WO2003080657A2

WO2003080657A2 - Yeast pheromones for the treatment of infectious diseases

Info

Publication number: WO2003080657A2
Application number: PCT/EP2003/002551
Authority: WO
Inventors: Fedor Severin; Anthony Hyman
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2002-03-25
Filing date: 2003-03-12
Publication date: 2003-10-02
Also published as: WO2003080657A3; AU2003227053A1

Abstract

Use of a yeast pheromone for the manufacture of a medicament for the treatment of yeast and other fungal infections.

Description

YEAST PHEROMONES FOR THE TREATMENT OF INFECTIOUS DISEASES

The present application relates to the field of medical treatment of yeast and/or other fungal infections. In addition, the present application relates to an invention in the context of programmed cell death (PCD). The invention disclosed and described herein further relates to yeast pheromones as fungicides. Moreover, the invention provides assays to identify substances interfering with and/or exerting a positive or negative effect on the development of PCD. A further aspect of the present invention is an assay to identify genes, RNA, and proteins involved in PCD.

Programmed cell death (PCD) is a ubiquitous process in multicellular organisms. The pathways by which cells in multicellular organisms trigger programmed cell death have been extensively characterized. A typical scenario for PCD development includes

(i) activation of the MAP kinase cascade, (ii) accumulation of reactive oxygen species (ROS),

(iii) release of cytochrome c from mitochondria into the cytoplasm as a consequence of opening the permeability transition pore (PTP) in the mitochondrial membrane, and

(iv) activation of caspases (reviewed in [1]).

Prior to the work of the present inventors, it had remained unclear whether single cell organisms have evolved such pathways, and if so, what benefit would accrue to a single cell organism undergoing PCD. One of the possible reasons for a unicellular organism to commit altruistic suicide is to benefit the cell community. Such a mechanism may improve the genetic fund of the community by actively eliminating the weak individuals. Thus, the inventors posed themselves the object to find out whether yeasts and other unicellular organisms exhibit a process such as PCD, and whether they would be able to develop methods/systems utilizing PCD in unicellular organisms, e.g., to combat infections caused by such unicellular organisms.

In fact, the inventors have found that yeast exhibits some features of PCD in higher organisms when treated with pheromone. As is well known in the state of the art, yeasts show aspects of communal behavior when they mate. There are two mating types in yeast: α and a. Cells of the α-mating type produce alpha- factor (α-factor, also termed a pheromone or a sexual pheromone), triggering cells of the α-type to mate. On the other hand, cells of the α-mating type produce ---factor (which is also a pheromone, or a sexual pheromone), triggering cells of the α-type to mate. Likewise, it has been noticed in the past that high doses of or prolonged exposure to α-factor is toxic for yeast (see [2] for review). Possibly, the altruistic death of yeast cells unable to mate after a long time in contact with cells of the opposite mating type would be beneficial for the cell community.

In fact, as depicted in row 2 of Table 1, the inventors confirmed cell death in yeast when subjecting α-cells to α-factor. The inventors therefore decided to test whether the phenomenon of cell death (toxicity of alpha-factor) shares similarities with programmed cell death as it occurs in multicellular organisms. As an outcome of the experiments conducted by the inventors, they concluded that there must exist a parallel between the pathways inducing PCD in higher eukaryotic cells and the pathways inducing cell death in unicellular organisms such as yeast, fungi, and bacteria Shouldn't we drop the bacteria for consistency reasons? Is the addition of fungi agreeable?.

One of the key mechanisms by which multicellular cells trigger PCD is by production of reactive oxygen species (ROS) (see [1] for review). 2',7'-dichlorodihydrofluorescein diacetate (H DCF- DA) can be used as a probe for ROS production; H₂DCF-DA is oxidized by ROS to DCF which fluoresces green [3]. Consequently, the inventors wanted to find out whether a corresponding mechanism could be detected in yeast as well. Therefore, they investigated whether alpha-factor addition to a cells could trigger DCF accumulation. In fact, the experiments demonstrated that addition of alpha-factor to cells of the opposite mating type could induce the formation of ROS, a marker of PCD in multicellular organisms (after 1.5 h exposure of α-cells to alpha-factor approximately 30% of α-type cells showed fluorescence, whereas no fluorescing cells were found either in the absence of pheromone (Fig. 1A), or when alpha-factor was added to α-cells (Table 1)).

To test whether α-factor induces other PCD markers in yeast α-cells - one such marker is degraded DNA demonstrated by a DNA-ladder - the inventors scored the accumulation of dead cells (as judged by phloxin B or methylene blue staining) and the appearance of degraded DNA. Fig. ID shows that approximately 30% of the α-cells are dead after 3.5 h of treatment with α- factor (100 μg/ml), and DNA is being degraded as shown by TUNEL (Fig. IB) and FACS (Fig. IB and C). Taken together, these data indicate that pheromone-induced yeast cell death shows some features of PCD in higher organisms.

Next, the inventors were interested in quantitative data and, therefore, titrated the concentration of pheromone and monitored the appearance of shmoos, morphological markers of mating. As a result, they found that ROS induction requires approximately 10-fold higher pheromone concentration than schmoo growth.

As mentioned previously, the pathway for development of pheromone-induced PCD in higher organisms goes through the MAP kinase pathway, of which a key component is the Ste20 kinase [2]. Interestingly, the inventors found that a deletion of ste20 in yeast α-cells prevents α-factor- induced death of such cells, and concomitantly prevents the formation of ROS (see Table 1, row 6). As homologues of components of the MAP kinase pathway have been shown to play a major role in PCD development in higher cells [4], it is highly likely that there is a parallel between the pathways inducing PCD in yeast and higher eukaryotic cells.

One of the consequences of MAP kinase cascade activation is the induction of the expression of a number of proteins [4]. To test whether this downstream effect of the pheromone action is important for PCD development, the present inventors checked whether inhibition of protein synthesis prevents pheromone-induced PCD in yeast. As shown in Table 1 (row 3), cycloheximide (which is well known in the art to inhibit protein synthesis in eukaryotes) inhibits both ROS production and the accumulation of dead cells.

Table 1 Pheromone causes mitochondrion-linked ROS production and death of yeast cells

Additions Effects

Cells Pheromone Other ROS-producing cells Dead cells (after (α-factor) (after 1.5 h) in % 3.5h)in%

ex + 0.8 + 0.8 0.9 + 1.0

2 a + - 26.9 ±3.3 26.3 ±4.0

3 a + cycloheximide 0 2.6 ±1.3

CO 4 a , no mitochondria + - 0 1.5 ±0.6 c CD CO 5 cyclΔ/cyc7Δ + -

— | a, 33.5 ± 12.7 2.6 ±3.6

H

C —1 6 a, ste20Δ + - 0.5 ±0.6 0.5 ±0.8 m

CO 7 a, cmdl-6 + - 54.9 ±4.4 51.6+1.0 m m

H 8 a + CsA 2.6 ±0.6 2.5 ±3.6

9 a + α - - 24.3 ±4.0 5.8 ±1.0 ro

10 a + α - chloroquine nd 33.7 ±8.7

11 a - chloroquine nd 1.4 ±0.2

12 a + chloroquine nd 32.9 ±3.4

13 a + α _ chloroquine nd 2.9 ±1.8

CsA

CsA = cyclosporin nd = not determined

The downstream events of PCD development in higher cells usually require mitochondria (see [1] for review). The inventors therefore tested whether yeast without functional mitochondria undergoes pheromone-induced PCD. Table 1 (row 4) shows that the destruction of mitochondrial DNA by ethidium bromide completely abolishes the effect of α-factor on both ROS formation and accumulation of dead cells. On the other hand, the disruption of mitochondrial functions does not prevent cell shmooing in response to α-factor addition, suggesting that the mating response is still intact. Thus, the presence of functional mitochondria is a common requirement both for yeast and higher (multicellular) organisms.

In multicellular organisms, the mitochondria-related PCD events generally include generation of ROS, opening of the mitochondrial permeability transition pore (PTP) and release of cytochrome c into the cytoplasm (see [1] for review). The inventors posed the question whether a similar pathway is involved in pheromone-induced PCD in yeast. As shown in Table 1 (row 5), yeast cells without cytochrome c are still generating ROS but fail to complete PCD. On the other hand, inhibition of PTP formation by cyclosporin A (CsA) prevents both ROS accumulation and cell death (Table 1, row 8).

It has been known that CsA inhibits not only PTP formation but also the calcineurin/calmodulin system [5], which was shown to regulate PCD development in animal cells [6]. To test how CsA interferes with PCD development in yeast, the inventors checked whether yeast cells with a compromised calcineurin/calmodulin system (a cmdl-6 mutant strain) undergo pheromone- induced cell death. As shown in Table 1 (row 7), both ROS accumulation and cell death are, as compared to wild-type α-cells plus alpha-factor, approximately doubled in the cmdl-6 mutant. Since earlier observations have shown that yeasts with mutations in the calmodulin and calcineurin genes are supersensitive to pheromone treatment [7], [8], it seems likely that the calcineurin/calmodulin system inhibits PCD. However, as CsA prevents both ROS accumulation and PCD (see the preceding para, and Table 1, row 8), CsA is unlikely to exert its effects via the calcineurin/cahnodulin system because a defective calcineurin/calmodulin system would trigger PCD rather than prevent PCD. Thus, it is evident that CsA inhibits PCD in yeast due to its effect on the PTP. The data on pheromone-induced PCD cascade, as obtained by the inventors, are schematically presented in Fig. 2. The cascade is assumed to include the calmodulin/calcineurin- controlled MAP kinase pathway, ROS generation and the formation of PTP triggering the release of cytochrome c and cell death. The inventors were also interested in whether pheromone-induced PCD occurs in native conditions. They mixed cells of α- and α-type and monitored the appearance of ROS-positive and dead cells. As shown in Table 1 (row 9) and Fig. ID, approximately 25%) of the cells were ROS-positive under these conditions, similar to the experiments with added pheromone. The percentage of dead cells was higher than that of control cells (cells without any pheromone additions) but significantly lower than α-type cells with α-factor added. Apparently the local concentrations of pheromones in the mating-induced agglutinates of α- and α-type cells are potentially high enough to induce cell death, but successful mating reverses or prevents PCD development. Indeed, it is logical to expect that the altruistic suicide of a yeast cell is a relatively rare event under normal physiological conditions, h line with such an explanation it was shown that once two cells fuse and form a diploid they lose their sensitivity to pheromones [2]. To check the possibility that cell fusion during mating prevents the PCD development, the inventors incubated a mixture of α- and α-type cells in the presence of 5 mM chloroquine; chloroquine at 5 mM does not significantly affect vegetative growth but does prevent zygote formation by inhibiting cell wall degradation, which is necessary for fusion [9]. As shown in Table 1, row 10 and Fig. ID, approximately 30% of mating cells die in the presence of chloroquine. Chloroquine is not toxic to non-mating cells (Table 1, row 11), nor does it affect the percentage of α-cells killed by α-factor (compare rows 12 and 2, Table 1). As expected, the anti-apoptotic agent CsA rescues the chloroquine-induced death in the mixture of α- and α-type cells (Table 1, row 13 and Fig ID). Thus, mating-induced cell agglutination without fusion leads to PCD in yeast. Taken together, the data on mixtures of α- and α-type cells suggest that PCD is a natural part of the yeast mating process. Interestingly, the plant antibiotic osmotin activates one of the components of the yeast mating kinase cascade [10] which leads to apoptosis-like cell death [11], suggesting that plants are able to use this natural process of yeast mating-linked PCD to defend themselves against pathogenic fungi.

The data accumulated by the inventors show that yeast, in particular yeast of the genus Saccharomyces, in particular of the species S. cerevisiae, can induce cell death which seems in outline to be similar to PCD in animal cells (although yeasts do not have caspases which are the components of the major apoptotic machinery in animal cells [12]). Thus, the data of the inventors are in line with data previously obtained (PCD has recently been described for plant cells although genes encoding typical caspases have not been found in plants [13]). Similarly, bacteria also lack caspases and still undergo PCD under certain conditions [14], [15]. On the other hand, PCD pathways exist that are without caspases involved. Based on the inventors' finding, a completely novel treatment of infections, both in humans and animals, caused by unicellular organisms such as yeast and other fungi has been developed. Accordingly, the invention in one of its aspects relates to the use of a yeast pheromone for the manufacture of a medicament for the treatment of yeast and fungal infections. According to a preferred embodiment of the invention, the infection is an infection caused by any of the following microorganisms: Absidia spp., Acremonium spp., Actinomadura spp., Alternaria spp. Apophysomyces spp., Arthrinium spp., Arthrographis spp., Aspergillus spp., Aureobasidium spp. Basidiobolus spp., Beauveria spp., Bipolaris spp., Blastomyces spp., Blastoschizomyces spp. Botrytis spp., Candida spp., Chaetomium spp., Chrysosporium spp., Cladophialophora spp. Cladosporium spp., Coccidioides spp., Conidiobolus spp., Cryptococcus spp., Cunninghamella spp., Curvularia spp., Dermatophytes, Epicoccum spp., Epidermophyton spp., Exophiala spp Fonsecaea spp., Fusarium spp., Geotrichum spp., Gliocladium spp., Graphium spp Helminthosporium spp., Histoplasma spp., Lacazia spp., Leptosphaeria spp., Madurella spp Malassezia spp., Malbranchea spp., Microsporum spp., Mucor spp., Neotestudina spp Nigrospora spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Paracoccidioides spp Penicillium spp., Phaeococcomyces spp., Phialophora spp., Phoma spp., Piedraia spp. Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp Saccharomyces spp., Scedosporium spp., Scopulariopsis spp., Sepedonium spp., Sporobolomyces spp., Sporothrix spp., Sporotrichum spp., Stachybotrys spp., Stemphylium spp., Streptomyces spp., Syncephalastrum spp., Trichoderma spp., Trichophyton spp., Trichosporon spp., Trichothecium spp., Ulocladium spp., Ustilago spp., Verticillium spp., and Zygomycetes. h particular, the infection is caused by Candida, more particularly by Candida albicans.

In accordance with a further preferred embodiment of the invention, the infection is caused by Candida, in particular Candida albicans, or Aspergillus.

In the alternative, according to a preferred embodiment the infection is blastomycosis, chromoblastomycosis, an eye infection, in particular one caused by Fusarium spp, Aspergillus spp, Acremonium, or Candida, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidiomycosis, sporotrichosis, zygomycosis, lobomycosis, mycetoma, a nail, hair, or skin disease, in particular onychomycosis (tinea unguium), piedra, pityriasis versicolor, tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea favosa, tinea nigra, tinea pedis, otomycosis, phaeohyphomycosis, or rhinosporidiosis. According to a still further preferred embodiment, the pheromone is a pheromone produced by any of the following: Absidia spp., Acremonium spp., Actinomadura spp., Alternaria spp., Apophysomyces spp., Arthrinium spp., Arthrographis spp., Aspergillus spp., Aureobasidium spp., Basidiobolus spp., Beauveria spp., Bipolaris spp., Blastomyces spp., Blastoschizomyces spp., Botrytis spp., Candida spp., Chaetomium spp., Chrysosporium spp., Cladophialophora spp., Cladosporium spp., Coccidioides spp., Conidiobolus spp., Cryptococcus spp., Cunninghamella spp., Curvularia spp., Dermatophytes, Epicoccum spp., Epidermophyton spp., Exophiala spp., Fonsecaea spp., Fusarium spp., Geotrichum spp., Gliocladium spp., Graphium spp., Helminthosporium spp., Histoplasma spp., Lacazia spp., Leptosphaeria spp., Madurella spp., Malassezia spp., Malbranchea spp., Microsporum spp., Mucor spp., Neotestudina spp., Nigrospora spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Paracoccidioides spp., Penicillium spp., Phaeococcomyces spp., Phialophora spp., Phoma spp., Piedraia spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsis spp., Sepedonium spp., Sporobolomyces spp., Sporothrix spp., Sporotrichum spp., Stachybotrys spp., Stemphylium spp., Streptomyces spp., Syncephalastrum spp., Trichoderma spp., Trichophyton spp., Trichosporon spp., Trichothecium spp., Ulocladium spp., Ustilago spp., Verticillium spp., and Zygomycetes.

According to a still further preferred embodiment, the pheromone is a yeast α-factor, in particular the α-factor of Saccharomyces cerevisiae.

According to a still further preferred embodiment, the pheromone is a yeast a-factor, in particular the a-factor of Saccharomyces cerevisiae.

A second aspect of the present invention relates to a method to identify substances inhibiting, stimulating, or interfering with PCD. Generally, the method comprises the steps of (i) contacting yeast or fungal cells in the presence of a sexual pheromone with the substance to be tested, and either (ii) determining the number of dead cells, in particular by Phloxin B or Methylene Blue staining, or by staining with dyes produced by Molecular Probes (Eugene) (FUN1, FU 2) or by any other vital dye or other means of assaying for cell death, or (iii) determining the number of cells that have survived.

The number of cells that have survived may be determined by the so-called plating method.

When applying this method, the culture to be analyzed is plated on agar. Subsequently, the colonies are counted, generally after a couple of days. Alternatively, the culture to be analyzed can be grown in liquid medium overnight and scored in the morning: if the culture is transparent, the cells did not survive. Quite conversely, the cells are survivors if the medium is murky. Quantitative data may be obtained by counting cells using microscopy-based methods, creating calibration curves using spectrophotometry and therefore quantifying the number of cells by the absorbance or transmission of light in a spectrophotometer, or by FACS.

Preferably, the assay according to the invention is a high-throughput assay for small molecules inducing apoptosis in unicellular organisms. Once such substance/small molecule has been identified to induce cell death, e.g., of yeasts, it may be used for the manufacture of a medicament for the treatment of infections caused by, e.g., yeast, in particular for the treatment of any of the diseases/infections mentioned previously.

The conditions for the assay are selected (which yeast/fungal strain to use and the dose/time of the pheromone/α-factor treatment) such that close to 100%) of the cells die. For example, one can use a fresh culture of a cmdl-6 mutant strain and treat it with 0.1 mg/ml of alpha-factor for 5 to 7 h). Under those conditions CsA should rescue the PCD by inhibiting the PCD cascade (by preventing the activation of the permeability transition pore (PTP)).

The to be tested substance is added to three tubes containing: 1. yeast without α-factor

2. yeast with α-factor

3. yeast with α-factor and with CsA

After 4 hours the samples are diluted with a 1000-fold excess volume of fresh medium without any additions. Samples are then grown overnight and scored next morning for being transparent (death) or murky (growth).

It should be noted that in this scenario and in all previous and subsequent scenarios, α-type yeast can be exposed to a-factor as an alternative to a-type yeast exposed to α-factor. Therefore, for the sake of clarity, pheromone refers to α-factor or a-factor and it is understood that when α-factor is used it is applied to a-type yeast and when a-factor is used, it is applied to α-type yeast.

Alternative 1:

Tubes 1 and 3 are murky: the cells survived and grew overnight; tube 2 is transparent: the cells were killed. Conclusion: the substance has no effect Alternative 2:

Tube 2 is murky: the cells survived and grew overnight. Conclusion: the substance is a potential anti-apoptotic/anti-necrotic agent.

Alternative 3:

At least tubes 2 and 3 are transparent: the cells were killed by the α-factor. Conclusion: the substance is a general poison.

Alternative 4:

Tubes 1 and 2 are transparent: the cells were killed; tube 3 is murky: the cells survived and grew overnight. Conclusion: the substance is a potential pro-apoptotic/pro-necrotic agent acting above the action of PTP in the cascade.

According to a preferred embodiment of the above method, the yeast cells are cells of Saccharomyces, in particular of Saccharomyces cerevisiae.

A further aspect of the present invention (third aspect) is directed to a method to identify genes, RNA, or proteins that are involved in PCD. The method comprises the steps of

(1) mutagenizing yeast/fungal cells;

(2) selecting for those cells that are resistant to pheromone-induced death;

(3) selecting for those cells that have retained the capacity to mate, thereby getting rid of the sterile mutants (those cells that are mutant in the mating pathway); and (4) analyzing the genome of the cells obtained in (3) for mutations.

The method according to the third aspect may be exemplified by describing the method to identify the yeast genes involved in PCD. For that purpose, yeast cells of α-mating type will have to be transformed with a library mutagenized by transposon insertions in bacteria [16]. A preferred yeast strain that can be used for the transformation step is a cmdl-6 mutation or a cnbl deletion strain. Both strains are hypersensitive to pheromone treatment. The principle of the method is that the transposon is randomly inserted into the yeast gene(s), thereby inactivating the gene(s) concerned (genes into which a transposon is inserted). After transformation of yeast cells with such a library, the genomic copy of the gene is replaced by the transposon-inactivated gene. As an outcome of this method, the transposon-inactivated gene can easily be identified by PCR [17].

It is accepted in the art that other methods exist to mutagenize yeast or other cells. For example, including but not limited to chemical mutagenesis, UN, and radiation, and these methods are also included in the scope of the invention.

The mutagenized cells are treated with the pheromone under conditions such that the cells die (more than 99%> of the cells die after a 4hr exposure to α-factor [7]). Before mutagenesis is started, the concentration of α-factor and the time of exposure is adjusted such that no more than 0.01%) of the cells survive. Treatment of the cells with the pheromone results in a selection for the survivors (primary selection). As most of the survivors are defective in the mating pathway, it will be necessary to get rid of those by a 2nd stage of selection, thereby selecting for those colonies which can still produce diploids, and are thus not defective for mating. The colonies which can still mate can be selected using the methods described in [18]. These colonies correspond to cells where an essential gene for PCD has been inactivated. The gene(s) can be identified using the methods described in [16,17].

A still further aspect (the fourth aspect) of the present invention utilizes the property of the pheromones to induce apoptosis in unicellular organisms. As fungal and yeast infections are not limited to higher eukaryotic organisms, the invention further relates to the use of yeast pheromones as a fungicide for plants, in particular crop (e.g., rice, maize, wheat, barley, rye), fruit (e.g., apple, pear, peach, cherry, pineapple, plum) and vegetables (e.g., cabbage, eggplant, squash, bean, pie).

h the accompanying figures,

Figure 1A are photographs showing the accumulation of ROS in the presence/absence of pheromone (α-factor) as visualized by the addition of H₂DCF-DA. The bar indicates a length of

10 μm, thereby determining the scale. Figure IB shows TUΝEL staimng illustrating DΝA breakage in pheromone-treated but not in the control cells. The bar indicates a length of 2 μm.

Figure 1C is a FACS analysis showing an accumulation of cells with degraded (less then lc)

DΝA in pheromone-treated cells.

Figure ID shows cell staining with phloxin B. Only the dead cells accumulate the dye. The bar indicates a length of 5 μm. Figure 2 shows a model for a PCD cascade in Saccharomyces cerevisiae, wherein PTP, ROS, and Ste20 stand for (opening the) permeability transition pore, reactive oxygen species, and Ste20 kinase, which is a key component of the MAP kinase pathway.

Materials and Methods

Cells were grown in YEPD medium [18] at 30°C to the density of 10⁶/ml, spun and resuspended in an equal volume of fresh medium with or without pheromone. For ROS visualization cells were incubated on a shaker at 30°C for 80 min, before H₂DCF-DA (Molecular Probes, Eugene) was added to 10 μM. After 10 min, the cells were photographed under a lOx objective lens in DIC and FITC channels. Cyclosporin A (Sigma-Aldrich Co) was used at 60 μg/ml, chloroquine (Sigma-Aldrich Co) at 5 mM, cycloheximide (Sigma-Aldrich Co) at 20 μg/ml. ROS accumulation in chloroquine-treated cells was not determined because chloroquine induces cellular auto-fluorescence.

To visualize dead cells, the culture was incubated with 0.4 mg/ml Phloxin B for 5 min, photographs were taken under a 40x objective lens. Vital staining of cells incubated with or without pheromone was also done with Methylene Blue - the commonly used reagent for yeast vital staining. The results were similar to the ones obtained with Phloxin B. Phloxin B has been chosen as the principal reagent for vital staining because Methylene Blue can not be used for staining of cells with compromised mitochondria.

The experiments were repeated at least twice for every experimental condition and more than 100 cells were counted each time. FACS analysis was done according to [19]. TUNEL staining was done as described in [20]. Approximately 20% of the cells treated with pheromone were TUNEL-positive, although a precise quantification was found to be impossible.

Yeast without mitochondrial DNA were generated by growing wild type on a plate with Ethidium Bromide [21]. References Skulachev, N. P.: Why are mitochondria involved in apoptosis? Permeability transition pores and apoptosis as selective mechanisms to eliminate superoxide-producing mitochondria and cells FEBS Lett 1996 397, 7-10 Kurjan, J.: The pheromone response pathway in Saccharomyces cerevisiae Annu Rev Genet 1993 27, 147-179 Possel, H., Νoack, H., Augustin, W., Keilhoff, G. and Wolf, G: 2, 7- Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation FEBS Lett 1997 416, 175-178 Dan, L, Watanabe, Ν. M. and Kusumi, A.: The StelO group kinases as regulators of MAP kinase cascades Trends Cell Biol 2001 11, 220-230 Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, I. and Schreiber, S. L.: Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP- FK506 complexes Cell 1991 66, 807-815 Lotem, J., Kama, R. and Sachs, L.: Suppression or induction of apoptosis by opposing pathways downstream from calcium-activated calcineurin Proc Νatl Acad Sci U S A 1999 96, 12016-12020 Cyert, M. S. and Thorner, J.: Regulatory subunit (CNB1 gene product) of yeast Ca²⁺/calmodulin- dependent phosphoprotein phosphatases is required for adaptation to pheromone Mol Cell Biol 1992 12, 3460-3469 Moser, M. J., Geiser, J. R. and Davis, T. Ν.: Ca²⁺ -calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca -calmodulin- dependent protein kinase Mol Cell Biol 1996 16, 4824-4831 Doi, S., Tanabe, K., Watanabe, M. and Yoshimura, M.: Chloroquine, a lysosomotropic agent, inhibits zygote formation in yeast Arch Microbiol 1989 151, 20-25 Yun, D. J., Ibeas, J. I., Lee, H., Coca, M. A., Narasimhan, M. L., Uesono, Y., Hasegawa, P. M., Pardo, J. M. and Bressan, R. A.: Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility Mol Cell 1998 1, 807-817 Narasimhan, M. L., Damsz, B., Coca, M. A., Ibeas, J. I., Yun, D. J., Pardo, J. M., Hasegawa, P. M. and Bressan, R. A.: A plant defense response effector induces microbial apoptosis Mol Cell 2001 8, 921-930 Aravind, L., Dixit, V. M. and Koonin, E. V.: Apoptotic molecular machinery: vastly increased complexity in vertebrates revealed by genome comparisons Science 2001 291, 1279-1284 Lam, E., Pontier, D. and del Pozo, O.: Die and let live - programmed cell death in plants Curr Opin Plant Biol 1999 2, 502-507 Raff, M.: Cell suicide for beginners Nature 1998 396, 119-122 Lewis, K.: Programmed death in bacteria Microbiol Mol Biol Rev 2000 64, 503-514 Burns, N., B. Grimwade, P.B. Ross-Macdonald, E.Y. Choi, K. Finberg, G.S. Roeder, and M. Snyder.: Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae Genes Dev. 1994 8, 1087-1105 Riley, J., R. Butler, D. Ogilvie, R. Finniear, D. Jenner, S. Powell, R. Anand, J.C. Smith, and A.F. Markham: A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones Nucleic Acids Res. 1990 18:2887-2890 Rose, M. D., Winston, F. and Hieter, P.: Laboratory course manuals for methods in yeast genetics, Cold Spring Harbor Laboratory Press, New York 1990 Piatti, S., Bohm, T., Cocker, J. H., Diffley, J. F. and Nasmyth, K.: Activation of S-phase- promoting CDKs in late Gl defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast Genes Dev. 1996 10, 1516-1531 Madeo, F., Frohlich, E. and Frohlich, K. U.: A yeast mutant showing diagnostic markers of early and late apoptosis J Cell Biol. 1997 139, 729-734 Pichler, S., Piatti, S. and Nasmyth, K.: Is the yeast anaphase promoting complex needed to prevent re- replication during G2 and M phases? EMBO J 1997 16, 5988-5997

Claims

1. Use of a yeast pheromone for the manufacture of a medicament for the treatment of yeast and other fungal infections.

2. Use of claim 1, wherein the infection is caused by Absidia spp., Acremonium spp., Actinomadura spp., Alternaria spp., Apophysomyces spp., Arthrinium spp., Arthrographis spp., Aspergillus spp., Aureobasidium spp., Basidiobolus spp., Beauveria spp., Bipolaris spp., Blastomyces spp., Blastoschizomyces spp., Botrytis spp., Candida spp., Chaetomium spp., Chrysosporium spp., Cladophialophora spp., Cladosporium spp., Coccidioides spp

Conidiobolus spp., Cryptococcus spp., Cunninghamella spp., Curvularia spp Dermatophytes, Epicoccum spp., Epidermophyton spp., Exophiala spp., Fonsecaea spp Fusarium spp., Geotrichum spp., Gliocladium spp., Graphium spp., Helminthosporium spp Histoplasma spp., Lacazia spp., Leptosphaeria spp., Madurella spp., Malassezia spp Malbranchea spp., Microsporum spp., Mucor spp., Neotestudina spp., Nigrospora spp

Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Paracoccidioides spp., Penicillium spp., Phaeococcomyces spp., Phialophora spp., Phoma spp., Piedraia spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsis spp., Sepedonium spp., Sporobolomyces spp., Sporothrix spp., Sporotrichum spp., Stachybotrys spp., Stemphylium spp., Streptomyces spp., Syncephalastrum spp., Trichoderma spp., Trichophyton spp., Trichosporon spp., Trichothecium spp., Ulocladium spp., Ustilago spp., Verticillium spp., and Zygomycetes. In particular, the infection is caused by Candida, more particularly by Candida albicans.

3. Use of claim 2, wherein the infection is caused by a microorganism of the genus Candida, in particular by a microorganism of the species Candida albicans, or by a microorganism of the genus Aspergillus.

4. Use of claim 2, wherein the infection is blastomycosis, chromoblastomycosis, an eye infection, in particular one caused by Fusarium spp, Aspergillus spp, Acremonium, or Candida, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidiomycosis, sporotrichosis, zygomycosis, lobomycosis, mycetoma, a nail, hair, or skin disease, in particular onychomycosis (tinea unguium), piedra, pityriasis versicolor, tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea favosa, tinea nigra, tinea pedis, otomycosis, phaeohyphomycosis, or rhinosporidiosis.

5. Use of any of the preceding claims, wherein the pheromone is a pheromone produced by Absidia spp., Acremonium spp., Actinomadura spp., Alternaria spp., Apophysomyces spp., Arthrinium spp., Arthrographis spp., Aspergillus spp., Aureobasidium spp., Basidiobolus spp., Beauveria spp., Bipolaris spp., Blastomyces spp., Blastoschizomyces spp., Botrytis spp., Candida spp., Chaetomium spp., Chrysosporium spp., Cladophialophora spp., Cladosporium spp., Coccidioides spp., Conidiobolus spp., Cryptococcus spp., Cunninghamella spp., Curvularia spp., Dermatophytes, Epicoccum spp., Epidermophyton spp., Exophiala spp., Fonsecaea spp., Fusarium spp., Geotrichum spp., Gliocladium spp., Graphium spp., Helminthosporium spp., Histoplasma spp., Lacazia spp., Leptosphaeria spp., Madurella spp., Malassezia spp., Malbranchea spp., Microsporum spp., Mucor spp., Neotestudina spp., Nigrospora spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Paracoccidioides spp., Penicillium spp., Phaeococcomyces spp., Phialophora spp., Phoma spp., Piedraia spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsis spp.,

Sepedonium spp., Sporobolomyces spp., Sporothrix spp., Sporotrichum spp., Stachybotrys spp., Stemphylium spp., Streptomyces spp., Syncephalastrum spp., Trichoderma spp., Trichophyton spp., Trichosporon spp., Trichothecium spp., Ulocladium spp., Ustilago spp., Verticillium spp., and Zygomycetes.

6. Use of claim 5, wherein the pheromone is a yeast α-factor, in particular the α-factor of Saccharomyces cerevisiae.

7. Use of claim 5, wherein the pheromone is a yeast a-factor, in particular the a-factor of Saccharomyces cerevisiae.

8. Method to identify substances inhibiting, stimulating, or interfering with PCD, the method comprising the steps of (i) contacting yeast or fungal cells in the presence of a sexual pheromone with the substance to be tested, and (ii) determining either (ii)(a) the number of dead cells or (ii) (b) the number of cells that have survived.

9. The method of claim 7, wherein step (ii)(a) is done by Phloxin B staimng, by Methylene Blue staining, by staining with a dye produced by Molecular Probes (Eugene; FUN1, FUN2), or by any other vital dye or other means of assaying for cell death.

10. The method of claim 8 or 9, wherein the yeast cells are cells of Saccharomyces, in particular of Saccharomyces cerevisiae.

11. Method to identify genes, RNA, or proteins that are involved in PCD, the method comprising the step(s) of RNA, or proteins that are involved in PCD. The method comprises the steps of

(1) mutagenizing yeast/fungal cells;

(2) selecting for those cells that are resistant to pheromone-induced death; (3) selecting for those cells that have retained the capacity to mate, thereby getting rid of the sterile mutants (those cells that are mutant in the mating pathway); and (4) cloning the cells obtained in (3) and analyzing the genome for mutations.

12. The method of claim 10, wherein the mutagenizing step (1) is by transposon mutagenesis.

13. The method of claim 12, wherein the mutagenizing step is by chemical mutagenesis, UN, radiation, or other means known in the art to generate mutant cells.

14. Use of a yeast pheromone as a fungicide for plants.

15. Use of claim 14, wherein the plant is in rice, maize, wheat, barley, rye, apple, pear, peach, cherry, pineapple, plum, cabbage, eggplant, squash, bean, or pie.