CZ200249A3 - Antimycotics and fungicidal agents, process of their preparation and 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, their preparation and their use

Technical field

The present invention relates to antimycotics 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 since infections caused by fungi or yeast in humans have become widespread in recent years and, to a greater extent, 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 fully functional level [Anaissie, 1992; Meunier et al., 1992; Vingard, 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 at particular risk of mycosis.

Λ [Left, 1993]. The antimycotics currently used for the therapy of 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 and thereby damage the infected host organism [Hector, 1993] . In addition, the application of conventional antifungals leads to a rapid increase in fluconazole resistance in a short period of time, which in human pathogenic microorganisms is rapidly increasing. The moss expands rapidly, posing 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 a high selectivity and that, as far as possible, they attack only fungi and yeasts pathogenic to humans. However, since most cellular processes in higher organisms are controlled by gene products that

They exhibit a high degree of functional homology in eukaryotes, and specific antifungal antibiotics have not yet been developed [Kurz, 1998; Komiyama et al., 1998].

The target area of selective antifungal agents are β-1,3-glucans of the yeast cell wall, which are essential for the mechanical and osmotic stability of the cell, but which do not occur in higher eukaryotes, so that they could be used as Achilles' heel. Achillesverse is likely to be involved in the eradication of pathogenic yeasts [Roemer et al., 1994]. Despite the fact that substances that selectively attack the cell wall structure of yeasts and fungi are of great interest, no similar substances have so far antibiotics used to treat mycoses. While bacterial antibiotic producers were discovered earlier this century, similar effects in yeast were only observed in the early 1960s when killer yeasts were identified and observed [Bevan and Makower, 1963]. Saccharomyces cerevisiae killer-strain-producing toxins produce and secrete proteins designated as killertoxins, which kill sensitive yeast in a receptor-dependent process (Rezeptor-abhángigen Prozess) [Bussey, 1991; Tipper and Schmitt, 1991]. The ability to produce a toxin rests in S. cerevisiae for infection with double-stranded viruses in S. cerevisiae. Reovirus-like RNAs (Reovirus-ähnliche Dopplestrang-RNA-Viren), which are stable in the yeast cytoplasm and persist in a large number of copies there, without appreciably damaging the eukaryotic host cell [Tipper and Schmitt, 1991]. Three known killer toxins (ΚΙ, K2, K28) from yeast S. cerevisiae are non-glycosylated α / β heterodimers, which are transferred from the infected cell as high-molecular preprotoxins and converted into intracellular secretory pathway by complex modifications to biologically active killer-proteins [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 ΚΙ, K2) or (as in killer toxin K28) in cell cycle arrest (Zellcycle-Arrest) with a targeted inhibition of DNA synthesis [Bussey, 1991; Schmitt and Compain, 1995; Schmitt et al., 1996]. Although killer toxins of classes ΚΙ, K2 and K28 differ greatly in their mode of action and in their physicochemical properties, they share a common narrow spectrum of activity 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 interact with each other in order to kill sensitive target cells. The primary yeast cell wall toxin receptors are either highly branched β-Ι, ό-D-glucans or outer mannotriose sidewalls of the cell wall mannoprotein [Bussey, 1991; Schmitt and Radler 1987, 1988].

In addition to the viral protein toxins of yeast S. cerevisiae, Hanseniaspora uvarum, Zygosaccaromyces bailii and Ustilago maydis, killer strains have also been described in the genera Debaromyces, Hansenula, Cryptococcus, Rhodotorula, Trichosporon, Ptchia, Kluyveromyli and Torulisis, Torulops, Torulops. Park et al., 1996; Schmitt and Neuhausen, 1994; Valker 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 [Schrůnder et al., 1994].

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

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

SUMMARY OF THE INVENTION

Surprisingly, toxins produced and secreted with high efficiency, namely the killer-toxin VICALTIN (also proteintoxin) from the wild yeast strain Williopsis californica strain 3/57 (DSM), have proved to be particularly suitable for destroying yeasts and / or fungi with pathogenic effects on humans or plants. 12865) and virus-encoded ZYGOCIN (also proteintoxin) from yeast Zygosaccharomyces bailia (DSM 12864). In addition, the feared harmful yeasts in food and feed have been killed by these means. 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 thus a process for both gene-engineering and expression (removal) has been developed in the culture of VICALTIN and ZYGOCIN.

· «

Accordingly, the present invention provides 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 under the Budapest Treaty on 9 June 1999. DSMZ - Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, 38124 Braunschweig, Mascheroder Veg lb (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), also killed numerous 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 toxigen functional unit, - are potential biopharmaceuticals which due to their specific receptor-mediated action, they only kill yeasts and / or fungi and are completely harmless to higher eukaryotes - and, of course, to human and mammalian cells - and also to plants, especially to crop plants [see Pfeiffer et al., 1988] .

The following human and plant-pathogenic as well as apatogenic yeasts and / or fungi were selectively killed:

Zygocin-sensitive yeast species: Saccharomyces cerevisiae, Candida albicans, Candida krusei, Candida glabrata, Candida vinii, Hanseniaspora uvarum, Kluyveromyces marxianus,

Metschnikowia pulcherrima, Ustilago maydis, Debaromyces hansenii, Pichia anomala, Pichia jadinii, Pichia membranefaciens, Yarowia lipolytica and Zygosaccharomyces rouxii.

Vicaltin-sensitive yeast species: Candida albicans,

Candida glabrata, Candida tropicalis, Debaromyces hansenii, Kluyveromyces lactis, Metschnikowia pulcherrima, Pichia anomala, Pichia jadinii, Saccharomyces cerevisiae, Sporthrix spec.

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 than other strains of the same yeast species. The killer-property of the zygocin-producing yeast strain DSM 12864 is due to infection with toxin-coding double-stranded RNA i (M ^^ - dsRNA) viruses that are stable, persist in the cytoplasm in high copy numbers and induce production in the respective yeast (DSM strain, 12864). and zygocin secretion [see Schmitt and Neuhausen, 1994]. Other strains of the same species showed no toxin production since they do not contain any toxin-coding dsRNA viruses in the cytoplasm, so they are

9 ^r f 9 9 · · · phenotypically classified as non-Killer types.

Another object of the present invention are proteintoxin-encoding nucleic acids - having the amino acid sequence of SEQ ID No 1 and No 2 and glucanase activity - or functional variants of these nucleic acids and portions thereof with at least 8 nucleotides, in particular at least 15 or 20 nucleotides, 100 nucleotides, preferably at least 300 nucleotides (hereinafter referred to as nucleic acids of the invention).

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 according to SEQ ID No 1 in yeast S. cerevisiae results in recombinant VICALTIN, 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 proteintoxin with glucanase activity in SEQ ID No 1 and, in the case of SEQ ID No 2, probably encode an O-glycosylated proteintoxin referred to as ZYGOCIN .

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 according to 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 the beginning and end of the coding according to the present invention. region, i.e. always the first and last amino acids in the respective reading frame (Leseraster).

As used herein, a functional variant is 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 of _f or allelic variants. The present invention also encompasses nucleic acid variants that can be derived from various yeast / yeast strains or other pathogens such as dermatophytes and fungi (according to the DHS system).

In another sense, variants of the invention are nucleic acids that 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 other 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 suitable as a primer in a PCR procedure, a nucleic acid of at least about 20 nucleotides is suitable for identifying additional variants and a nucleic acid consisting of at least about 100 nucleotides is suitable as a probe.

In a further preferred embodiment of the invention, the nucleic acid according to the invention comprises one or more of 9 in the sequence of the invention. Multiple non-coding sequences and / or poles (A) - sequence, one or more (-). for intracellular pro-protein processing of the necessary Kex2β-endopeptidase recognition sequences (Endopeptidase Erkennungssequenzen) as well as one or more potential N-glycosylation sites. The non-coding sequences are control sequences, such as a promoter or enhancer sequence, for the controlled expression of a coding toxingen comprising the nucleic acids of the invention.

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

For example, in the case of the nucleic acids of SEQ ID No 2, the expression vectors 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 heterologically expressed protein toxin is toxic to bacterial cells. Cloning of the VITCALTIN-encoding nucleic acids of SEQ ID No 1 in E. coli is only possible with plasmids that carry no promoter (eg, by using plasmid derivatives pBR322). An example of a prokaryotic vector that allows heterologous expression of the ZYGOCIN-encoding nucleic acid of SEQ ID No 2 is the commercially available vector pGEX-4T-1, which allows glutathione-S-transferase-ZYGOCIN-fusion protein to be expressed in E. coli. Another vector for the r expression of ZYGOCIN in E. coli is, for example, the T7 expression vector pGM10 (Martin, 1996), which encodes the N-terminal Met-Ala-His6-tag (tag), allowing convenient purification of the expressed protein on the Ni- NTA. Suitable eukaryotic expression vectors for expression in Saccharomyces cerevisiae are, for example, vectors such as <RTIgt; vectors. </RTI> P426Met25 or p426GAL1 [Mumberg et al., (1994) Nucl. Acids Res., 22, 5767], for expression in insect cells, for example, Baculovirus vectors such as EP-B1-0127839 or EP-B1-0549721 are useful, and for expression in mammalian cells, for example, SV40-vectors that are commercially available.

Expression vectors also generally contain 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 _Λ promoter for expression in yeast [Russel spol.

(1983). J.Biol.Chem 2588, 2674], baculovirus-polyhedrin- * promoter for expression in insect cells (see, for example,

EP-B1-0127839) or the older SV40-promoter or LTR-promoters eg from MMTV (Mammary Tumor Virus; Lee et al., (1981) Nature, 214, 228).

Examples of gene-therapeutically effective vectors are virusvectors, in particular adenovirusvectors, in particular replication-deficient adenovirusvectors, or adeno-associated virusvectors, e.g. an adeno-associated virusvector, which consists exclusively of two inserted terminal repeat sequences (ITRs).

Suitable adenoviral vectors are described, for example, in McGroy, V.J. et al. (1988) Virol. 163, 614; Gluzman, Y. et al. (1982) in 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;

WO95 / 00655.

Suitable adeno-associated virus vectors are described, for example, in Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol.

44 · 4 · 4 · 4 · 4

158, 97; WO95 / 23867; Samulski, R.J. (1989) J. Virol., 63, 3822; WO95 / 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 suitable for this purpose are described, for example, in Felgner, P.L. et al., (1987) Proc. Nati. Acad. Sci. USA 84, 7413; Behr, J.P. et al.

(1989) Proc. Nati. 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, DNA binds ionically to the surface of the liposomes in such a proportion that a positive charge is maintained on the surface and that the DNA is completely complexed with the liposomes.

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 described killer-toxins VICALTIN and ZYGOCIN have a broad spectrum of activity and also kill phytopathogenic yeasts and fungi, it is possible to obtain such transgenic plants which are resistant, for example, to infection caused by maize by the pathogen Ustilago maydis. Similar experiments have already been carried out on tobacco plants which, after heterologous expression of naturally virally encoded killertoxin KP4 from U.maydis, have the ability to secrete the respective protein toxin and thereby obtain specific protection against infections with certain phytopathogenic U.maydis strains (Park et al., 1996; et al., 1995; Bevan, 1984). Based on commercially available transformation systems based on modified natural derivatives.

- ···· · · · ·

The thi plasmids from Agrobacterium tumefaciens can be cloned into the so-called bi-directional pBI vectors (CLONTECH) and used to obtain transgenic plants. The respective WCT and ZBT toxins are introduced under the transcriptional control of the potent cauliflower mosaic virus promoter (CaMV-P). Further details of the construction of this engineered vector are shown schematically in Example 9.

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

synthesized chemically using the sequences of SEQ ID No 1 'and No 2 or using the peptide sequences of SEQ ID No and No 2 using the genetic code, eg, by the phosphotriester method (see, eg, Uhlman, E. and Peyman, A. (1990) Chemical Reviews, 90,543, No.4). Another possibility for 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, for example, single-stranded DNA fragments of about 100 to 1000 nucleotides in length, in particular 200 to 500 nucleotides in length, in particular about 300 to 400 nucleotides in length, the sequence of which can 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 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 especially 309 amino acids (SEQ. ID No 1) and having 99 amino acids (SEQ ID No 2) (hereinafter referred to as polypeptides of the invention). For example, a polypeptide with a length of approx

6-12, in particular about 8 amino acids in length, contain an epitope which, upon binding to a carrier, serves to prepare specific pole or monoclonal antibodies (see, eg, US 5,656,435). Polypeptides of at least about 65 amino acids in length can be used directly or without a carrier to prepare polys or monoclonal antibodies.

The term functional variant within the meaning of the present invention is understood to mean polypeptides that are related to the peptide of the present invention, i.e., exhibit glucanase activity.

Variants include allelic variants or polypeptides,

- which may also originate from different yeast / yeast strains or other agents of infections such as dermatophytes or fungi (according to the DHS system).

In a further aspect, polypeptides which exhibit sequence homology, in particular sequence identity of about 70%, especially about 80%, especially about 90%, and especially about 95% with the polypeptide having the amino acid sequence of Figure 2, are also included in the invention. Also included are cleavage of the polypeptide in the range of about 1-60, especially 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 above-described 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. These include, in particular, fusion proteins having a proportion of particularly 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, eg, from E.coli galactosidase, such as galactosidase from E. coli. Or a so-called histidine-tag, e.g.

Met-Ala-Hisg-chain (Tag). A fusion protein with a so-called histidine chain is particularly suitable for purifying the expressed protein on a metal ion column, e.g. using o.

Ni-NTA columns. NTA is a chelator which is nitrilotriacetic 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, for example, epitopes that can be specifically recognized by antibodies.

Polypeptides of the invention may be prepared, for example, by expressing the nucleic acids of the invention in a suitable expression system using methods previously described known to those skilled in the art. Only eukaryotic organisms, in particular budding yeast (Sprosshefe) of Saccharomyces cerevisiae and fission yeast (Spalthefe) of Schizosaccharomyces pombe, are suitable as host cells for the preparation of correctly processed and thus biologically active protein toxins.

These portions of the polypeptides can also be synthesized by classical peptide synthesis (Merrifield technique). These portions are particularly useful for obtaining antisera by which suitable genexpression banks can be screened to obtain additional functional variants of the polypeptides of the invention.

Therefore, a further object of the present invention relates to a process for preparing the polypeptides of the present invention, wherein the polypeptide of the present invention is: 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 The nucleic acid of the invention is expressed and optionally isolated in a suitable host cell.

Particularly preferred are yeast dividing Schizosaccharomyces pombe, since these yeasts exhibit natural resistance to VICALTIN and ZYGOCIN and have been successfully used several times for heterologous expression of foreign proteins [Giga-Hama and Kumagai (1997) in Foreign Gene Expression in Fission Yeast genes in dividing yeast): Schizosaccharomyces pombe, nakl. Springer]. As shown in Example 11, the toxin-encoding nucleic acids of SEQ ID No 1 and No 2 can be cloned into, e.g., the S. pombe-vector pREP1 [Maundrell (1990), J. Biol. Chem. 265: 10857-10864], in which they are under transcriptional control of the thiamine-regulated nmt1 promoter of 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 may optionally be temporally separated from the foreign protein production phase, so that in principle the expression of proteins which are toxic to the yeast can also be expressed. In order to perform simultaneous secretion and thereby substantially facilitate the purification of heterologously expressed toxins VICALTIN and ZYGOCIN in S.pombe, we constructed an expression / / secretion vector [vector ρΤΖατ; see Example 11], which contains the secretion and process signal of viral K28-preprotoxingene [Schmitt and Tipper, 1995], thereby allowing efficient secretion of the respective in-frame inserted foreign protein.

Another aspect of the invention also relates to antibodies that specifically react with a polypeptide of the invention

The above-mentioned portions of the polypeptide are either immunogenic themselves or their immunogenicity can be induced or increased by binding to a suitable carrier, such as bovine serum albumin.

These antibodies are either polyclonal or monoclonal. Their preparation, which is also an object of the invention, is carried out, for example, according to 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, eg, Diamond, BA et al (1981)

The New England Journal of Medicine, 1344). Finally, polyclonal antibodies produced in an animal by an immunological reaction can be readily isolated from the blood by means of generally known methods and purified, for example, by column chromatography. Affinity purification of antibodies is preferred, wherein, for example, an appropriate antigen (VICALTIN 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 Vinter and Milstein (Vinter, 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 the polypeptides of the invention (alone or in combination) and optionally suitable additives or excipients and a process for preparing the medicament for the treatment of mycoses such as superficial, cutaneous and subcutaneous dermatomycoses, mucosal mycoses and systemic mycoses, in particular Candida mycoses,

Wherein the nucleic acid of the invention or polypeptide of the invention is combined with pharmaceutically acceptable excipients and / or excipients.

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

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

Particularly suitable for gene-therapeutic use in humans is a medicament which comprises the nucleic acid of the present invention in its 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.

Suitable additives and / or excipients are, for example, saline, stabilizers, proteinase inhibitors, nuclease inhibitors, etc.

The invention further provides 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 diagnosing mycoses such as superficial, cutaneous and subcutaneous dermatomycoses. mucosal mycoses and systemic mycoses, in particular Candida mycosis, wherein the nucleic acid of the invention, the polypeptide of the invention or the antibodies of the invention are combined with suitable excipients and / or excipients.

Thus, for example, according to the invention, using the nucleic acids of the invention, one can obtain diagnostics based on polymerase chain reaction (PCR-diagnostics, e.g. according to EP-0200362), or Northern and / or Southern Blot analysis, as described in more detail in Example 13. These assays are based on the specific hybridization of a nucleic acid of the invention with a complementary counter strand (Gegenstrang) typically corresponding to mRNA. The nucleic acids according to the invention can also be modified in the manner described, for example, in EP0063879. In particular, this DNA fragment according to the invention labeled by known methods with suitable reagents such as e.g. aP ^J radioactive or non-radioactive biotin -dATP and incubated with isolated RNA, generally attached to a suitable membrane, e.g. cellulose or nylonu.-It is it is preferred to separate the isolated RNA prior to hybridization and membrane binding, depending on its size, for example by agarose gel electrophoresis. Thus, with the same amount of RNA of interest from each tissue sample, the amount of mRNA specifically labeled with the probe can be determined.

For further diagnosis, the polypeptide of the invention and the invention comprise, respectively. immunogenic portions thereof. This polypeptide, respectively. For example, portions thereof bound primarily to a solid phase, such as nitrocellulose or nylon, may be contacted in vitro with the body fluid to be examined, such as blood, for example, to react with the antibody. This antibody-peptide complex can then be detected, for example, with a labeled anti-human-IgG-antibody or anti-human-IgI-antibody. This labeling is, for example, an enzyme such as

is a peroxidase that catalyses the color reaction. Thus, the presence and amount of autoimmune antibodies present can be easily and rapidly detected by a color reaction.

Other diagnostics include 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 with, for example, an enzyme as described previously. Thus, the specific antibody-peptide complex can be detected easily and rapidly 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 (singly or in combination) and optionally a suitable additive or excipient and a process for preparing a fungicide for controlling harmful yeasts and harmful fungi combining a nucleic acid of the invention or a polypeptide of the invention with agriculturally acceptable ingredients and / or excipients.

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

A further object of the invention relates to an assay for identifying functional interactions, such as inhibitors or stimulators 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.

A suitable test for the identification of functional interactions, especially those that react with protein toxin ZYGOCIN according to SED ID No 2 in a sensitive yeast cell, is the so-called Two-Hybrid System (Fields, S. and Sternglanz, R. (1994) Trends in Genetics, 10, 286). In this assay, a cell, eg, a yeast cell, is transformed or transferred 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, eg, 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 reportergen, e.g., the LacZ gene from E. coli, Green Fluorescence Protein or yeast amino acid biosynthesis genes (AS-Biosynthesegene der Hefe) His3 or Leu2, which, through regulatory sequences such as IexA- the promoter / operator or the so-called Upstream Activation Sequence (UAS) controls the yeast. For example, an unknown polypeptide is encoded by a DNA fragment derived from a gene bank, such as 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 the trans. • • · «« «« «« «« «* * * * * * * * * * * * * * * ··· ·· ···· formed 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-activation domain, so that a fusion protein from an unknown polypeptide and the Gal4-transcriptional-activation domain is expressed in transformed yeast. The yeast transformed with both of these vectors, e.g. Leu2 ', also contains a nucleic acid encoding Leu2, 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 through 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. Green Fluorescence Protein ”-reportergen instead of the amino acid biosynthesis gene can be shown by activation of transcription in blue, resp. green fluorescing colonies. Peace blue respectively. green fluorescence can be easily quantified spectrophotometrically, for example in the case of a blue color at 585 nm.

In this way, the presence of polypeptides that react with a 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 agents, such as chemical compounds. This method also makes it possible to easily find a new chemically synthesizable active substance which can be used as therapeutics. Therefore, the present invention is directed not only to a procedure for screening for polypeptide interactions but also to a process for screening for substances that may react with the above-mentioned protein-protein complex. These peptide and chemical interactions are referred to within the meaning of the invention 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, eg by ultrafiltration and cation exchange chromatography and / or Laminarin-Sepharose affinity chromatography and / or Mannoprotein-Sepharose [see Example 1 and Appendix to Examples]. In the case of VICALTIN produced and secreted by DSM 12865 strain, further increase in toxin production can be achieved by adding vegetable (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 stimulates the formation of VICALTIN, which is induced by induction of transcription according to Northern analysis.

Synthetic B-medium can be used to form ZYGOCIN secreted DSM 12864 [see Radler et al.

1993].

The examples below 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 anti-Candida toxin VICALTIN from the killer-yeast H ⁷ culture supernatant. californica strain 3/57 Q) SM 12865)

Killer-toxin VICALTIN secreted by W. californica 3/57 killer-yeast showed an optimal inhibitory effect on sensitive yeast at pH 4.7 and 20 ° C in an methylene blue agar diffusion test (Methylenblau-Agar). In synthetic liquid medium, killer-yeast W. californica 3/51 show maximum toxin production when cultured in BAVC-medium (pH 4.7). To increase toxin production, the killer yeast was first incubated with shaking for 24 hours in 5 ml YEPD-medium at 30 ° C and then quantitatively transferred to 200 ml BAVC-medium and cultured again for 48 hours at 20 ° C on the 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, the cell-free supernatant at +4 ° C and 1 bar pressure was 200-fold concentrated by ultrafiltration on polysulfonic acid membranes (EasyFlow [fa.Sartorius]; exclusion limit / Ausschlussgrenze / 10 kDa) to a volume of 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 limit 10-20 kDa) against 5 mM citrate-phosphate buffer (pH 4.7). To store the toxin concentrate, the dialyzed preparation was sterile filtered through a 0.2 µm membrane and frozen in portions of 1 ml at -20 ° C.

Detection and calibration of toxin activity was performed in a methylene blue agar diffusion test (MBA;

i pH 4.7) against the sensitive indicator yeast Saccharomyces cerevzsiae 192.2d. Logarithmic dilution steps in 0.1 M citrate-phosphate buffer (pH 4.7) were prepared from the toxin concentrate and 100 µl each was pipetted into pre-punched holes (9 mm hole diameter) in an MBA plate inoculated with sensitive indicator yeast (2x10 6). $ 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 inhibition court diameter and the logarithm of the toxin concentration. To the diameter of the inhibition court (corrected for hole diameter) of 20 mm, toxin activity was 1 x 10 6 units / ml.

Purification of concentrated VITCALTIN toxin was performed either by cation exchange chromatography on Bioscale-S (FPLC) or by affinity chromatography on epoxy-activated Sepharose-68-Matrix (Pharmacia) to which the plant β-1,6-D-glucan pustulan was pre-bound . This toxin preparation, which showed a specific activity corresponding to the 625-fold enrichment (Table 1), appeared to be pure in gel electrophoresis and showed only separate discrete bands after SDS-PAGE (in a 10-22% gradient gel) at ca. 37 kDa, which was detected by both Coomassie-Blau blue (protein staining) and Schiff's periodic acid reagent (Perjodsáure-Schiffs-Reagent - PAS; carbohydrate staining). Positive PAS staining indicates potential N-glycosylation of arzti-Candida toxin VITCALTIN.

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 · »» «4 - 9» »4

Reaction of the purified toxin with endoglycases-H revealed that VITCALTIN contains an N-glycosidically linked carbohydrate moiety of about 3 kDa, which, due to its size in yeast, also points to a single N-glycosylation site in proteintoxin. Since deglycosylated VITCALTIN exhibits substantially reduced toxicity, it can be concluded that the saccharide portion of VITCALTIN 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 VITCALTIN toxin from supernatant of killer-yeast culture medium Williopsis californica [UF, ultrafiltration]

Preparation Volume [ml] Celk.protein [mg] Total toxin activity [Ε], Specific activity toxin [E / mg] Activity yield [%] Factor cleanup (Reini- gungsf.) Culture supernatant. media 10000 24600 7,9x10 ⁵ 3,2x10 100 ALIGN! 1 UF-retentate 50 162 6,3x10 ⁵ 3,9x10 ³ 80 122 Ivofil.dialyzát 25 45.8 3,1x10 ⁵ 6,8x10 ³ 39 213 Bioscale S (cation exchanger) 64 1,28 2,5x10 ⁴ 2,0x10 ⁴ 3.2 625

Example 2

Determination of NH ₂ -terminal amino acid sequence of VICALTIN 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 shown in Fig. 1, the N-terminus of VICALTIN exhibits significant homology to the amino-terminus of the endo-β-β-glucanase encoded by the yeast RG-2 gene.

Saccharomyces cerevisiae.

Based on this homology of VICALTIN and BgI2, 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 test using β-1,3-D-glucan laminarin as well as in the fluorescent test using substrate

4-methyl-umbelliferyl-β-D-glucoside (MUC), VICALTIN preparations showed significant β-1,3-D-glucanase activity; concomitantly tested β-1,6-D-glucan pustulan was not hydrolyzed by VICALTIN.

Example 3

Survival rate of yeast cells after treatment with VICALTIN in the presence and absence of cell wall glucan: competition analysis

Sensitive yeast cells of strain 5. cerevisiae 192.2d, which were cultured in liquid medium YEPD (pH 4.7) at 20 ° C in the presence of purified VICALTIN with an activity of 1x10 $ E / ml, showed killing kinetics as shown in Fig. 2. of plant β-1,6-D-glucan of 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 VICALTIN. Unlike pustulan, β-1,3-D-glucan laminarin did not show the ability to increase the survival rate of toxin-modified yeast cells (Figure 2).

It can be concluded that the action of VICALTIN requires binding to β-1,6-ϋ ^ 1η27 forged, which acts as the primary binding site (Andockstelle) (toxin receptors) on the yeast cell wall. Accordingly, yeasts cleaved at the chromosomal gene center (Genlocus) have been shown to have toxinresistant behavior and, once retransformed with the KRE1-bearing episomal vector, are again toxinsensitive (Fig. 3).

In Kre-mutants, the toxin resistance relies 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.

Example 4

Spectrum of efficacy and killing by VICALTIN

In the agar diffusion test, purified W. californica VICALTIN toxin showed significant yeast toxicity shown in Table 2. Except for three Candida crusei strains, all 22 clinical patient isolates studied as well as all other control strains of human pathogenic Candida species were demonstrated very effective destruction by VICALTIN. In 14 toxinsensitive yeast species from a total of 10 different genera, VICALTIN has shown an extremely wide spectrum of efficacy as a killer-toxin.

·· ··· ·· · ···

Table 2:

The spectrum of activity of VICALTIN on pathogenic and apatogenic yeasts of different genera. All strains were tested in an agar diffusion assay (MBA; pH 4.7) against purified VICALTIN. The activity of the applied toxin was in all cases 1 x 10 µE / ml. The strain C. tropicalis (patient no. 541965) came from the Institute of Forces of Medizinische Microbiologie und Hygiene from the University Clinic in Mainz.

Yeast strain Phenotype Diameter of inhibition 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 22nd K. marxianus C 8.1 R 0 Metchnikowia pulcherrima K / 3I B6 WITH 8 Pichia anomala WITH 17 P. farinosa R 0 P. jardinii R 0 P. klyveri ATCC 64301 R 0 P. membranaefaciens NCYC 333 R 0 Saccharomyces cerevisiae 192.2d WITH 30 381 WITH 23 ATCC 42017 (K1) WITH 19 Dec NCYC 738 (Killer) WITH 14 452 (NCYC 1006) WITH 16 Saccharomyces ludwigii 240 R 0 Schizosaccharomyces pombe CBS 1042 R 0 Sporothríx spec. 1129 WITH 11 Torulospora delbrueckii 208 WITH 18 T. pretoriensis WITH 10 Yarrowia lipolytica WITH 8 Zygosaccharomyces bailii WITH 23

• · * · · · · · ··· · · · · · · · · ·

Example 5

Cloning, sequencing and molecular characterization

VICALTIN-encoding VCT yeast V. californica strain 3/57 (DSM 12865)

Based on the N-terminal amino acid sequence of VICALTIN, specific DNA-oligonucleotides were prepared which led to identification and cloning as well as molecular-biological characterization of the chromosomally localized toxin of WCT. The WCT DNA sequence (SEQ ID No. 1) shows a single open reading frame that encodes a potentially N-glycosylated protein of 309 amino acids and a calculated molecular weight of 34,017 Da.

Monitoring of the efficacy of WCT-coded killer-toxin has shown that VICALTIN is an extremely toxic yeast to the yeast, the primary target of which is the β-1,3-D-glucans found in the yeast cell wall . Its selective toxicity to yeasts and fungi is that VICALTIN disrupts the structure and / or integrity of the cell wall in a sensitive target cell, thus attacking the yeast at its most sensitive site and thereby destroying it.

Example 6

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

Of the Z. bailii yeast strain 412 was from the supernatant of the killer-yeast culture medium according to the method described by the method described above.

• · · · · · · · · · ·

Radler et al. (1993) isolated the virus-encoded killer-toxin ZYGOCIN, which was further concentrated by ultrafiltration and then purified by affinity chromatography. The one-step purification of ZYGOCIN described in the cited study utilizes the natural affinity of the toxin to the cell wall mannoproteins of sensitive yeast. According to the method of Schmitt and Radler (1997), isolated and partially purified mannoprotein from S. cerevysia strain 192.2d was covalently bound to an epoxyactivated Sepharose-6B-matrix (Pharmacia) and used for FPLC toxin purification by column chromatography. According to SDS-PAGE, this 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

The activity spectrum of viral ZYGOCIN from yeast Z. bailii 412 (DSM 12864), determined in the agar diffusion test, includes pathogenic and apatogenic yeast genera of which Candida albicans and Sporothrix schenkii are feared as causative agents in humans and animals and Ustilago maydis and Debaromyces hansenii as harmful yeasts in agriculture and food industry (tab.3) · · ····· · · ····· · · ····· · · · · ··· * · É · · · · · · ·

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 &lt; 6 &gt; 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 ++

Example 8

Cloning and sequencing of ZYGOCIN-coding

ZBT-gemi (ZBT) yeast Z.bailIi strain 412 (DSM 12864) cDNA-synthesis of the toxin-coding double-stranded RNA genome of the z.bailIi 412 was performed according to the method described by Schmitt with purified and methylmercuryhydroxide denatured M-dsRNA as a matrix and with various hexanucleotides as primers. After ligation into the EcoRI-restricted pUC18 vector, transformation into E. coli, and 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 having a potential Kex2-endopeptidase cleavage site at the amino acid position of RR. In vivo Kex2-mediated conversion of Pro-ZYGOCIN in the late Golgi stage results in 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 ZUT-cDNA in yeast S. cerevisiae, 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 Schizosaccharomyces pombe, since - as already mentioned by the viral K28-toxin example - these dividing yeast are particularly suitable for the expression and secretion of foreign proteins.

Example 9

Toxin expression of WCT and ZBT in transgenic plants

Since the described killer toxins VICALTIN and ZYGOCIN have a broad spectrum of activity and also kill phytopathogenic yeasts and fungi, it should be possible to prepare such transgenic plants which are resistant, for example, to maize infection caused by the pathogen Ustilago maydis. Similar experiments have already been performed with tobacco plants which have the ability to secrete upon heterologous expression of the naturally virally encoded killer toxin KP4 from U. maydis.

Příslušný «« příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný příslušný k příslušný příslušný příslušný příslušný příslušný toxin and thus 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, cloned WCT and ZBT toxingenes could be cloned into so-called bi-directional pBI vectors (Clontech) and used to obtain transgenic plants. The respective WCT and ZBT toxingenes were under transcriptional control of the potent cauliflower mosaic virus (CaMV-P) promoter. The construction of the constructed vectors is shown schematically in FIG. 5.

Example 10

Heterological expression of the VICALTIN-coding WCT gene of W. californica 3/57 (DSM 12865) in S. cerevisiae

For heterologous expression of the WCT gene in S. cerevisiae yeast, the VICALTIN-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 toxin under the transcriptional control of its own yeast triose phosphate isomerase promoter (ΤΡΓ), thus allowing the constitutive expression of VITCALTIN to be performed after transformation in yeast (S. cerevisiae). The gel electrophoresis of the culture medium supernatant in the yeast transformants thus obtained showed that recombinant VITCALTIN secreted into the culture medium and showed β-1,3-D-glucanase activity (Fig. 6) corresponding to homologous VITCALTIN (from wild strain DSM 12865).

»♦ *........

Example 11

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

Since the dividing yeast, both in the form of an intact cell and as a cell wall spheroplast, are resistant to VITCALTIN 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 accultivation medium, a vector (ρΤΖ / ατ; Figure 7) has been constructed to carry a functional signal for secretion and processing in S.pombe (S / P), which originates from the cDNA of the viral K28-preprotoxin of yeast S. cerevisiae [see Schmitt, 1995; Schmitt and Tipper, 1995]. This secretion and process signal ensures that the always in-frame foreign protein in the yeast divider is imported into the lumen of the endoplasmic reticulum and thus enters the yeast secretory pathway. Through the Kex2p-cleavage site at the C-terminus of the S / P region, the desired foreign protein in the late Golgi space in the cells (in the Golgi-Compartiment) is cleaved by its own yeast Kex2p-endopeptidase from its intracellular transporter (Transport-Vehikel). finally secreted as a biologically active protein (ZYGOCIN and / or VITCALTIN) into the external culture medium.

Example 12

Comparison of biological activity of purified VITCALTIN and topical antifungal agents Clotrimazole and Miconazole 4

Since purified VITCALTIN exhibits a broad spectrum of activity and effectively kills even human pathogenic yeast and / or fungi, it is also of great importance as a potential antifungal agent. Therefore, studies have been conducted comparing VITCALTIN with the very commonly used topical antifungal agents Clotrimazole and Miconazole. Initially, the toxic effects of Clotrimazole and Miconazole were tested in the MBA-agar diffusion test using 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 μΐ each in an MBA test at concentrations ranging from 0.1 to 10 mg / ml. The diameter of the inhibition court (Hemmhof) was 10 at the amount used

- 50 µg Clotrimazole between 12 and 32 mm. A 100 µg / ml stock solution in DMSO (100%) was prepared from Miconazole and monitored in the same way as Clotrimazole for biological activity against Sporothrix spec. Use 0,08

- 0.3 µg of Miconazole resulted in Bioassay inhibition yards with a diameter of between 22 and 36 mm. Biological activity of 10 pg of Clotrimazole resp. 0.08 g of Miconazole corresponded to the toxicity of 2 g of purified VICALTIN. Comparison of the three test compounds, based on their molecular weight, shows that VICALTIN already exhibits the same activity as 0.2 pmol of Miconazole and 29 pmol of Clotrimaziol at a concentration of 0.07 pmol; that is, VICALTIN is highly effective as an antifungal agent (Fig. 8).

Example 13

Detection of the VICALTIN-encoding UCT gene of W. californica 3/57 (DSM 12865) by Southern hybridization with a gene-specific DNA probe

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

The results of the agarose gel electrophoresis shown in FIG. 9 and the corresponding Southern-Blot analysis undoubtedly demonstrated that the VICALTIN-encoding WCT gene could be detected using a nucleic acid probe.

Example 14

Northern blot analysis to demonstrate transcriptional induction of the VICALTIN-coding UCT gene of Williopsis californica 3/57 (DSM 12865) with β-1,3-D-glucan

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 containing 0.03% natural β-1,3-D-glucan laminarin for 48 hours at 20 ° C with gentle shaking (60 rpm) and was gradually used at various intervals to prepare total RNA . All samples (10 ml) were adjusted to the same number of cells before RNA isolation

Q

1.8 x 10 cells / ml and then these samples were electrophoretically separated in denaturing agarose-formaldehyde gels. As shown in FIG. 10, both the non-induction conditions (BAVC medium without addition) and the laminarin-enriched BAVC medium showed that the UCT transcript was 1,100 bases in size. Maximum glucose expression was achieved without the addition of glucan • ·

WCT towards 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 HUT transcript showed significantly greater intensity after 10 hours than in the non-inducing culture medium, suggesting that this transcription of the VICALTIN encoded WCT gene could 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-jectate 20 g / l trinatriumcitrát 0.5 g / l (nh ₄ ) ₂ Sat ₄ 1.5 g / l MgS0 ₄ 1.0 mg / l myoinosit 0.04 g / l amino acid stock solution (10x) 200 ml / l trace element basic solution (100x) 10 ml / l Vitamin Essential Solution (100x) 20 ml / l

b) amino acid base 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 serine threonine tryptophan 0.4 g / l 0.5 g / l 2 g / l 0.4 g / l c) trace element basic solution (100x) boric acid 200 mg / l FeCl ₃ .6 H ₂ 0 200 mg / l ZnSO ₄ .7 H ₂ 0 200 mg / l aici ₃ 200 mg / l CuSO ₄ .5 H ₂ 0 100 mg / l Na ₂ Mo0 ₄ .2 H ₂ 0 100 mg / l li ₂ Sat ₄ .h ₂ o 100 mg / l KJ 100 mg / l potassium hydrogen tartrate 2 g / l

d) vitamin solution (100x)

4-Aminobenzoic acid 20 May mg / l biotin 2 mg / l folic acid 2 mg / l nicotinic acid 100 ALIGN! mg / l pyridoxol hydrochloride 100 ALIGN! mg / l riboflavin 50 mg / l thiaminium dichloride 50 mg / l Ca-D-hinge othenate 100 ALIGN! mg / l

Biotin: was dissolved in a solution of 5 g KH ₂ PO ₄ in 50 ml distilled water · · · ot 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 • · · · · ··· ·· ····

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

WCT-encoded protein toxins VICALTIN from yeast Williopsis californica strain 3/57.

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

Fig. 1: N-terminal amino acid sequences of W.

californica-toxin VICALTIN and endo-β-1,3-glucanase BgI2 from yeast S. cerevisiae. Individual deviations of otherwise identical sub-sequences are printed in bold (BgI2p-sequences as reported by Klebl and Tanner, 1989).

Figure 2: Kinetics of S.cerevisiae sensitive yeast cells 192.2 by VICALTIN in the presence of (2a)

and the absence (2b) of laminarin (L) and pustulan (P) β-D-glucan. 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.

Giant. 3 (a, b, c, d): Agar diffusion test for the detection of sensitivity / resistance to WICALTIN in Krel Krel ⁺ and ^- strains of the yeast S. cerevisiae. After transformation of the VICALTIN-resistant Cre-null mutant S. cerevisiae SEY6210 [krel} with the KRE1-carrying vector pPGK [KR £7], full sensitivity to VICALTIN was restored.

Giant. 4: (A) Gel electrophoresis (SDS-PAGE) of ZYGOCIN produced and secreted by yeast Z. bailii strain 412 (DSM 12864) after affinity chromatography on mannoprotein-Sepharose. (B) An agar diffusion assay to demonstrate the biological activity of purified ZYGOCIN killer-toxin.

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

[Explanation: RB, LB: right and left border sequences of natural TI plasmids from Agrobacterium tumefaciens; CaMV-P: Cauliflower Mosaic Virus 35S promoter; NOS-P, NOS-T: nopaline synthase transcriptional promoter and terminator; kan: kanamycm-resistant Streptococcus gene for selection in E. coli; NPT-II:

neomycin phosphotransferase gene from transposon Tn5 for selection in plant].

Giant. 6:

(A) Partial restriction card of the episomal vector pSTH2 for heterologous expression of VICALTIN-encoding WUT toxin in yeast Saccaromyces cerevisiae. The vector pSTH2 is a commercially engineered plasmid

of the available 2μ Multi-Copy pYX242 vector, 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 VICALTIN upon transformation in S. cerevisiae.

(B) Gel electrophoresis (SDS-PAGE; 10-22.5% gradient gel) of concentrated culture medium supernatant

S. cerevisiae after transformation with engineered

VICALTIN-expression vector pSTH2 (record / Spur / 1).

VICALTIN heterologously expressed in S. cerevisiae is indicated by an arrow.

(C) Detection of extracellular β-1,3-glucanase activity of S. cerevisiae yeast after transformation with VICALTIN-expressing yeast vector pSTH2. To determine exo-β-β-glucanase activity, yeast colonies grown on non-leucine SC-agar were sprayed with 0.04% 4-methylumbelliferyl-4-D-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 MUG-hydrolysis.

[Explanations: 1 and 4, S. cerevisiae transformed with a vector (pEP-VCT) that expresses the VICALTIN-encoding UCT gene under the action of its own promoter; 2, wild-type W. californica 3/57 yeast (DSM 12865); 3. wild yeast W. californica 3/111; 5, S. cerevisiae after transformation with the VICALTIN-expressing vector pYX-VCT; 6, S. cerevisiae transformed with base vector pYX242 (no toxingen)].

Figure 7: Schematic structure of the vector pTZ α / τ for heterologous expression and secretion of foreign proteins (especially VICALTIN and ZYGOCIN) in dividing yeast Schizosaccaromyces pombe.

[Explanation: T _nnrt i, transcriptional promoter and terminator of the nmt1 gene of the dividing yeast S. pombe regulated by thiamine; S / P secretory and process sequences of viral K28-preprotoxin of budding yeast S. cerevisiae, arsl, auto-replicating sequences from chromosome 1 of dividing yeast; leu2, a leucine-2-marker gene for the selection of leucine-prototrophic transformants S.pombe].

Figure 8: comparison of biological activity of purified VICALTIN, 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.

Giant. 9: Detection of the VICALTIN-coding WCT-yeast gene

W. californica 3/57 (DSM 12865) cloned in pSTH1 (derivative of pBR322) by agarose gel electrophoresis (A) and Southern hybridization with a DIG-labeled WCT probe (B).

[Legend: DIG-labeled DNA-length standard II;

Entry 1, pSTH1 restricted with EcoRI and Sa / I; record 2, DNA-marker Smart-ladder]

Giant. 10: Northern analysis of transcriptional induction

VICALTIN-coding WCT gene of W. californica 3/57 (DSM 12865) under non-inducing culture conditions in BAVC-medium (A) and under inducing conditions in BAVC-medium ₉ · ₉ · I · 4 · 4 · Add 0.03 % laminarin (B). Electrophoretic separation of total RNA isolated from strain DSM 12865 was performed in denaturing agarose / formaldehyde gel at a constant voltage (7V / cm). RNA was hybridized on a nylon membrane against a VICALTIN-specific DIG-labeled probe (630 bp) and detected by chemiluminescence.

[Legend: M, DIG-labeled DNA-length standard I; records 1-8 correspond to sampling times * for total RNA isolation; 1, 10 h, 2, 15 h, 3, 19 h, 4, h, 5, 33 h, 6, 38 h, 7, 43 h, 8, 48 h.

Abbreviations used in the text

VCT Villiopsis Californica Toxin

ZBT Zygosaccharomyces Bailii Toxin

ZYGOCIN name; toxin secreted from DSM 12864

VICATIN name; toxin secreted from DSM 12865

The following microorganisms used according to the present 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 Veg lb, 38124 Braunschweig, Germany, according to the Budapest Treaty on International Recognition (number of deposit, date of deposit):

Wi lliopsis californica strain 3/57 (DSM 12865) (09/06/1999) Zygosaccharomyces bailii strain 412 (DSM 12864) (09/06/1999)

Claims (26)

Proteintoxins obtainable from Milliopsis californica and / or Zygosaccharomyces bažii. Protein toxins according to claim 1, obtainable from DSM 12864 and / or DSM 12865. Proteintoxins according to claims 1 and 2, characterized in that they have antifungal and / or fungicidal effects. Proteintoxin according to any one of claims 1 to 3 with glucanase activity. Proteintoxin according to claim 4, characterized in that it binds to β-1,6-D-glucan and exhibits β-1,3-D-glucanase activity and / or β-1,3-D-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 N-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 according to claims 1 to 5 and 13, characterized in that the nucleic acid according to 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. A method for producing an antibody according to claim 15, wherein the mammal is immunized with the polypeptide of claim 7 and the optionally formed antibody is isolated. A medicament comprising 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, 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 Candida mycosis, characterized in that the nucleic acid according to any one of claims 6 to 10 or the polypeptide according to any one of claims 1 to 5 and 13 or the antibody of claim 15 is combined with a pharmaceutically acceptable excipient or excipient. 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. 20. A method of preparing a diagnosis for the diagnosis of mycoses, such as the mycosis, such as 9; superficial, cutaneous and subcutaneous dermatomycosis, mucosal mycosis and systemic mycosis, in particular Candida mycosis, characterized in that the nucleic acid according to any one of claims 6 to 10 or the polypeptide according to any one of claims 1 to 5 and 13 or the antibody according to claim 15 combined with a pharmaceutically acceptable carrier. An assay for identifying functional interactions comprising 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 and optionally suitable excipients 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 23 10 for variant searches, characterized in that the gene bank is searched by the nucleic acid and the variant found is isolated. Use of the polypeptide of 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 process is carried out in synthetic B- and / or BAVC-medium. Use of nucleic acids according to any one of claims 6 to 26 11 for the preparation of transgenic plants and plant cells.