wild-type cells transformed with a control vector or the vector containing the ccr1+ gene were streaked onto each YPD plate, as indicated, and incubated for 4 days at 27°C. (B) TAM treatment inhibited NADPH-cytochrome P450 reductase activities. Wild-type cells were cultured to log phase in YPD medium with or without TAM for 8 h, as indicated, and the Ccr1 activity of wild-type cells with or without TAM treatment was determined as described in Materials and Methods. Data from three independent experiments are expressed as the mean ± SD. prot, protein. **, P < 0.01; ***, P < 0.001.
To further investigate whether TAM interacts with Ccr1 in fission yeast, we tested the effect of TAM on NADPH-cytochrome P450 reductase activities. As shown in Fig. 4B, the results showed that TAM inhibited NADPH-cytochrome P450 reductase activities in a dose-dependent manner (Fig. 4B), further supporting the suggestion that Ccr1 is a molecular target of TAM.
TAM-treated wild-type cells and cells with the Ccr1 deletion displayed similar phenotypes.Since Ccr1 is a true target of TAM, we hypothesized that TAM treatment of wild-type cells would result in pleiotropic phenotypes, similar to those of cells lacking Ccr1. To this end, we first examined whether TAM treatment could alter the resistance of wild-type cells to cell wall damage. We used β-glucanase sensitivity as a direct quantitative readout of cell wall modifications, as described above. Wild-type cells were assayed for β-glucanase sensitivity with or without the addition of TAM. As shown in Fig. 5A, TAM treatment significantly enhanced the sensitivity of wild-type cells to β-glucanase in a dose-dependent manner, suggesting that TAM treatment resulted in cell wall defects. Notably, the overexpression of Ccr1 partially rescued the β-glucanase sensitivity caused by TAM treatment of wild-type cells (Fig. 5A), indicating that TAM caused defects in yeast cell wall integrity by targeting Ccr1.
TAM-treated wild-type cells and cells with the Ccr1 deletion displayed similar phenotypes. (A) Effect of TAM on cell wall digestion by β-glucanase. Wild-type cells containing the vector or ccr1+ were cultured to log phase in EMM and then incubated with 100 μg/ml of β-glucanase (Zymolyase 20T) and the indicated concentrations of TAM at 27°C with vigorous shaking. Cell lysis was monitored by measuring the optical density at 660 nm (the value before adding the enzyme was taken as 100%). The data shown are representative of those from multiple experiments. (B) TAM treatment inhibited vacuole fusion. Wild-type (wt) cells containing the vector or ccr1+ were grown to log phase in EMM. Then, the cells were harvested, resuspended in water with or without TAM as indicated for 90 min at 27°C, and examined by DIC microscopy. (Left) Photographs were taken after resuspension in water for 0 min and 90 min. (Right) Quantification of vacuole fusion in wild-type cells with or without the addition of TAM to the medium. At least 100 cells were observed at one time in each experiment. The data represent the mean ± SD from five determinations. (C) TAM treatment induced a dose-dependent response of the 3хCDRE::luc(R2.2) reporter in wild-type cells. Wild-type cells harboring the multicopy plasmid 3хCDRE::luc(R2.2) reporter vector were incubated with d-luciferin and treated with various concentrations of TAM, as indicated, and were then analyzed as described in Materials and Methods. Data from three independent experiments are expressed as the mean ± SD. (D) TAM caused higher Ca2+ concentrations in wild-type cells in a dose-dependent manner than in wild-type cells without TAM treatment. Wild-type cells transformed with plasmid GFP-19-AEQ were incubated with coelenterazine and treated with various concentrations of TAM, as indicated, and then the aequorin assay was performed as described in Materials and Methods. The data from the area under the curve (AUC) from three independent experiments are expressed as the mean ± SD. (E) TAM treatment stimulates the translocation of GFP-Prz1 from the cytosol to the nucleus. (Left) The cellular localization of Prz1 in wild-type cells harboring the plasmid GFP-Prz1 with or without the addition of 10-μg/ml TAM for 30 min was examined by fluorescence microscopy. (Right) Quantification of the nuclear accumulation of GFP-Prz1 in wild-type cells with or without the addition of TAM to the medium. At least 100 cells were observed at one time in each experiment. The data represent the mean ± SD from five determinations. **, P < 0.01; ***, P < 0.001.
We also tested the effects of TAM treatment on the vacuole fusion of wild-type cells. The results showed that, when the cells were transferred from medium to water without the addition of TAM, a number of much larger vacuole structures appeared. However, vacuoles remained small and numerous when the cells were transferred to water with the addition of TAM (Fig. 5B). On the other hand, larger vacuoles were observed when cells overexpressing Ccr1 were transferred to water in spite of TAM treatment (Fig. 5B), suggesting that TAM treatment inhibited vacuole fusion by targeting Ccr1.
Given that Ccr1 deletion exhibited enhanced calcineurin activity as well as resulted in a higher Ca2+ concentration, we then investigated the effects of TAM treatment on the CDRE response and the intracellular Ca2+ levels of the wild-type cells. As expected, TAM treatment induced a dose-dependent response of the 3хCDRE::luc(R2.2) reporter in the wild-type cells (Fig. 5C). Meanwhile, TAM caused higher Ca2+ concentrations in yeast cells in a dose-dependent manner (Fig. 5D). Consistent with the marked stimulation of the 3хCDRE::luc(R2.2) reporter response by TAM, the translocation of GFP-Prz1 to the nucleus was also observed by the addition of TAM to the medium (Fig. 5E).
TAM damaged cell wall integrity and inhibited the NADPH-cytochrome P450 reductase activities of Candida albicans.To further verify that TAM regulates cell wall integrity by targeting NADPH-cytochrome P450 reductase in pathogenic fungi, we determined the β-glucanase sensitivity of Candida albicans with or without the addition of TAM, as described above. As shown in Fig. 6A, TAM treatment also enhanced the sensitivity of wild-type cells to β-glucanase in a dose-dependent manner, which was similar to the findings for the fission yeast (Fig. 6A). We further tested the NADPH-cytochrome P450 reductase activities of Candida albicans with TAM treatment, as described in Materials and Methods. The results showed that TAM also dose dependently inhibited the NADPH-cytochrome P450 reductase activities of Candida albicans (Fig. 6B). These results further suggest that NADPH-cytochrome P450 reductase is a target of TAM in the pathogenic fungus Candida albicans, similar to the findings for fission yeast.
TAM treatment damages the cell wall integrity and inhibits the NADPH-cytochrome P450 reductase activities of Candida albicans. (A) Effect of TAM on cell wall digestion by β-glucanase. Candida albicans cells were cultured to log phase in LB medium and then incubated with 100 μg/ml of β-glucanase (Zymolyase 20T) and the indicated concentrations of TAM at 37°C with vigorous shaking. Cell lysis was monitored by measuring the optical density at 660 nm (the value before adding the enzyme was taken as 100%). The data represent the mean ± SD from three determinations. (B) TAM treatment inhibited the NADPH-cytochrome P450 reductase activities of Candida albicans. Candida albicans cells were cultured to log phase in LB medium with or without TAM for 14 h, as indicated, and the activity of wild-type cells with or without TAM treatment was determined as described in Materials and Methods. Data from three independent experiments are expressed as the mean ± SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
DISCUSSION
Ideally, a new therapeutic strategy for fungal infectious disease would enhance the efficacy of existing antifungal drugs, have fungicidal activity, and have broad efficacy against diverse fungal pathogens (1). In addition, drug repurposing would be an outstanding strategy to obtain agents active against fungal disease (4). As a gold standard in the treatment of estrogen receptor-positive breast cancer for almost 50 years, TAM fulfills almost all of these criteria. However, the molecular details of the mechanisms of its antifungal activity are far from understood. Here, we found that TAM caused defects in cell wall integrity in fission yeast by targeting Ccr1. To our knowledge, this is the first report of the involvement of Ccr1 in the antifungal action of TAM through the regulation of cell wall integrity.
Multiple lines of evidence support the hypothesis that Ccr1 regulates cell wall integrity. First, Δccr1 cells were sensitive to the 1,3-β-d-glucan synthase inhibitor micafungin and β-glucanase. Second, overexpression of the constitutively active calcineurin suppressed the phenotypes of Δccr1 cells. Third, Δccr1 cells displayed a higher CDRE response and higher Ca2+ levels than wild-type cells. Fourth, deletion of Ccr1 promoted the nuclear accumulation of GFP-Prz1. As calcineurin regulates 1,3-β-d-glucan synthesis and is involved in cell wall integrity (24), it is possible that the deletion of Ccr1 induced calcineurin activity which, when increased, functionally compensated for the regulation of cell wall integrity. On the other hand, overexpression of the constitutively active calcineurin complemented the defects in cell wall integrity caused by the deletion of Ccr1. Thus, Ccr1 and calcineurin are required in parallel for the regulation of cell wall integrity. In addition to cell wall integrity, we also demonstrate that Ccr1 is required for vacuole fusion, although the mechanism in Ccr1 underlying vacuole fusion is not clear.
More importantly, we identified that Ccr1 is a novel molecular target of TAM. First, overexpression of the ccr1+ gene conferred resistance to TAM but not the other drugs tested here. Second, TAM inhibited Ccr1 activity in a dose-dependent manner. Third, wild-type cells treated with TAM displayed phenotypes similar to those of cells with the Ccr1 deletion treated with TAM, such as defects in cell wall integrity and vacuole fusion, higher Ca2+ levels, a higher CDRE response, and higher rates of nuclear accumulation of GFP-Prz1. Notably, overexpression of Ccr1 reduced the defects in cell wall integrity and vacuole fusion caused by TAM treatment. Finally, TAM treatment also damaged the cell wall integrity and inhibited the NADPH-cytochrome P450 reductase activity of Candida albicans. These data suggest that TAM inhibits Ccr1, thereby disrupting cell wall integrity.
It has been reported that TAM binds to calmodulin and inhibits the binding of calmodulin to calcineurin in Cryptococcus and Saccharomyces cerevisiae. Overexpression of calmodulin suppressed the sensitivity of Cryptococcus and S. cerevisiae to TAM (5, 9). However, in fission yeast, we previously reported that the overexpression of calmodulin slightly increased the sensitivity to TAM but did not suppress the sensitivity to TAM, suggesting that calmodulin is related to the antifungal action of TAM but is not the molecular target of TAM (7).
In conclusion, the present study strongly suggests that Ccr1 is a novel target of TAM and that TAM interacts with Ccr1, thereby causing cell wall damage. Given that the fungal cell wall not only is essential for the survival and growth of fungal cells but also is unique to fungi and not present in higher eukaryotes, the fungal cell wall is a prime target for the development of antifungal drugs. It is worth mentioning that TAM treatment resulted in disruption of the cell wall, which makes TAM an ideal candidate as an antifungal agent. Moreover, identifying the protein target as well as understanding the molecular mechanisms of existing drugs is critical because it allows researchers to repurpose existing drugs for new indications and develop next-generation drugs with improved pharmacological properties. Thus, our study provides important evidence that targeting the Ccr1 function as an antifungal strategy holds considerable promise. Our findings pave the way for further optimization of TAM as an antifungal drug as well as the development of novel drugs targeting Ccr1.
MATERIALS AND METHODS
Strains, media, and genetic and molecular biology methods.The S. pombe strains used in this study are listed in Table 2. The Candida albicans strain was obtained from the China Pharmaceutical Culture Collection (strain CPCC400616). The complete medium (YPD), the minimal medium (Eagle’s minimal medium [EMM]), and Luria-Bertani-agarose (LB) medium have been described previously (17). Standard S. pombe genetic and recombinant DNA methods were performed, except where noted, as described previously (25). Gene disruptions are denoted by lowercase letters representing the disrupted gene followed by two colons and the wild-type gene marker used for disruption (for example, ccr1::ura4+). Gene disruptions are abbreviated by the gene preceded by Δ (for example, Δccr1). Proteins are denoted by roman letters, and only the first letter is capitalized (for example, Ccr1). All chemicals and reagents were from commercial sources.
Strains used in this study
Isolation of the ccr1-1 mutant.The cls1/ccr1-1 mutant (strain CM49) was isolated in a screen of cells that had been mutagenized with nitrosoguanidine as described previously (26, 27). Mutants were spread on YPD plates to give ∼1,000 cells/plate, and the plates were incubated at 27°C for 4 days. The colonies were then replica plated onto YPD plates containing 0.01 μg/ml clotrimazole and incubated at 27°C for 2 days. Mutants that showed high levels of clotrimazole sensitivity were selected. The original mutants isolated were backcrossed three times to wild-type strains HM123 and HM528.
Cloning of the ccr1+ gene.To clone the ccr1+ gene, the CM49 cls1/ccr1-1 mutant was grown at 27°C and transformed with an S. pombe genomic DNA library constructed in the vector pDB248 (28). The Leu+ transformants were replica plated onto YPD plates containing 0.01 μg/ml clotrimazole and incubated at 27°C, and plasmid DNA was recovered from the transformants that showed a plasmid-dependent rescue. These plasmids complemented the clotrimazole sensitivity of the CM49 cls1/ccr1-1 mutant. By DNA sequencing, the suppressing plasmids were identified to contain the ccr1+ gene.
To investigate the relationship between the cloned ccr1+ gene and the CM49 cls1/ccr1-1 mutant, linkage analysis was performed as follows. The entire ccr1+ gene was subcloned into a pUC-derived plasmid containing the S. cerevisiae LEU2 gene and was integrated by homologous recombination into the genome of wild-type strain HM123. The integrant was mated with the cls1/ccr1-1 mutant. The resulting diploid was sporulated, and tetrads were dissected. A total of 30 tetrads were dissected. In all cases, only parental ditype tetrads were found, indicating the allelism between the ccr1+ gene and the cls1/ccr1-1 mutation (our unpublished data).
Knockout of the ccr1+ gene.To knock out the ccr1+ gene, a one-step gene disruption was performed by homologous recombination (29). The ccr1::ura4+ disruption was constructed as follows. The open reading frame (ORF) of ccr1+ was amplified by PCR (forward primer, 5′-CGCGGATCCATGAAGACTTATG-3′; reverse primer, 5′-CGCGGATCCTTACCAGGTATCTTC-3′) from the genomic DNA and was subcloned into the BamHI site of BlueScriptSK(+). Then, a HindIII fragment containing the ura4+ gene was inserted into the HindIII site of the previous construct. The construct containing the disrupted ccr1+ gene was digested with BamHI, and the ccr1::ura4+ fragment was used to transform the diploids (5A/1D; Table 2). Stable integrants were subsequently cloned on plates containing medium lacking uracil, and gene disruption by the ccr1+ derivative containing the ura4+ insertion was verified by PCR and Southern blotting (our unpublished data).
Determination of MICs.The MICs for the fission yeast strains were determined by a microdilution method previously used and published for fission yeast (14, 30). In brief, strains were inoculated into 96-well microtiter plates containing 100 μl of YPD medium and different concentrations of drugs to yield 105 cells/well. The plates were incubated at 27°C and examined by using a microplate reader (iMark; Bio-Rad) at 48 h. MIC endpoints were recorded as the lowest concentrations of the drugs causing at least 50% inhibition compared to the growth of the growth control. Each MIC was determined in triplicate.
CDRE-dependent reporter assay.Real-time monitoring of calcineurin-dependent response element (CDRE) reporter activity was performed using a firefly luciferase reporter assay as described previously (22). To assess the effect of the Ccr1 deletion on CDRE activity, the multicopy reporter plasmid pKB5723 [3×CDRE::luc(R2.2)] was transformed into wild-type and Δccr1 cells and treated with various concentrations of CaCl2. d-Luciferin sodium salt monohydrate (50 mM in sterile water as a stock solution; Biosynth Corporation, Switzerland) was used as a substrate for firefly luciferase and was added to the cell suspension at a 1:100 dilution. Light emission levels, expressed as relative light units, were measured at 1-min intervals for 3 h using a luminometer (model AB-2350; Atto Co., Tokyo, Japan) at 27°C.
Measurement of cytoplasmic Ca2+ levels using GFP-19-AEQ.Cytoplasmic Ca2+ levels were determined using a previously described method with minor modifications (31). In brief, cells expressing GFP linked by 19 amino acids to aequorin (GFP-19-AEQ) were resuspended in fresh EMM containing 10 mM coelenterazine (catalog number S200A; Promega), and the optical density at 660 nm was adjusted to 0.3. To convert apoaequorin to aequorin (AEQ), the cells were incubated for 4 h at 27°C. The cells were washed three times and resuspended in fresh EMM. Then, the luminescence from aequorin that remained in the cells was determined after treating the cells with 20% Triton X-100 (50 μl) and CaCl2 (50 mM to 200 mM). The light emission levels, expressed as the area under the curve (AUC), were measured using a luminometer at 15-s intervals for 10 min.
Determination of NADPH-cytochrome P450 reductase activity.The NADPH-cytochrome P450 reductase Ccr1 activity of wild-type cells with or without TAM treatment was determined using a previously described method with minor modifications (32, 33). In brief, wild-type cells were cultured to log phase in YPD medium with or without a series of concentrations of TAM and harvested. Then, microsomal enzymes were isolated from the cells and prepared by using an NADPH-cytochrome c reductase assay kit (Solarbio Science & Technology, China). The protein amounts in the microsomal enzyme solution were quantified by a bicinchoninic acid assay (Beyotime Biotechnology, China). Then, based on the ability of the NADPH-cytochrome P450 reductase to reduce bovine heart cytochrome c, the Ccr1 activity in the microsomal enzyme solution was determined by using an NADPH-cytochrome c reductase assay kit (Solarbio Science & Technology, China). After the enzyme reaction resulting from the addition of cytochrome c and NADPH to the microsomal enzyme solution, the increase in the absorption of the microsomal enzyme solution at 550 nm was recorded for 2 min using a UV/visible spectrophotometer (model TU-1901; Persee, China). A molar extinction coefficient of 0.0211 mM−1 cm−1 was used for the calculation, and the Ccr1 activity was expressed in nanomoles of cytochrome c reduced per minute per milligram of microsomal protein. Ccr1 relative activity (in nanomoles per minute per milligram of protein) was calculated as ΔA550/ε × Vtotal/Cpr/Vsample/T = 104 × ΔA/Cpr, where ΔA550 is the increase in absorption of the microsomal enzyme solution at 550 nm in 2 min, Vtotal is the total reaction system volume, Cpr is the amount of protein in microsomal enzyme solution (in milligrams per milliliter), Vsample is the microsomal enzyme solution volume, and T is the means reaction time (2 min).
Microscopy and miscellaneous methods.Methods in light microscopy, such as fluorescence microscopy and differential interference contrast (DIC) microscopy, were performed as described previously (18, 27) by using a Nikon Eclipse Ni-U microscope equipped with a DS-Qi2 camera (Nikon Instruments Inc., Japan). Cell wall digestion by β-glucanase (Zymolyase 20T; Seikagakukogyo, Tokyo, Japan) was performed as described previously (17). Database searches were performed using the National Center for Biotechnology Information BLAST network service and the fission yeast S. pombe database search service (http://www.pombase.org/).
Statistical analyses.Quantitative data are expressed as the mean ± standard error of the mean (SEM). Student’s t test was used to assess the statistical significance of the differences in the results of multiple comparisons. The difference was considered statistically significant when the P value was less than 0.05. All statistical analyses were performed using the SPSS (version 16.0) software package (SPSS, Inc., Chicago, IL, USA).
ACKNOWLEDGMENTS
We thank Toshiaki Kato for critical reading of the manuscript.
This work was supported by grants from the Natural Science Foundation of Liaoning Province, China (grant no. 2019-MS-384), to Yue Fang, and was supported by research funds from the Doctoral Start-up Foundation of Liaoning Province, China (grant no. 2019-BS-284), and the Young Scholar Support Program of China Medical University (grant no. QGZ2018081) to Si Chen. This work was also supported by the National Natural Science Foundation of China (grant no. 81974418 to Fan Yao).
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We declare no competing financial interests in relation to the work.
FOOTNOTES
- Received 13 January 2020.
- Returned for modification 23 March 2020.
- Accepted 13 June 2020.
- Accepted manuscript posted online 22 June 2020.
- Copyright © 2020 American Society for Microbiology.
