SK122002A3 - Novel antifungal agents and fungicides, method for the production thereof and their use - Google Patents

Abstract

The invention relates to the preparation of protein toxins from yeasts so-called killer yeasts using genetic technology in order to control human pathogenic and plant pathogenic yeasts and/or fungi, whereby these are selectively destroyed. The high specificity enables the protein toxins to be used as an antifungal agent and/or fungicide. In addition, protein toxins of this type can be used for protecting plants.

Description

ANTIMYCOTICS AND FUNGICIDES, METHOD OF PREPARATION AND USE

Technical field

The present invention relates to antifungals and fungicides obtainable from yeast, to a process for their preparation and to their use.

BACKGROUND OF THE INVENTION

Selective antifungals are of great importance because infections caused by fungi or yeasts in humans have become widespread in recent years, and there is an increasingly undesirable contamination of food and feed. Mycoses have particularly severe consequences in immunosuppressed patients whose cellular and humoral defense systems must be maintained at a not quite functional level (Anaissie, 1992; Meunier et al., 1992; Wingard, 1995). In particular, HIV-1-infected persons (AIDS), who in the advanced stage of the disease die very often from opportunistic infections caused by human pathogenic fungi and / or yeast, are particularly at risk for mycosis (Levy, 1993). The antimycotics that are currently used to treat these infections (such as Amphotericin B, Fluconazole, Itraconazole, Ketoconazole) have significant side effects because they disrupt the structural integrity of the eukaryotic membrane of the cytoplasm, thereby damaging even the infected host organism (Hector, 1993) . Furthermore, the application of conventional antifungal agents leads to a rapid increase in fluconazole resistance in a short time, which is rapidly expanding in human pathogenic microorganisms and is thus an increasing problem (Cameron et al., 1993; Chavenet et al., 1994; Maenza et al., 1996) Pfaller et al., 1994; Rex et al., 1995; Troillet et al., 1993). It is therefore important to develop antimycotics which, like bacterial antibiotics, are characterized by high selectivity and that they attack only fungi and yeasts pathogenic to humans. However, since most cellular processes in higher organisms are driven by gene products that exhibit a high degree of functional homology in eukaryotes, it has not been possible to date.

31854 / H to develop "specific antifungal antibiotics" (Kurz, 1998; Komiyama et al., 1998).

The target area of selective antifungal agents are yeast cell wall β-1,3-glucanes, which are essential for mechanical and osmotic cell stability, but which do not occur in higher eukaryotes, so that they could be used as an "Achilles heel". .: Achillesverse, supposed to be Achillesferse) in the control of pathogenic yeast (Roemer et al., 1994). Although substances that selectively attack the cell wall structure of yeast and fungi are of great interest, no antibiotics-like substances have been used to date to treat mycoses. While bacterial antibiotic producers appeared as early as this century, similar effects in yeast were not observed until the early 1960s, when so-called "yeasts" were identified and observed. Killer yeast (Bevan and Makower, 1963). Killer-producing toxins of baker's yeast Saccharomyces cerevisiae produce and secrete proteins designated as "killertoxins" that kill sensitive yeast in a receptor-dependent process (Rezeptor-abhängigen Prozess) (Bussey, 1991; Tipper and Schmitt, 1991). The ability to produce a toxin resides in S. cerevisiae from infection with reovirus-like double-stranded RNA viruses (Reovirus-ähnliche Dopplestrang-RNA-Viren), which are stable in yeast cytoplasm and persist in large numbers without the eukaryotic host they were noticeably damaged (Tipper and Schmitt, 1991). The three hitherto known killer toxins (K1, K2, K28) from yeast S. cerevisiae are non-glycosylated α / β heterodimers, which are transmitted from the infected cell as high-molecular preprotoxins and converted into intracellular secretory pathway by complex modifications to biologically active killer (Hanes et al., 1986; Dignard et al., 1991; Schmitt and Tipper, 1995). The toxic action of the S. cerevisiae toxin consists either in disrupting membrane integrity (toxins Κ1, K2) or (as in killertoxin K28) in cell cycle arrest (Zellcyclus-Arrest) with targeted inhibition of DNA synthesis (Bussey, 1991; Schmitt and Compain) , 1995; Schmitt et al., 1996). Killer toxins of classes K1, K2 and K28, although in their mode of action a

31854 / H in their physicochemical properties differ considerably from one another, but they share a narrow spectrum of activity in common and furthermore that they kill predominantly sensitive yeasts of closely related species. This limited spectrum of efficacy is due to the fact that the previously characterized baker's yeast killer toxins with different receptor populations at yeast cell wall and cytoplasmic membrane levels must react to each other in order to kill sensitive target cells. The primary yeast cell wall toxin receptors are either highly branched β-Ι, θ-D-glucans, or outer mannotriose cell wall mannoprotein side chains (Bussey, 1991; Schmitt and Radler, 1987, 1988).

In addition to the yeast viral protein toxins of S. cerevisiae, Hanseniaspora uvarum, Zygosaccharomyces bailii and Ustilago maydis, the killer strains have also been described in the genera Debaromyces, Hansenula, Cryptococcus, Rhodotorula, Pichia, Kluyveromyen and Parkis, Colis, Colis. et al., 1996, Schmitt and Neuhausen, 1994; Walker et al., 1995). However, the genetic basis of this killer phenomenon in these yeasts lies not in viral genomes, but either in linear DNA plasmids or in chromosomal yeast genes (Schrunder et al., 1994).

Intensive molecular-biological studies of various toxin-producing killer-yeasts have shown that the secretion of toxic proteins (killer-toxins) in yeast is widespread and that it can be overlooked in the development of selective antifungals (Walker et al., 1995, Hodgson et al. , 1995; Polonelli et al., 1986; Schmitt and Neuhausen, 1994; Neuhausen and Schmitt, 1996; Schmitt et al., 1997), but to date these protein toxins have not been prepared.

It is therefore an object of the present invention to provide suitable highly potent protein toxins with antifungal or fungicidal effects on the control of yeast and / or fungi with a pathogenic effect on humans or plants.

31854 / H

SUMMARY OF THE INVENTION

Surprisingly, toxins produced and secreted with high efficiency, namely the killer-toxin WICALTIN (also proteintoxin) from the wild yeast strain Williopsis californica strain 3/57 (DSM), have been found to be particularly suitable for destroying yeasts and / or fungi with pathogenic effects on humans or plants. 12865) and virus-encoded ZYGOCIN (also protein toxin) from yeast Zygosaccharomyces bailii (DSM 12864). In addition, the detrimental harmful yeasts in food and feed can be killed by these agents. Both of these protein toxins can therefore potentially be used as an antifungal and / or fungicide for the control of yeast and / or fungal infections, in particular for the control of mycoses. These indications are verified in the present invention by investigating the mode of action of these agents. Within the scope of the present invention, the toxins have been appropriately cloned and sequenced, and a gene-preparation and expression procedure (Clberexpression) has been developed in the culture of WICALTIN and ZYGOCIN.

Accordingly, the present invention relates to protein toxins obtainable from Williopsis californica, in particular from strain DSM 12865 and Zygosaccharomyces bailii, in particular from strain DSM 12864. Both strains were deposited in the German Collection of Microorganisms and Cell Cultures DSMZ under the Budapest Treaty on 9 June 1999. -Deutsche Sammlung von Microorganism und Zellkulturen GmbH, 38124 Braunschweig, Mascheroder Weg 1b (www.dsmz.de).

In particular, strains DSM 12864 and DSM 12865 secreted biologically highly active protein toxins which, due to their broad spectrum of activity (see Examples 4 and 7), killed many human and plant pathogenic yeasts and fungi. Therefore, the present invention also relates to selective antifungals or fungicides in the sense that the protein toxins - and the polypeptides of the invention and the nucleic acids encoded by them of the invention, in particular in the functional unit of the toxin - mentioned below, are potential biopharmaceuticals which receptor-mediated agents kill exclusively yeast and / or fungi and pro

31854 / H higher eukaryotes - and of course for human and mammalian cells - as well as for plants, especially for crop plants, are quite harmless (see Pfeiffer et al.,

1988).

It was possible to selectively kill the following human and plant-pathogenic as well as apatogenic yeasts and / or fungi:

ZYGOCIN-sensitive yeast species: Saccharomyces cerevisiae, Candida albicans, Candida krusei, Candida glabrata, Candida vinii, Hanseniaspora uvarum, Kluyveromyces marxianus, Metschnikowia pulcherrima, Ustilago maydis, Debaromyichia nuclia, Pichosomyiaiaia, Pichosiaiaia, Pichia .

WICALTIN-sensitive yeast species: Candida albicans, Candida glabrata, Candida tropicalis, Debaromyces hansenii, Kluyveromyces lactis, Metschnikowia pulcherrima, Pichia anomala, Pichia jadinii, Saccharomyces cerevisiae, Sporthhysa sp.

The particularly high activity of the wicaltin-producing strain DSM 12865 is likely to be due to the pronounced secretory efficacy of this strain, which is significantly higher compared to other strains of the same yeast species. The "killer" -property of the zigocin-producing yeast strain DSM 12864 consists in infection with toxin-encoding double-stranded RNA viruses (Mzb-dsRNA) that are stable, persist in the cytoplasm in a high copy number and produce the appropriate yeast (DSM 12864in production strain) (See Schmitt and Neuhausen, 1994). Other strains of the same species showed no toxin production because they do not contain any toxin-coding dsRNA-viruses in the cytoplasm, so they are phenotypically classified as "non-killer" types.

A further object of the invention are nucleic acids encoding protein toxin - having the amino acid sequence of SEQ ID No 1 and No 2 and glucanase activity - or functional variants of these nucleic acids, and

31854 / H portions thereof of at least 8 nucleotides, in particular of at least 15 or 20 nucleotides, in particular of at least 100 nucleotides, in particular of at least 300 nucleotides (hereinafter referred to as "nucleic acids of the invention").

The complete nucleic acids encode protein toxins which, after intracellular processing and secretion, have a 309 amino acid size and a molecular weight of 34 kDA (SEQ ID No 1), respectively. 99 amino acids and a molecular weight of 10 kDA (SEQ ID No 2). Expression of this nucleic acid of SEQ ID No 1 in yeast S. cerevisiae results in recombinant WICALTIN, which is secreted in the yeast culture supernatant as a glycosylated protein with significant β-1,3-D-glucanase activity (see Example 10). Further experiments carried out according to the invention confirmed that the nucleic acids of the invention are nucleic acids which encode a protein toxin with glucanase activity in SEQ ID No 1 and, in the case of SEQ ID No 2, probably encode an O-glycosylated protein toxin referred to as ZYGOCIN in vivo. Nucleic acids of the invention can be obtained from DSM 12865 (SEQ ID No 1) and from DSM 12864 (SEQ ID No 2).

In one preferred embodiment, the nucleic acids of the invention are DNA or RNA, in particular double stranded DNA, and in particular DNA having the nucleic acid sequence of SEQ ID No 1 from position 1 to position 947 and according to SEQ ID No 2 from position 1 to position 713. Both of these positions determine, according to the invention, the beginning and the end of the coding region, i always the first and last amino acids in the respective reading frame (Leseraster).

By "functional variant" is meant according to the invention a nucleic acid that is functionally related to the nucleic acids of the invention. For example, related nucleic acids are nucleic acids from different yeast cells, respectively. strains and cultures or allelic variants. The present invention also encompasses nucleic acid variants that may be derived from various yeast / yeast strains or other infectious agents such as dermatophytes and fungi (according to the DHS system).

In another sense, the term "variants" according to the invention is understood

31854 / H nucleic acids which exhibit homology, in particular sequence identity of about 60%, preferably about 75%, especially about 90%, and especially about 95%.

The nucleic acid portions of the invention can be used, for example, to prepare individual epitopes, as probes to identify additional functional variants, or as antisense nucleic acids. For example, a nucleic acid of at least about 8 nucleotides is suitable as an antisense nucleic acid, a nucleic acid of at least about 15 nucleotides is useful as a primer in a PCR procedure, a nucleic acid of at least about 20 nucleotides is suitable for identification other variants, and a nucleic acid consisting of at least about 100 nucleotides is suitable as a probe.

In another preferred embodiment of the invention, the nucleic acid according to the invention comprises one or more non-coding sequences and / or a poly (A) sequence, one or more (necessary for intracellular protein processing) Kex2β-endopeptidase recognition sequences (Endopeptidase Erkennungssequenzen) and one or more potential N-glycosylation sites. Non-coding sequences are control sequences, such as promoter or enhancer sequences, for the controlled expression of a coding toxin comprising nucleic acids of the invention.

Thus, in another embodiment, the nucleic acid is contained in a vector, preferably an expression vector or a gene-therapeutically effective vector.

For example, the expression vectors for the nucleic acids of SEQ ID No 2 may be prokaryotic and / or eukaryotic expression vectors, respectively. in the case of the nucleic acids of SEQ ID No 1, exclusively eukaryotic expression vectors. Expression of the toxin-encoding nucleic acids of SEQ ID No 1 in Escherichia coli is not possible because the heterologously expressed protein toxin is toxic to bacterial cells. The cloning of the WICALTIN-encoding nucleic acids of SEQ ID No 1 in E. coli is only possible with plasmids that do not carry any promoter (e.g., using derivatives of plasmid pBR322). An example of a prokaryotic vector that

31854 / H allows heterologous expression of the ZYGOCIN-encoding nucleic acid of SEQ ID No 2, is a commercially available vector pGEX-4T-1 that allows the expression of glutathione-S-transferase-ZYGOCIN-fusion protein in E. coli. Another vector for the expression of ZYGOCIN in E. coli is, for example, the T7 expression vector pGM10 (Martin, 1996), which encodes an N-terminal Met-AlaHis6-tag (tag), allowing for convenient purification of the expressed protein on the Ni ²⁺ -NTA column. Suitable eukaryotic expression vectors for expression in Saccharomyces cerevisiae are, for example, vectors p426Met25 or p426GAL1 (Mumberg et al., 1994, Nucl. Acids Res., 22, 5767), for example, Baculovirus vectors such as EP- B10127839 or EP-B1-0549721 and for expression in mammalian cells, for example, SV40-vectors that are commercially available can be used.

Expression vectors contain general regulatory sequences suitable for the host cell, such as e.g. the trp promoter for expression in E. coli (see e.g. EP-B1-0154133), the ADH-2 promoter for expression in yeast (Russel et al., 1983, J. Biol. Chem., 2588, 2674), baculovirus a polyhedrin promoter for expression in insect cells (see for example EP-B1-0127839) or the older SV40 promoter or LTR promoters e.g. from MMTV (Mouse Mammary Tumor Virus, Lee et al., 1981, Nature, 214, 228).

Examples of gene-therapeutically effective vectors are virvectors, particularly adenovirus convectors, in particular replication-deficient adenovirus convectors or adeno-associated virus convectors, e.g. adeno-associated virusvector consisting exclusively of two inserted terminal repeat sequences (Wiederholungssequenz) (ITR).

Suitable adenovirus convectors are described e.g. in McGroy, W. J. et al., 1988, Virol., 163, 614; Gluzman, Y. et al., 1982, "Eukaryotic Viral Vectors" (Gluzman, Y. ed.) 187, Cold Spring Harbor Press, Cold Spring Harbor, New York; Chroboczek, J. et al., 1992, Virol., 186, 280; Karlsson, S. et al., 1986, EMBO J., 5, 2377 or in WO 95/00655.

Suitable adeno-associated virusvectors are described e.g. in Muzyczka, N., 1992, Curr. Top. Microbiol. Immunol., 158, 97; WO 95/23867;

31854 / K

Samulski, R. J., 1989, J. Virol., 63, 3822; WO 95/23867; Chiorini, J. A. et al.,

1995, Human Gene Therapy, 6, 1531 or Kotin, R. M., 1994, Human Gene

Therapy, 5, 793.

Gene-therapeutically effective vectors can also be obtained by binding a nucleic acid of the invention complexed with liposomes. Lipid mixtures which are described in e.g. in Felgner, P. L. et al., 1987, Proc. Natl. Acad. Sci. USA 84, 7413; Behr, J. P. et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 6982; Felgner, J. H. et al., 1994, J. Biol. Chem., 269, 2550 or Gao, X. and Huang, L., 1991, Biochim. Biophys. Acta, 1189, 195. In the preparation of these liposomes, the DNA is bound ionically to the surface of the liposomes, in a bird ratio to maintain a positive charge on the surface and to fully bind the DNA to the liposomes in complex.

In another embodiment, the nucleic acids of the invention are contained in a vector, preferably an expression vector for the preparation of transgenic plants. Since the disclosed killer-toxins WICALTIN and ZYGOCIN have a broad spectrum of activity and also kill phytopathogenic yeasts and fungi, transgenic plants that are resistant to e.g. against infection caused by the pathogen Ustilago maydis in maize. Similar experiments have already been carried out on tobacco plants which, after heterologous expression of wild-type virally encoded killertoxin KP4 from U. maydis, have the ability to secrete the respective protein toxin and thus obtain specific protection against infections with certain phytopathogenic U. maydis strains (Park et al., 1996; Kinal et al. et al., 1995; Bevan, 1984). On the basis of commercially available transformation systems based on modified derivatives of natural Ti-plasmids from Agrobacterium tumefaciens, the nucleic acids of the present invention, also present in the WCT and ZBT toxins, can be cloned into the so-called. bi-directional pBIectors (CLONTECH) and used to obtain transgenic plants. The respective WCT and ZBT toxins are inserted under the transcriptional control of the strong cauliflower mosaic virus (CaMV-P) promoter. Further details on the construction of this engineered vector are shown schematically in Example 9.

The nucleic acids of the invention may e.g. synthesize

31854 / H chemically using the sequences of SEQ ID No 1 and No 2 or using the peptide sequences of SEQ ID No 1 and No 2 using the genetic code e.g. by the phosphotriester method (see, e.g., Uhlman, E. and Peyman, A., 1990, Chemical Reviews, 90, 543, No. 4). Another possibility of obtaining nucleic acids of the invention is isolation from a suitable gene bank using a suitable probe (see, e.g., Sambrook, J. et al., 1989, Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York) ). Suitable probes are e.g. single-stranded DNA fragments of about 100 to 1000 nucleotides in length, especially about 200 to 500 nucleotides in length, particularly about 300 to 400 nucleotides in length, the sequence of which may be derived from the nucleic acid sequence of SEQ ID No 1 and No 2.

A further object of the invention are polypeptides per se having the amino acid sequence according to SEQ ID No 1 and No 2 or functional variants thereof and portions thereof with at least six amino acids, in particular at least 12 amino acids, in particular at least 65 amino acids and in particular 309 amino acids. No. 1) and having 99 amino acids (SEQ ID No 2) (hereinafter referred to as "polypeptides of the invention"). So eg. For example, a polypeptide of about 6-12, in particular about 8 amino acids, may contain an epitope that serves, upon binding to a carrier, to produce specific poly- or monoclonal antibodies (see, e.g., US 5,656,435). Polypeptides of at least about 65 amino acids in length can be used directly or without a carrier to prepare poly- or monoclonal antibodies.

The term "functional variant" within the meaning of the invention is understood to mean polypeptides that are related to the peptide of the invention, i.e., exhibit glucanase activity. Variants are also to be understood as allelic variants or polypeptides which may also originate from different yeast / yeast strains or other agents of infection, such as dermatophytes or fungi (according to the DHS system).

In another aspect, polypeptides that exhibit sequence homology, particularly about 70% identity, in particular about 80%, particularly about 90%, and especially about 95%, of the polypeptide having the amino acid sequence of FIG. 2. In addition, cleavage is also included

31854 / H polypeptide in the range of about 1 - 60, especially about 1 - 30, especially about 1 - 15, and especially about 1 - 5 amino acids. For example, the first amino acid methionine may be absent without substantially altering the function of the polypeptide. In addition, fusion proteins comprising the aforementioned polypeptides of the present invention are included, wherein the fusion proteins themselves already have a glucanase function or can only obtain this specific function after cleavage of the fusion moiety. In particular, these include fusion proteins having a proportion of mainly non-human sequences comprising about 1 - 200, especially about 1-150, especially about 1 - 100 and especially about 1 - 50 amino acids. Examples of non-human peptide sequences include prokaryotic peptide sequences, e.g. from galactosidase from E. coli or so-called. a histidine chain (Histidine-Tag), e.g. Met-Ala-His _6- chain (Tag). Fusion protein with so-called. the histidine chain is particularly suitable for purifying the expressed protein on a metal ion column, e.g. using a Ni ²⁺ -NTA column. "NTA" refers to a chelator which is nitrile acetic acid (Qiagen GmbH, Hilden). The invention also encompasses polypeptides of the invention that are hidden as a pro-protein or, in the broadest sense, as a Pre-drug.

Portions of the polypeptides of the invention are e.g. epitopes that can be specifically recognized by antibodies.

Polypeptides of the invention may be prepared e.g. expressing the nucleic acids of the invention in a suitable expression system using the methods described above, which are known to those skilled in the art. Only eukaryotic organisms, particularly germinating yeast (Sprosshefe) of Saccharomyces cerevisiae and splitting yeast (Spalthefe) of Schizosaccharomyces pombe, are suitable as host cells for the preparation of properly processed and thus biologically active protein toxins.

Said portions of the polypeptide can also be synthesized by classical peptide synthesis (Merrifield technique). These portions are particularly suitable for obtaining antisera by means of which suitable gene expression banks can be examined in order to obtain other functional variants of the polypeptide of the invention.

31854 / H

Accordingly, a further aspect of the invention relates to a process for preparing a polypeptide of the invention, wherein the nucleic acid of the invention is expressed and optionally isolated in a suitable host cell.

Particularly preferred are yeast dividing Schizosaccharomyces pombe because these yeasts exhibit natural resistance to WICALTIN and ZYGOCIN and have been successfully used several times for heterologous expression of foreign proteins (Giga-Hama and Kimagai, 1997, in the publication Foreign Gene Expression in Fission foreign genes in dividing yeasts: Schizosaccharomyces pombe, e.g. Springer). As shown in Example 11, the toxin-encoding nucleic acids of SEQ ID No 1 and No 2 can be cloned e.g. to S. a vector pREP1 (Maundrell, 1990, J. Biol. Chem., 265, 10857-10864) in which they are under transcriptional control of the thiamin-regulated nmt1 promoter of the dividing yeast (nmt = no message with thiamine). The yeast transformed with this vector expresses the respective foreign gene depending on the concentration of thiamine in the yeast culture medium. In this way, the yeast growth phase can optionally be separated in time from the foreign protein production phase, so that in principle the expression of proteins which are toxic to the yeast can also be carried out. In order to perform concomitant secretion, thereby substantially facilitating the purification of heterologously expressed toxins WICALTIN and ZYGOCIN in S. pom, we constructed an expression / secretion vector (ρΤΖατ vector; see Example 11) containing the secretion and process signal of viral K28-pre- protoxingen (Schmitt and Tipper, 1995), thereby allowing efficient secretion of the respective inframe of the included foreign protein.

Another aspect of the invention also relates to antibodies that specifically react with a polypeptide of the invention, wherein the above portions of the polypeptide are either immunogenic alone or their immunogenicity can be induced or increased by binding to a suitable carrier, such as e.g. bovine serum albumin.

These antibodies are either polyclonal or monoclonal. Their preparation, which is likewise an object of the present invention, is carried out e.g. by

31854 / H by well-known methods by immunizing a mammal, such as a rabbit, with a polypeptide or a portion thereof as described herein, optionally in the presence of e.g. Freund's adjuvant and / or aluminum hydroxide gels (see, e.g., Diamond, B.A. et al., 1981, The New England Journal of Medicine, 1344). Finally, polyclonal antibodies generated in an animal by an immunological reaction can be readily isolated from the blood by known methods and purified, for example, by column chromatography. Affinity purification of antibodies is preferred, wherein, for example, an appropriate antigen (WICALTIN or ZYGOCIN) is covalently bound to a generally available CnBr-activated Sepharose matrix and used to purify the respective toxin-specific antibodies.

Monoclonal antibodies can be prepared, for example, according to the known method of Winter and Miistein (Winter, G. and Milstein, C., 1991, Nature, 349, 293).

A further object of the invention is also a medicament comprising the nucleic acids of the invention or polypeptides of the invention (alone or in combination) and optionally suitable additives or excipients and a process for the preparation of the medicament for the treatment of mycoses such as superficial, cutaneous and subcutaneous dermatomycoses, mucosal mycoses and systemic mycoses, in particular Cand / c-α-mycoses, wherein the nucleic acid of the invention or the polypeptide of the invention are combined with pharmaceutically acceptable excipients and / or excipients.

Example 12 shows that purified WICALTIN toxin produced by DSM 12865 strain has even significantly higher yeast toxicity than the compared antifungal agents Clotrimazole and Miconazole, which are often used to treat mycoses.

Therefore, the present invention also relates to a medicament in the above sense comprising an antifungal or protein toxin isolated from DSM 12864 and / or DSM 12865 and / or polypeptides having antifungal activity according to the invention.

31854 / H

In particular, for gene therapy in humans, a drug comprising a nucleic acid of the present invention in a basic form (nackte Form) or in the form of one of the aforementioned gene therapeutically effective vectors or in the form of a complex with liposomes is suitable.

Suitable additives and / or excipients can be used e.g. saline, stabilizers, proteinase inhibitors, nuclease inhibitors, etc.

A further object of the invention is a diagnostic composition comprising a nucleic acid of the invention, a polypeptide of the invention or an antibody of the invention, and optionally suitable additives and / or excipients, and a process for preparing the diagnostic composition for diagnosis of mycoses such as superficial, cutaneous and subcutaneous dermatomycoses. , mucosal mycoses and systemic mycoses, in particular Cand / da-mycoses, in which the nucleic acid of the invention, the polypeptide of the invention or the antibodies of the invention are combined with suitable additives and / or excipients.

Thus, for example, according to the invention, using the nucleic acids of the invention, a polymerase chain reaction (PCR diagnostics, for example according to EP-0 200 362) or Northern and / or Southern Blot analysis can be obtained as described in more detail in the example. 13. These assays are based on the specific hybridization of a nucleic acid of the invention with a complementary counter-strand (Gegenstrang) as a rule corresponding to mRNA. The nucleic acids according to the invention can also be modified in the manner described, for example, in EP 0 063 879. In particular, the DNA fragment according to the invention is labeled according to known methods with suitable reagents such as radioactive aP ³² -dATP or non-radioactive biotin and incubated with isolated RNA, typically bound to a suitable membrane of, for example, cellulose or nylon. In this case, it is advantageous to separate the isolated RNA, depending on its size, for example by electrophoresis on an agarose gel, before hybridization and membrane binding. Thus, with the same amount of RNA of interest from each tissue sample, it is possible to determine the amount of mRNA

31854 / H specifically labeled with a probe.

Another diagnostic comprises a polypeptide of the invention, respectively. the above immunogenic portions thereof. This polypeptide, respectively. for example, parts thereof bound primarily to the solid phase, for example nitrocellulose or nylon, can be contacted in vitro with the body fluid to be examined, for example blood, for example to react with the antibody. This antibody-peptide complex can then be detected using, for example, a labeled anti-human-IgG antibody or an anti-human-IgM antibody. This label is, for example, an enzyme such as peroxidase, which catalyzes the color reaction. Thus, the presence and amount of autoimmune antibodies present can be easily and rapidly detected by a color reaction.

Another diagnostic comprises the antibodies of the invention as such. With these antibodies, for example, human tissue samples can be easily and rapidly examined for the presence of the polypeptide of interest. In this case, the antibodies of the invention are labeled e.g. enzyme as described above. Thus, the specific antibody-peptide complex can be easily and rapidly detected by an enzymatic color reaction.

A further object of the invention relates to a fungicide comprising the nucleic acids of the invention and / or the polypeptides of the invention (individually or in combination) and optionally suitable additives or excipients and to a process for preparing a fungicide for controlling harmful yeasts and harmful fungi. which combines a nucleic acid of the invention or a polypeptide of the invention with agriculturally acceptable additives and / or excipients.

As described above, in one of the above embodiments, a transgenic plant that expresses a protein toxin of the invention is obtained. Therefore, the present invention also relates to plant cells as well as to a transgenic plant as such comprising the polypeptides and / or protein toxins of the present invention.

Another object of the invention relates to a functional identification test

31854 / H interactors, such as e.g. inhibitors or stimulators comprising a nucleic acid of the invention, a polypeptide of the invention or an antibody of the invention, and optionally suitable excipients and / or excipients.

A suitable assay for identifying functional interactions, particularly those that react with the protein toxin ZYGOCIN of SEQ ID No 2 in a sensitive yeast cell, e.g. called. The "Two-Hybrid System" (Fields, S. and Sternglanz, R., 1994, Trends in Genetics, 10, 286). In this assay, a cell, e.g. the yeast cell transforms or transfers with one or more expression vectors expressing a fusion protein comprising a polypeptide of the invention and a DNA-binding domain of a known protein, e.g. from Gal4 or LexA from E. coli and / or with vectors that express a fusion protein that contains an unknown polypeptide and one transcriptional activation domain, e.g. from Gal4, herpesvirus VP16 or B42. In addition, the cell contains a reporter gene, e.g. The LacZ gene from E. coli " Green Fluorescence Protein " or the yeast amino acid biosynthesis genes (AS-Biosynthesegene der Hefe) His3 or Leu2, which via regulatory sequences such as e.g. lexApromotor / operator or so-called. The upstream Activation Sequence (UAS) controls the yeast. An unknown polypeptide is e.g. encodes a DNA fragment derived from a gene bank, e.g. from a human gene bank. In yeast, a cDNA gene bank is typically prepared using the expression vectors described, so that the assay can be performed immediately thereafter.

For example, in a yeast expression vector, the nucleic acids of the invention are cloned in a functional unit into a nucleic acid encoding a LexA-DNA-binding domain such that the fusion protein from the polypeptide of the invention and the LexA-DNA-binding domain are expressed in transformed yeast. In another yeast expression vector, cDNA fragments from a cDNA gene bank in a functional unit are cloned into a nucleic acid encoding a Gal4-transcriptional-activating domain, so that the fusion protein from an unknown polypeptide and the Gal4-transcriptional-activating domain is expressed in transformed yeast. Yeast transformed with both of these vectors, e.g. Leu2 ', contains in addition to

31854 / H of that Leu2 encoding nucleic acid, which is under the control of the LexA promoter operator. In the case of a functional interaction between a polypeptide of the invention and an unknown polypeptide, the Gal4-transcriptional-activation domain binds via the LexA-DNA-binding domain to a LexA-promoter operator that activates and expresses the Leu2 gene. As a result, the Leu2 'yeast can grow on minimal medium containing no leucine.

When using LacZ- resp. The Green Fluorescence Protein of the " receptor-gene " instead of the amino acid biosynthesis gene, activation of transcription can be demonstrated by the formation of blue and blue cells respectively. green fluorescing colonies. Blue, respectively. green fluorescence can be easily quantified spectrophotometrically, e.g. in the case of blue at 585 nm.

In this way, the presence of polypeptides that react with the polypeptide of the invention can be readily and rapidly detected in expression gene banks. The novel polypeptide can then be isolated and further characterized.

Another possibility of using the "Two-Hybrid System" is to influence the interaction between a polypeptide of the invention and a known or unknown polypeptide by the action of other substances, such as e.g. chemical compounds. This method also makes it possible to easily find a new chemically synthesized active substance which can be used as a therapeutic. Therefore, the present invention is not only directed to a procedure for screening for polypeptide interactions, but also to a procedure for screening for substances that may react with the above protein-protein complex. For the purposes of the present invention, these peptide and chemical interactions are referred to as functional interactions that may have inhibitory or stimulatory effects.

Another object of the present invention relates to a process for the preparation of protein toxins comprising culturing and secreting protein toxins in a medium that is a synthetic culture medium (BAVC-medium), chromatographic purification of a secreted toxin, e.g. by ultrafiltration and cation exchange chromatography and / or affinity chromatography on Laminarin-Sepharose and / or on:

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Mannoprotein-Sepharose (see Example 1 and Example Supplement). For WICALTIN produced and secreted by DSM 12865 strain, further increase in toxin production can be achieved by adding plant (and generally available) β-1,3-D-glucan laminarin to a final concentration of 1%. As shown in Example 14, the addition of laminarin to the culture medium induces the formation of WICALTIN, which is induced by induction of transcription according to Northern analysis.

Synthetic B-medium can be used to generate ZYGOCIN toxin secreted by DSM 12864 (see Radler et al., 1993)

The following examples serve to illustrate the invention and do not limit the invention to these examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Isolation, concentration and purification of WICALTIN anti-Can / D-toxin from supernatant of W. californica killer-yeast culture solution strain 3/57 (DSM 12865)

Killer-toxin WICALTIN secreted by killer-yeast l / V. californica 3/57 showed an optimal inhibitory effect on sensitive yeasts at pH 4.7 and 20 ° C in an methylene blue agar diffusion test (Methylenblau-Agar). In a synthetic liquid medium, killer-yeast 1 / V californica 3/57 exhibits maximum toxin production when cultured in BAVC-medium (pH 4.7). To increase toxin formation, the killer yeast was first incubated with shaking for 24 hours in 5 ml YEPD medium at 30 ° C, then quantitatively transferred to 200 ml BAVC medium and cultured again for 48 hours at 20 ° C to shaker (140 rpm). The four main cultures (Hauptkultur) were inoculated with a pre-cultured culture (Vorkultur) (1% inoculum) in 2.5 L of BAVC medium (pH 4.7 in a 5 L Erlenmeyer flask) and incubated for 5 days with gentle shaking (60). rpm.). To concentrate the secreted killer-toxin, a cell-free supernatant is added

31854 / H at a temperature of +4 ° C and a pressure of 1 bar, 200-fold concentrated by ultrafiltration on polysulfonic acid membranes ("EasyFIow" (Sartorius); exclusion limit (Ausschlussgrenze / 10 kDA) to 50 ml) to remove low molecular weight compounds. and to remove salts from the concentrate thus obtained, the toxin was dialyzed overnight at 4 ° C in a dialysis tube (exclusion threshold of 10-20 kDA) against 5 mM citrate-phosphate buffer (pH 4.7). filtered through a 0.2 µm membrane and frozen at -20 ° C in 1 ml portions.

Detection and calibration of toxin activity was performed in a methylene blue (MBA, pH 4.7) agar diffusion assay against the sensitive indicator yeast Saccharomyces cerevisiae 192.2d. Logarithmic dilution steps were prepared from the toxin concentrate in 0.1 M citrate phosphate buffer (pH 4.7) and pipetted 100 μΙ into pre-prepared holes (9 mm hole diameter) in an MBA plate inoculated with sensitive indicator yeast (2). x 10 ⁵ cells / ml). After three days incubation of the plates at 20 ° C, well visible inhibition wells (Hemmhof) were measured. It has been shown that there is a linear relationship between the mean inhibition court and the logarithm of the toxin concentration. The diameter of the inhibiting court (corrected for hole diameter) of 20 mm was assigned a toxin activity of 1 x 10 ⁴ units / ml.

Purification of the concentrated WICALTIN toxin was carried out either by cation exchange chromatography on Bioscale-S (FPLC) or by affinity chromatography on epoxy-activated Sepharose-68-Matrix (Pharm. Pharmacia) to which the plant β-Ι, β-D-glucan pustulanin was pre-bound. . This toxin preparation, which showed a specific activity corresponding to a 625-fold enrichment in this way (Table 1), appeared to be pure in gel electrophoresis and showed separate discrete bands after SDS-PAGE (in a 10-22% gradient gel). at about 37 kDA, which was detectable by both blue Coomassie-Blau (protein staining) and Schiff's periodic acid reagent (Perjodsäure-Schifffs-Reagent-PAS; carbohydrate staining). Positive PAS staining indicates the potential Nglycosylation of the anti-Can / Da-toxin WICALTIN. Reaction of purified toxin

31854 / H with endoglycase-H, WICALTIN has been shown to contain an approximately 3 kDA Nglycosidically linked carbohydrate moiety, which, due to its size in yeast, also points to a single N-glycosylation site in proteintoxin. Since deglycosylated WICALTIN exhibits substantially reduced toxicity, it can be concluded that the carbohydrate portion of WICALTIN appears to be necessary for binding to a sensitive target cell, i.e., it indirectly affects the biological activity of the toxin.

Table 1

Concentration of WICALTIN toxin from supernatant of Williopsis californica killer yeast culture medium (UF, ultrafiltration)

preparation volume (Ml) Tot. protein (Mg) total activity toxin (E) specific activity toxin (E / mg) active yield (%) factor cleaning (Reinigungsf.) The supernatant cultivate. the media 10 000 24 600 7.9 x 10 * 3,2x10 100 1 UF-retentate 50 162 6,3x10 3,9x10 80 122 Lyofil. dialysate 25 45.8 3.1 x 10 6.8 x 10 39 213 Bioscale S (Cation exchanger) 64 1.28 2.5 x 10 ⁴ 2.0 x 10 ⁴ 3.2 625

Example 2

Determination of NH ₂ -terminal amino acid sequence of WICALTIN and evidence of enzymatic β-1,3-gluconase activity

The first ten amino acids were determined by sequencing the N-terminal amino acids in the purified killer toxin. As can be seen from FIG. 1, the end of WICALTIN shows significant homology to the amino terminus of the endo-β1,3-glucanase encoded by the yeast Saccharomyces cerevisiae BGL2 gene.

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Based on this homology of WICALTIN and Bgl2, it was determined whether glucanase activity was detectable in the unpurified toxin concentrate as well as in the purified toxin preparation. Both in the enzymatic assay using β-1,3-D-glucan laminarin as substrate, and in the fluorescent assay using 4-methyl-umbelliferyl-β-D-glucoside (MUC) substrate, WICALTIN preparations were possible. demonstrate significant β-β 1,3-D-glucanase activity; concurrently tested?> 1,6-Duclucan pustulan was not hydrolyzed by WICALTIN.

Example 3

Survival rate of yeast cells after WICALTIN treatment in the presence and absence of cell wall glucans: competition analyzes

Yeast sensitive cells of strain S. cerevisiae 192.2d, which were cultured in liquid YEPD medium (pH 4.7) at 20 ° C in the presence of purified WICALTIN with an activity of 1 x 10 ⁵ E / ml, showed the kinetics of killing shown in FIG. 2. By adding plant β-Ι, β-D-glucan to pustulan, the survival rate of yeast cells after toxin treatment could be significantly increased. This addition at concentrations of 10 mg / ml caused complete suppression of the toxicity of WICALTIN. In contrast to pustulan, β-1,3-D-glucan laminarin did not show the ability to increase the survival rate of toxin-modified yeast cells (Fig. 2).

From the above, it can be concluded that the action of WICALTIN requires binding to β-1,6-D-glucans, which act as the primary binding site (Andockstelle) (toxin receptors) on the yeast cell wall. Accordingly, yeasts cleaved at the chromosomal KRE1-gene center (Genlocus) have been shown to have toxin-resistant behavior and, once retransformed with the KRE1-bearing episomal vector, are again toxinsensitive (Fig. 3). In cre1-mutants, the toxin resistance rests on a substantially reduced β-1,6-D-glucan content and thereby conditionally reduces the binding of the toxin to the yeast cell surface required for the lethal effect.

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Example 4

Spectrum of efficacy and killing by WICALTIN

In the agar diffusion test, purified WICALTIN zl / K califomica toxin showed significant yeast toxicity listed in Table 2. Except for three Candida crusei yeast strains, all 22 clinical patient isolates studied as well as all other control strains of human pathogenic Candida species has shown very effective destruction by WICALTIN. In 14 toxin-sensitive yeast species from a total of 10 different genera, WICALTIN has shown an unusually broad spectrum of efficacy as a killer toxin.

Table 2

Spectrum of WICALTIN activity on pathogenic and apatogenic yeasts of different genera. All strains were tested in an agar diffusion assay (MBA; pH 4.7) against purified WICALTIN. The activity of the applied toxin was in all cases 1 x 10 ⁶ E / ml. The strain of C. tropicalis (patient # 541965) was from the Institute of Fur Medizinisché Microbiology and Hygiene from the University Clinic of Mainz).

Yeast strain phenotype Inhibitory diameter court (mm) Candida albicans ATCC 10231 WITH 11 C. glabrata NCYC 388 WITH 12 C. krusei R 0 C. tropicalis patient no. 541965 WITH 11 Debaromyces hansenii 233 WITH 16 Hanseniaspora uvarum ATCC 64295 R 0 Hasegawaea japonica var. Versatilis 191 R 0 Kluyveromyces lactis CBS 2359/152 WITH 22

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Yeast strain phenotype Inhibitory diameter court (mm) K. marxianus C 8.1 R 0 Metchnikowia pulcherrima K / 31 B6 WITH 8 Pichia anomala WITH 17 P. farinosa R 0 P. jardinii 258? R 0 P. klyveri ATCC 64301 R 0 P. membranaefaciens NCYC 333 R 0 Saccharomyces cerevisiae 192.2 WITH 30 381 WITH 23 ATCC 42017 (K1 Superkiller) WITH 19 NCYC 738 (K2-Killer) WITH 14 452 (NCYC 1006) WITH 16 Saccharomyces ludwigii 240 R 0 Schizosaccharomyces pombe CBS 1042 R 0 Sporothrix spec. 1129 WITH 11 Toruiospora delbrueckii 208 WITH 18 T. pretoriensis WITH 1θ Yarrowia lipolytica WITH 8 Zygosaccharomyces bailii WITH 23

Example 5

Cloning, sequencing and molecular characterization of the WICALTIN coding WCT gene of W. califomica strain 3/57 (DSM 12865)

Based on the N-terminal amino acid sequence of WICALTIN, specific DNA-oligonucleotides were prepared that led to identification and cloning as well as molecular-biological characterization of the chromosomally localized toxin of WCT. DNA sequence WCT (SEG ID

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No 1) shows a single open reading frame that encodes a potentially N-glycosylated protein of 309 amino acids and with a calculated molecular weight of 34,017 Da.

Monitoring the efficacy of WCT-encoded killer-toxin has shown that WICALTIN is a glycoprotein that is extremely toxic to yeast, whose primary "target"_; are β-1,3-D-glucans that occur in the yeast cell wall. Its selective toxicity to yeasts and fungi lies in the fact that WICALTIN disrupts the structure and / or integrity of the cell wall in a sensitive target cell, thus attacking the yeast at their most sensitive site and thereby destroying it.

Example 6

Treatment and purification of ZYGOCIN virus-toxin obtained from the supernatant after cultivation of Z. ba /// killer-yeast strain 412 (DSM 12864)

From Z. bailii yeast strain 412, from the supernatant of the killer-yeast culture medium according to the method of Radler et al. (1993) isolated the virus-encoded killer-toxin ZYGOCIN, which was further concentrated by ultrafiltration and then purified by affinity chromatography. The single-step purification of ZYGOCIN described in the cited study utilizes the natural affinity of the toxin to sensitive yeast cell wall proteins. According to the method described by Schmitt and Radler (1997), isolated and partially purified mannoprotein from S. cerevisiae strain 192.2d was covalently coupled to an epoxyactivated Sepharose-6B-matrix (Pharmacia) and used for FPLC purification of the toxin by column chromatography. According to SDS-PAGE, the purified biologically highly active ZYGOCIN showed separate protein bands with an apparent molecular weight of about 10 kDa (FIG. 4).

Example 7

Spectrum of efficacy and killing by ZYGOCIN

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The spectrum of viral ZYGOCIN from yeast Z. bailii 412 (DSM 12864), determined in the agar diffusion test, includes the pathogenic and apatogenic yeast genera of which Candida albicans and Sporothrix schenkii are believed to be causative agents in humans and animals and Ustilago Debaromyces hansenii for harmful yeasts in agriculture and food (Table 3).

Table 3

Spectrum of ZYGOCIN activity against pathogenic and apatogenic yeasts of different genera. All strains were tested in an agar diffusion assay (MBA, pH 4.5) for ZYGOCIN preparation with an activity of 1 x 10 ⁴ E / ml.

ZYGOCIN-sensitive yeast Relative degree of sensitivity Saccharomyces cerevisiae ++ Candida albicans + Candida krusei ++ Candida glabrata ++ Candida vinii + Hanseniaspora uvarum ++ Kluyveromyces marxianus + Metchnikowia pulcherrima + Ustilago maydis ++ Debaromyces hansenii ++ Pichia anomala ++ Pichia jadinii + Pichia membranefaciens + Yarrowia lipolytica + Zygosaccharomyces rouxii ++

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Example 8

Cloning and sequencing of the ZYGOCIN-encoding ZBT-gene (ZBT) of yeast Z. bailii strain 412 (DSM 12864) cDNA synthesis of the toxin-coding double-stranded RNA genome of the Z: bailii 412 yeast was performed according to the method of qroxymortortuted with purified and methyl dsRNA as a matrix and with various hexanucleotides as "primers". After ligation into the EcoRI-restricted pUC18 vector, transformation into E. colia by isolation of the recombinant plasmid, several cDNA clones could be identified and sequenced. The cDNA sequence of the ZYGOCIN encoded reading frame (SEQ ID No 2) contains genetic information for the protein (Vorlauferprotein) (Pro-Toxin) of 238 amino acids. which has a potential Kex2 endopeptidase cleavage site at amino acid position RR ¹³⁹ . The in vivo conversion of ProZYGOCIN in late Golgi-mediated Kex2 mediated biologically active ZYGOCIN whose molecular weight (10 kDA; 99 amino acids) and the N-terminal amino acid sequence correspond exactly to the values corresponding to the purified ZYGOCIN.

The heterologous expression of ZBT-cDNA in S. cerevisiae yeast, due to the toxicity of ZYGOCIN, resulted in the transformed yeast being killed by its own toxin. In the future, heterologous expression of ZYGOCIN will be directed to the toxin resistant dividing yeast of Schizosaccharomyces pombe because, as already mentioned in the viral K28-toxin example, these dividing yeasts are particularly suitable for the expression and secretion of foreign proteins.

Example 9

Toxin toxin expression of WCT and ZBT in transgenic plants

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Since the described killer toxins WICALTIN and ZYGOCIN have a broad spectrum of activity and also kill phytopathogenic yeasts and fungi, it should be possible to prepare such transgenic plants that are resistant to e.g. against maize infection caused by the pathogen Ustilago maydis. Similar experiments have already been performed with tobacco plants which, when heterologously expressing the naturally virus-encoded killer toxin KP4 from U. maydis, had the ability to secrete the killer toxin in question and thereby obtain specific protection against infections caused by certain phytopathogenic U. maydis strains (Park et al. , 1996; Kinal et al., 1995; Bevan, 1984). Based on commercially available transformation systems based on modified derivatives of natural Ti-plasmid from Agrobacterium tumefaciens, it was possible to clone the cloned WCT and ZBT toxin genes into the so-called. bi-directional pBI vectors (Clontech) and used to obtain transgenic plants. The respective WCT and ZBT toxins were under the transcriptional control of the potent cauliflower mosaic virus (CaMVP) promoter. The construction of the constructed vectors is shown schematically in FIG. 5th

Example 10

Heterological expression of the WICALTIN-encoding WCT yeast gene IV. califomica 3/57 (DSM 12865) in S. cerevisiae

Due to heterologous expression of the WCT gene in S. cerevisiae yeast, the WICALTIN-encoding WCT gene as a 930 bp EcoR / SmaI fragment was cloned into the generally available 2 μ vector of pYX242. The resulting pSTH2 vector (Fig. 6) contains the toxin under the transcriptional control of its own yeast triose phosphate isomerase promoter (TPI), thus allowing constitutive expression of WICALTIN after yeast transformation (S. cerevisiae). Gel electrophoresis of the culture medium supernatant in the yeast transformants thus obtained showed that recombinant WICALTIN secreted into the culture medium and showed β-1,3-D-glucanase activity (Fig. 6) corresponding to homologous WICALTIN (from "wild" strain DSM 12865) .

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Example

Experiments with heterologous expression of WICALTIN and ZYGOCIN in dividing yeast Schizosaccharomyces pombe

Since the dividing yeast, both in the form of an intact cell and as a cell wall-free sphere, are resistant to WICALTIN and ZYGOCIN, they are suitable ^; as host cells for heterologous expression of these toxins. To ensure that these recombinant toxins are not only expressed in dividing yeast, but also enter the intracellular secretory pathway and are secreted into the external culture medium, a vector (ρΤΖ / ατ, Fig. 7) was constructed which is carrier of a functional signal for S. pombe secretion and processing (S / P) and which is derived from the yeast S. cerevisiae viral K28 preprotoxin cDNA (see Schmitt, 1995; Schmitt and Tipper, 1995). This secretory and process signal ensures that the in-frame foreign protein in the dividing yeast is always imported into the lumen of the endoplasmic reticulum, and thus enters the yeast secretory pathway. Through the Kex2β-cleavage site at the C-terminus of the S / P region, the desired foreign protein in the late Golgi space in cells (in back Golgi-Compartiment) is cleaved by its own yeast Kex2β-endopeptidase from its intracellular "Transport-Vehicle". and can ultimately be secreted as a biologically active protein (ZYGOCIN and / or WICALTIN) into an external culture medium.

Example 12

Comparison of biological activity of purified WICALTIN and topical antifungal agents Clotrimazole and Miconazole

Since purified WICALTIN exhibits a broad spectrum of activity and effectively kills even human pathogenic yeasts and / or fungi, it is also of great importance as a potential antifungal agent. Therefore, studies have been conducted comparing WICALTIN with the very commonly used topical antifungal agents Clotrimazole and Miconazole. The toxic action of Clotrimazole a was first tested

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Miconazole in an MBA-agar diffusion assay in which Sporothrix spec. Clotrimazole was dissolved to a concentration of 10 mg / ml in ethanol (96%); this stock solution was diluted with redistilled water and used at 100 μΙ in each case in the MBA test at concentrations ranging from 0.1 to 10 mg / ml. The diameter of the Inhibitory Court (Hemmhof) was between 12 and 32 mm, using an amount of 10 - 50 µ9 Clotrimazole. From Miconazole, a stock solution of 100 μρ / ηιΙ in DMSO (100%) was prepared which; in the MBA assay was followed in the same manner as Clotrimazole for biological activity against Sporothrix spec. The use of 0.08-0.3 μ9 of Miconazole resulted in inhibition wells with a diameter of between 22 and 36 mm at Bioassay. Biological activity of 10 μς Clotrimazole, resp. 0.08 g of Miconazole corresponded to the toxicity of 2 g of purified WICALTIN. A comparison of the three test compounds, based on their molecular weights, shows that WICALTIN already exhibits the same activity as 0.2 pmol of Miconazole and 29 pmol of Clotrimazole at a concentration of 0.07 pmol; this means that WICALTIN is highly effective as an antifungal agent (Fig. 8).

Example 13

Detection of the WICALTIN-coding WCT gene of W. califomica 3/57 (DSM 12865) by Southern hybridization with a gene-specific DNA probe

As evidence that the nucleic acid of SEQ ID No 1 can be used to prepare a WICALTIN-specific DNA probe for subsequent Southern hybridization, a DIG-labeled, 930 bp long DNA probe was used as evidence of the WCT gene cloned into the pSTH1 vector . The engineered vector pSTH1 is a derivative of the generally available prokaryotic cloning vector pBR322.

The agarose gel electrophoresis results shown in FIG. 9 and the respective Southern-Blot analyzes undoubtedly demonstrated that the WICALTIN encoding WCT gene can be detected using a nucleic acid probe.

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Example 14

Northern blot analysis to demonstrate transcriptional induction of the WICALTIN-coding WCT gene of Williopsis califomica 3/57 (DSM 12865) by β-1,3-Dglucan

To demonstrate WCT-transcription induced by β-1,3-D-glucan, the yeast strain DSM 12865 was cultured in 300 ml of BAVC-medium, respectively. in BAVC-? medium with the addition of 0.03% natural β-1,3-D-glucan laminarin for 48 hours at 20 ° C with gentle shaking (60 rpm) and was successively used at various intervals to prepare total RNA. All samples (10 ml) were adjusted to an equal cell count of 1.8 x 10 ⁸ cells / ml prior to RNA isolation and then electrophoretically resolved in denaturing agarose-formaldehyde gels. As can be seen from FIG. 10, both the non-induction conditions (BAVC-medium without addition) and the laminarin-enriched BAVC-medium could be shown to be 1,100 bases in the WCT transcript. Without the addition of glucan, maximal expression of WCT was achieved at the end of the exponential growth phase (after 19 hours); substantially weaker hybridization signals in the stationary phase of growth indicate attenuated transcription. Under inducing culture conditions (in the presence of laminarin), the WCT transcript showed significantly greater intensity after 10 hours than in the non-inducing culture medium, suggesting that this transcription of the WICALTIN-encoded WCT gene can be induced by the addition of β-1,3-D-glucan.

Addendum to the Examples

Media and solutions used in the examples:

a) BAVC medium glucose 50 g / l

D, L-malate 20 g / l trisodium citrate 0,5 g / l (NH ₄ ) ₂ SO ₄

MgSO ₄

1.5 g / l 1.0 mg / l

31854 / H myoinositis 0.04 g / l amino acid base solution (10 x) 200 ml / l trace element base (100 x) 10 ml / l vitamin base (100 x) 20 ml / l

b) amino acid stock solution (10x) alanine 0.75 g / l arginine monohydrochloride 3.5 g / l aspartic acid 0.5 g / l glutamic acid 3 g / l histidine monohydrochloride 0.2 g / l methionine 0.4 g / l serine 0.5 g / l threonine 2 g / l tryptophan 0.4 g / l

(c) trace element stock (100 x) boric acid 200 mg / l

FeCl3.6 H2O 200 mg / l

ZnSO4.7 H2O 200 mg / l

AlCl3 200 mg / L

CuSO4.5 H2O 100 mg / L

Na2MoO4.2 H2O 100 mg / L

Li2SO4. H 2 O 100 mg / L

KJ 100 mg / l potassium hydrogen tartrate 2 g / l

d) vitamin solution (100 x)

4-aminobenzoic acid 20 mg / l biotin 2 mg / l folic acid 2 mg / l nicotinic acid 100 mg / l pyridoxol hydrochloride 100 mg / l

31854 / H riboflavin thiaminium dichloride

Ca-D-pantothenate mg / l 50 mg / l 100 mg / l

Biotin: was dissolved in a solution of 5 g KH2PO4 in 50 ml distilled water.

Folic acid: was dissolved in 50 ml of distilled water with the addition of a few drops of dilute NaOH.>

Riboflavin: was dissolved with heating in 500 ml of distilled water with a few drops of HCl.

Other vitamins are soluble in a small amount of distilled water.

The pH of the BAVC medium was adjusted to pH 4.7 by addition of KOH. Glucose and stock solutions were sterilized separately. Amino acid, vitamin, and trace element solutions were sterilized for 20 minutes with a steam stream at 100 ° C and then autoclaved BAVC-medium was added.

Explanation of the drawings and the most important sequences

SEQ ID No 1: DNA- and deduced amino acid sequence of WCT-encoded protein toxin WICALTIN from yeast Williopsis califomica strain 3/57.

SEQ ID No 2: cDNA- and deduced amino acid sequence of ZBT-encoded protein toxin ZYGOCIN from yeast Z. bailii.

Fig. 1: N-terminal amino acid sequences of W. califomica-ioxin WICALTIN and endo-β-1,3-glucanase Bg12 from yeast S. cerevisiae. Individual deviations of otherwise identical sub-sequences are bold (Bg12 sequences as reported by Klebl and Tanner, 1989).

Fig. 2: Kinetics of S. cerevisiae sensitive yeast 192.2 cells by WICALTIN in the presence (2a) and absence (2b) of laminarin-β-D-glucan (L) and pustulan (P). The toxin used had a total activity of 4.0 x 10 ⁵ E / ml at a specific activity of 4.2 x 10 ⁵ E / mg protein.

Fig. 3 (a, b, c, d): Agar diffusion test as evidence of sensitivity / resistance

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WICALTIN in Kre1 ⁺ and Kreel of yeast strains of S. cerevisiae. After transformation

WICALTIN-resistant cre1-nullmutant of S. cerevisiae SEY6210 [kre1] with KRE1-carrying vector pPGK [KRE1] restored full sensitivity to

WICALTIN.

Fig. 4: (A) Gel electrophoresis (SDS-PAGE) of ZYGOCIN produced and secreted by yeast Z. bailii strain 412 (DSM 12864) _; after affinity chromatography on mahoprotein-Sepharose. (B) Agar diffusion assay as evidence of the biological activity of purified ZYGOCIN killer-toxin.

Fig. 5: Scheme structure of ZBT-, resp. WCT-bearing expression vector for the preparation of transgenic plants.

(Explanatory notes: RB, LB: right and left border of the natural T1 plasmid from Agrobacterium tumefaciens', CaMV-P: 35S cauliflower mosaic virus promoter; NOS-P, NOS-T: transcriptional promoter and nopaline synthase terminator kan ^R : kanamycin-resistant gene from Streptococcus for selection in E. coli; NPT-II: neomycin phosphotransferase gene from transpranone Tn5 for selection in plant).

Fig. 6:

(A) Partial restriction card of the pSTH2 episomal vector for heterologous expression of WICALTIN-encoding toxin WCT in yeast Saccharomyces cerevisiae. The vector pSTH2 is a constructed plasmid based on the commercially available 2μ Multi-Copy vector pYX242, in which the WCT gene is cloned from the DSM 12865 strain as a 930 bp EcoRI / SmaI fragment. The respective toxin undergoes transcriptional control of its own yeast TPI-promoter, thus allowing intensive and constitutive expression of WICALTIN upon transformation in S. cerevisiae.

(B) Gel electrophoresis (SDS-PAGE; 10-22.5% gradient gel) of concentrated supernatant of S. cerevisiae culture medium after transformation with the constructed WICALTIN-expression vector pSTH2 (record / Spur / 1). WICALTIN heterologically expressed in S. cerevisiae is indicated by an arrow.

31854 / H (C) Demonstration of extracellular β-1,3-glucanase activity of S. cerevisiae yeast after transformation with WICALTIN-expressing yeast vector pSTH2. To determine exo-β-1,3-glucanase activity, yeast colonies grown on leucine SC-agar were sprayed with 0.04% 4-methylumbelliferyl-4-glucoside (MUG) in 50 mM sodium acetate buffer (pH 5.2). After incubation at 37 ° C for 30 minutes, the agar plates were irradiated with UV light (254 nm wavelength). Glucanase activity was demonstrated by fluorescence based on MUGhydrolysis.

(Explanations: 1 and 4, S. cerevisiae transformed with a vector (pEP-WCT) that expresses the WICALTIN-encoding WCT gene under the action of its own promoter; 3. "wild" yeast W. californica 3/111; 5. S. cerevisiae after transformation with the WICALTINexpressing vector pYX-WCT; 6. S. cerevisiae transformed with the base vector pYX242 (non-toxin).

Fig. 7: Schematic structure of the pTZ cdx vector for heterologous expression and secretion of foreign proteins (especially WICALTIN and ZYGOCIN) in dividing yeast Schizosaccharomyces pombe.

(Explanation: P _nmt1 , T _nm n, transcriptional promoter and terminator of the nmt1 gene of the dividing yeast S. pombe regulated by thiamine; S / P secretory and processing sequence of viral S. cerevisiae viral K28-preprotoxin, ars1, autonomously replicating chromosome sequence Ieu2, a leucine-2-marker gene for the selection of leucine prototrophic transformants (S. pombe).

Fig. 8: Comparison of biological activity of purified WICALTIN, Clotrimazole and Miconazole; said molar amounts elicited in the "Bioassay" (agar diffusion test) against the sensitive indicator yeast Sporothrix spec. diameter of inhibition court 12 mm.

Fig. 9: Detection of WICALTIN-coding WCT gene of W. californica 3/57 (DSM 12865) cloned in pSTHI (derivative of pBR322) by agarose gel electrophoresis (A) and Southern hybridization with WCT-labeled DIG31854 / H (B) ).

(Legend: DIG-labeled DNA-length standard II; record 1, pSTH1 restricted with EcoRI and Sa / I; record 2, DNA marker "Smart-ladder").

Fig. 10: Northern analysis of transcriptional induction of the WICALTIN-coding WCT gene of W. califomica 3/57 (DSM 12865) under non-inducing culture conditions in BAVC medium (A) and under inducing conditions in BAVC medium with an addition of 0.03% lamininarin (B) ). Electrophoretic separation of the length RNA isolated from the DSM 12865 strain was performed on a denaturing agarose / formaldehyde gel at a constant voltage (7 V / cm). RNA on the nylon membrane was hybridized against a WICALTIN-specific DIG-labeled probe (630 bp) and detected by chemiluminescence.

(Explanation: M, DIG-labeled DNA-length standard I; records 1-8 correspond to sampling times for total RNA isolation; record 1, 10 hr. Record 2, 15 hr, record 3, 7 hr, record 4, 24 hours, recording 5, 33 hours, recording 6, 38 hours, recording 7, 43 hours, recording 8, 48 hours).

Abbreviations used in the text

WCT Williopsis Califomica Toxin

ZBT Zygosaccharomyces Bailii Toxin

ZYGOCIN name: toxin secreted from DSM 12864

WICALTIN name: toxin secreted from DSM 12865

The following microorganisms used according to the invention were deposited in the internationally recognized German Collection of Microorganisms and Cell Cultures of the Deutsche Sammlung von Microorganisms and Zellkulturen GmbH (DSMZ), Maschenroder Weg 1b, 38124 Braunschweig, Germany, according to the requirements of the Budapest Treaty on International Recognition for (save number, save date).

Williopsis califomica strain 3/57 (DSM 12865) (09/06/1999)

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Zygosaccharomyces bailii strain 412 (DSM 12864) (09/06/1999).

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Sequence protocol · <110> Aventis Research & Technologies GmbH & Co KG <120> Antimycotics and fungicides, process for their preparation and use <130> 99F028 <140> 19930959.0 <14l> 1999-07-05 <160> 4 <170> Patentln Ver. 2.1 <210> 1 <211> 930 <212> DNA <213> Williopsis californica <220>

<221> CDS <222> (2). . (930) <400> 1

atg cgt Met Arg 1 ttc Phe act Thr aca Thr 5 CTC Leu gtt wall gcc Ala CTC Leu GCA Ala 10 GGT Gly gcc Ala att Ile tcc tca gtc 48 Ser Ser 15 wall cag gcc ATC GGC CAA CTA get ttt aac TTG GGT GTC aag gat aac tCA 96 gin Ala Ile Gly gin Leu Ala Phe same time Leu Gly wall Lys Asp same time Ser 20 25 30 GGT cag TGC aag act gcc tCA gag tac aag gat GAC TTG TCT acc CTT 144 Gly gin Cys Lys Thr Ala Ser Glu Tyr Lys Asp Asp Leu Ser Thr Leu 35 40 45 tCA GGC tac aca TCT aag gtt aga GTC tac get gcc tCA GAC tgt aac 192 Ser Gly Tyr Thr Ser Lys wall Arg wall Tyr Ala Ala Ser Asp Cys same time 50 55 60 act TTG cag act TTG GGT ca. gtt GTC gaa gag get GGC ttc tCA ttt 240 Thr Leu gin Thr Leu Gly for wall wall Glu Glu Ala Gly Phe Ser Phe 65 70 75 80 ttc gtt GGT att TGG ca. aac gat gat get CAC ttc cag gaa gag CAA 288 Phe wall Gly Ile Trp for same time Asp Asp Ala His Phe gin Glu Glu gin

90 95

31854 / H

3 /

gac get TTG Leu aaa Lys 100 act Thr tat Tyr ttg approx aag att aag Lys aga Arg TCC Ser aca Thr 110 GTG wall gag Glu 336 Asp Ala Leu Pro Lys 105 Ile gcc ttc act gtt GGT tet gag gcc TTG tat aga gat gat atg act get 384 Ala Phe Thr wall Gly Ser Glu Ala Leu Tyr Arg Asp Asp Met Thr Ala 115 120 125 CAA gag TTG get GAC aga ATC aaa act att aga gag TTG gtt gcc act 432 gin Glu Leu Ala Asp Arg Ile Lys Thr Ile Arg Glu Leu wall Ala Thr 130 135 140 att GAC GAC TCC gaa GGT aac tCA tat get GGT att ca. gtt GGT ttc 480 Ile Asp Asp Ser Glu Gly same time Ser Tyr Ala Gly Ile for wall Gly Phe 145 150 155 160 gtt GAC TCC TGG aac gtt TTG gtt gat GGT get tet CAC ca. get att 528 wall Asp Ser Trp same time wall Leu wall Asp Gly Ala Ser His for Ala Ile 165 170 175 gtt gag get gat gtt GTG ttc gcc aat get ttc tet tac TGG CAA GGT 576 wall Glu Ala Asp wall wall Phe Ala same time Ala Phe Ser Tyr Trp gin Gly 180 185 190 cag act cag cag aac TCG tCA tac tet ttc ttt GAC GAC . att atg CAA 624 gin Thr gin gin same time Ser Ser Tyr Ser Phe Phe Asp Asp Ile Met gin 195 200 205 get TTG CAA acc att CAA act get aag GGT gag aca gat ATC act ttc 672 Ala Leu gin Thr Ile gin Thr Ala Lys Gly Glu Thr Asp Ile Thr Phe 210 215 220 TGG gtt GGT gag acc GGC TGG ca. acc gat GGT act CAC ttt gaa GAC 720 Trp wall Gly Glu Thr Gly Trp for Thr Asp Gly Thr His Phe Glu Asp 225 230 235 240 tet GTC ca. tet gtt gag aat get cag acc ttc TGG aaa gat gcc GTC 768 Ser wall for Ser wall Glu same time Ala gin Thr Phe Trp Lys Asp Ala wall 245 250 255 tgt gcc att aga GGT TGG GGT ATC aat gtt att gcc ttt gag gcc ttt 816 Cys Ala Ile Arg Gly Trp Gly Ile same time wall Ile Ala Phe Glu Ala Phe 260 265 270 GAC gaa get TGG aag ca. gat acc tet GGT acc tet gat GTG gaa aag 864 Asp Glu Ala Trp Lys for Asp Thr Ser Gly Thr Ser Asp wall Glu Lys 275 280 285 tac TGG GGT gtt TGG GAC tet aac age aag TTG aag tat gat TTG TCC 912 Tyr Trp Gly wall Trp Asp Ser same time Ser Lys Leu Lys Tyr Asp Leu Ser

31854 / H si

290 295 300 tgt GAC ttt acc TCT tag 930 Cys Asp Phe Thr Ser 305 310

<210> 2 <211> 309 <212> PRT <213> Williopsis californica

Met 1 Arg Phe Thr Thr 5 Leu Val Ala Leu Ala 10 Gly Ala Ile Ser Ser 15 wall gin Ala Ile Gly gin Leu Ala Phe same time Leu Gly wall Lys Asp same time Ser 20 25 30 Gly gin Cys Lys Thr Ala Ser Glu Tyr Lys Asp Asp Leu Ser Thr Leu 35 40 45 Ser Gly Tyr Thr Ser Lys wall Arg wall Tyr Ala Ala Ser Asp Cys same time 50 55 60 Thr Leu gin Thr Leu Gly for wall wall Glu Glu Ala Gly Phe Ser Phe 65 70 75 80 Phe wall Gly Ile Trp for same time Asp Asp Ala His Phe gin Glu Glu gin 85 90 95 Asp Ala Leu Lys Thr Tyr Leu for Lys Ile Lys Arg Ser Thr wall Glu 100 105 110 Ala Phe Thr wall Gly Ser Glu Ala Leu Tyr Arg Asp Asp Met Thr Ala 115 120 125 gin Glu Leu Ala Asp Arg Ile Lys Thr Ile Arg Glu Leu wall Ala Thr 130 135 140 Ile Asp Asp Ser Glu Gly same time Ser Tyr Ala Gly Ile for wall Gly Phe 145 150 155 160 wall Asp Ser Trp same time wall Leu wall Asp Gly Ala Ser His for Ala Ile 165 170 175 wall Glu Ala Asp wall wall Phe Ala same time Ala Phe Ser Tyr Trp gin Gly

31854 / H

180 185 190 gin Thr gin gin same time Ser Ser Tyr Ser Phe Phe Asp Asp Ile Met gin 195 200 205 Ala Leu gin Thr Ile gin Thr Ala Lys Gly Glu Thr Asp Ile Thr Phe 210 215 220 Trp wall Gly Glu Thr Gly Trp for Thr Asp Gly Thr His Phe Glu Asp 225 230 235 240 Ser wall for Ser wall Glu same time Ala gin Thr Phe Trp Lys Asp Ala wall 245 250 255 Cys Ala Ile Arg Gly Trp Gly Ile same time wall Ile Ala Phe Glu Ala Phe 260 265 270 Asp Glu Ala Trp Lys for Asp Thr Ser Gly Thr Ser Asp wall Glu Lys 275 280 285 Tyr Trp Gly wall Trp Asp Ser same time Ser Lys Leu Lys Tyr Asp Leu Ser 290 295 300 Cys Asp Phe Thr Ser 305

<210> 3 <211> 718 <212> DNA <213> Zygosaccharomyces bailii <220>

<221> CDS <222> 1) .. (717) <400> 3 atg aaa gca gcc caa ata tta aca gca agt ata gta agc tta ttg approx.

10 15 ata tat act agt get aga aac tta gac aga gaa tac aca gca aac ile Tyr Thr Ser Ala Arg Asn ile Leu Asp Arg Glu Tyr Thr Ala Asn

25 30 gaa tta aaa act get ttt

Glu Leu Lys Thr Gly Asp Glu Glu White Phe Thr Asp Leu Thr

144

31854 / H

35 40 45 tat CAC att CAC gtt aac GTC agt GGC gaa att GAC tet tac tat cat 192 Tyr His Ile His wall same time wall Ser Gly Glu Ile Asp Ser Tyr Tyr His 50 55 60 aat TTA GTC aat ttt GTC gat aac get CTA GCA aac aaa gat att aat 240 same time Leu wall same time Phe wall Asp same time Ala Leu Ala same time Lys Asp Ile same time 65 70 75 80 aga tat ata tac get ata ttt aca cag cag aca aac tat aca gag gat 288 Arg Tyr Ile Tyr Ala Ile Phe Thr gin gin Thr same time Tyr Thr Glu Asp 85 90 95 ggg CTC att gag tac TTA aat cat tac gat tCA gag act TGC aaa gat 336 Gly Leu Ile Glu Tyr Leu same time His Tyr Asp Ser Glu Thr Cys Lys Asp 100 105 110 ATC att act cag tat aat gtt aac gta GAC act agt aac tgt ata age 384 Ile Ile Thr gin Tyr same time wall same time wall Asp Thr Ser same time Cys Ile Ser 115 120 125 aat act aca gat CAA get aga CTC CAA CGT ege GGA ggg TGG GTG aac 432 same time Thr Thr Asp gin Ala Arg Leu gin Arg Arg Gly Gly Trp wall same time 130 135 140 ca. cat tgt agt GGT gat aac TTA gcc gat act age gat tgt tgt aac 480 for His Cys Ser Gly Asp same time Leu Ala Asp Thr Ser Asp Cys Cys same time 145 150 155 160 TTG get tat aac aag att aac ccc tet tCA aac TTA cag tCA TGG aat 528 Leu Ala Tyr same time Lys Ile same time for Ser Ser same time Leu gin Ser Trp same time 165 170 175 tat gtt GTC ggg cag tgt CAC tat att tet CAC get aat GGA aag gta 576 Tyr wall wall Gly gin Cys His Tyr Ile Ser His Ala same time Gly Lys wall 180 185 190 tgt agt GGT get GAC agg CAA cag TTA get gaa aat gta tgt aac TGG 624 Cys Ser Gly Ala Asp Arg gin gin Leu Ala Glu same time wall Cys same time Trp 195 200 205 tgt cag gtt aac GGT GGT gtt age get ttt get age agt agt tet GCA 672 Cys gin wall same time Gly Gly wall Ser Ala Phe Ala Ser Ser Ser Ser Ala 210 215 220 cat ca. GGT get TGC atg agt gat gta ggg ttc TGC tat QCT tag 717 His for Gly Ala Cys Met Ser Asp wall Gly Phe Cys Tyr Ala 225 230 235

31854 / H

<210> 4 <211> 238 <212> PRTs <213> Zygosaccharomyces bai <400> 4 Met Lys Ala Ala Gin White Leu 1 5

Iii il Tyr Thr Ser Ala Arg Asn 20

Glu Leu Lys Thr Ala Phe Gly

Tyr His White His Val Asn Val 50 55

Asn Phu Val Asp 65 70

Arg Tyr ile Tyr Ala ile Phe 85

Gly Leu Clay Glu Tyr Leu Asn 100 Clay Thr Gin Tyr Asn Val 115

Asn Thr Thr Asp Gin Ala Arg

Pro His Cys Ser Gly Asp Asn 145,150

Leu Ala Tyr Asn Lys White Asn 165

Tyr Val Val Gly Gin Cys His 180

Cys Ser Gly Ala

Cys Gin Val Asn

Thr Ala Ser il Val Ser Leu Leu 10 15 il Leu Asp Arg Glu Tyr Thr Ala 25 30

Asp Glu Glu White Phe Thr Asp Leu 40 45

Ser Gly Glu White Asp Ser Tyr Tyr 60

Asn Ala Leu

Thr Gin Gin Thr Asn Tyr Thr Glu 90

His Tyr Asp Ser Glu Thr Cys Lys

Asn Val Asp Thr Ser Asn Cys White 120 125

Leu Gin Arg Arg

Leu Ala Asp Thr Ser Cys Cys 155

Pro Ser Ser Asn Leu Gin Ser Trp 170 175

Tyr ile Ser His Ala Asn Gly Lys 185,190

Gin Leu Ala Glu Asn Val Cys Asn 200 205

Ser Ala Phe Ala Ser Ser Ser 220

for

same time

Thr

His

same time

Asp

Asp

Ser

same time

same time

160

same time

wall

Trp

Ala

31854 / H

Y3

His Gly Ala Cys Met Ser

Claims (26)

PATENT CLAIMS Protein toxins obtainable from Williopsis califomica and / or Zygosaccharomyces bailii. Protein toxins according to claim 1, obtainable from DSM 12864 and / or DSM 12865.; Protein toxins according to claims 1 and 2, characterized in that they have antifungal and / or fungicidal effects. A protein toxin according to any one of claims 1 to 3 with glucanase activity. Protein toxin according to claim 4, characterized in that it binds to β1,6-D-glucan and exhibits β-1,3-D-glucanase activity and / or β-1,3-lu-glucanosyltransferase activity. A nucleic acid encoding a glucanase and / or a protein toxin according to any one of claims 1 to 5 having an amino acid sequence according to SEQ ID No 1 or SEQ ID No 2 or a functional variant thereof and portions thereof with at least 8 nucleotides, wherein SEQ ID No 1 or SEQ ID No 2 is part of this claim. Nucleic acid according to claim 6, characterized in that the nucleic acid is DNA or RNA, preferably double-stranded DNA. Nucleic acid according to claim 6 or 7, characterized in that the nucleic acid is a DNA having the amino acid sequence according to SEQ ID No 1 having a base position 1 to 951 or SEQ ID No 2 having a base position 1 to 717, wherein SEQ ID No 1 or SEQ ID No 2 is part of this claim. Nucleic acid according to claim 8, characterized in that the nucleic acid comprises one or more regulatory regions, such as a promoter, enhancer, terminator and / or 3'-terminal PolyA-sequence and / or a Kex2β-endopeptidase cleavage site necessary for intracellular processing of the pro-toxin and / or one or more potential N31854 / H glycosylation sites. Nucleic acid according to any one of claims 8 to 9, obtainable from DSM 12864 and / or DSM 12865. Nucleic acid according to one of Claims 6 to 10, characterized in that the nucleic acid is contained in a vector, preferably in an expression vector or in a gene-therapeutically active vector. A method for preparing a nucleic acid according to any one of claims 6 to 10, characterized in that the nucleic acid is synthesized chemically or isolated by a probe from a gene bank. 13. A polypeptide having the amino acid sequence of SEQ ID No 1 or SEQ ID No 2, or a functional variant thereof, and portions thereof with at least six amino acids. A method for preparing a polypeptide of claims 1 to 5 and 13, wherein the nucleic acid of any one of claims 6 to 11 is expressed in a suitable host cell. The anti-peptide antibody of any one of claims 1 to 5 and 13. 16. The method of preparing an antibody of claim 15, wherein the mammal is immunized with the polypeptide of claim 7 and the optionally formed antibody is isolated. A medicament comprising a nucleic acid according to any one of claims 6 to 10 or a polypeptide according to any one of claims 1 to 5 and 13 or an antibody according to claim 15 and optionally pharmaceutically acceptable excipients or excipients. Method for the preparation of a medicament for the treatment of mycoses such as superficial, cutaneous and subcutaneous dermatomycosis, mucosal mycosis and systemic mycosis, in particular Cand / c / α-mycosis, characterized in that the nucleic acid according to any one of claims 6 to 10 or the polypeptide according to any of claims 1 to 5 and 13 or the antibody of claim 15 is combined with a pharmaceutically acceptable excipient or excipient. 31854 / H Η Diagnostics comprising a nucleic acid according to any one of claims 6 to 10 or a polypeptide according to any one of claims 1 to 5 and 13 or an antibody according to claim 15 and optionally suitable additives and / or excipients. A method of preparing a diagnosis for the diagnosis of mycoses such as superficial, cutaneous and subcutaneous dermatomycosis, mucosal mycosis and systemic mycosis, in particular Cand / da-mycosis, characterized in that the nucleic acid of any one of claims 6 to 10 or the polypeptide of any one of claims 1 to 5 and 13 or the antibody of claim 15 is combined with a pharmaceutically acceptable carrier. An assay for identifying functional interactions comprising a nucleic acid according to any one of claims 6 to 10 or a polypeptide according to any one of claims 1 to 5 and 13 or an antibody according to claim 15, and optionally suitable additives and / or excipients. Use of a nucleic acid according to any one of claims 6 to 10 or a polypeptide according to any one of claims 1 to 5 and 13 for identifying functional interactions. Use of a nucleic acid according to any one of claims 6 to 10 for searching for variants, characterized in that the gene bank is searched by the nucleic acid and the variant found is isolated. Use of a polypeptide according to any one of claims 1 to 5 and 13 for controlling harmful yeasts and fungi in food and feed. 25. A method of culturing DSM 12864 and DSM 12865, wherein the method is carried out in synthetic B- and / or BAVC medium. Use of nucleic acids according to any one of claims 6 to 11 for the preparation of transgenic plants and plant cells.