KR20020059581A - Novel antifungal agents and fungicides, method for the production thereof and their use - Google Patents

Abstract

The present invention relates to a process for the production of protein toxins from yeast (commonly referred to as killer yeast) using genetic techniques to control yeast and / or fungi in hospitals for humans and plants, where yeast and / ). ≪ / RTI > High specificity allows the use of protein toxins as antifungal drugs and / or fungicides. In addition, these protein toxins can be used for crop protection.

Description

TECHNICAL FIELD [0001] The present invention relates to antifungal drugs and fungicides, methods for producing the same, and uses thereof.

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

Selective antifungal drugs are very important because fungal and / or yeast infections are increasing in humans in recent years and consistently cause undesirable contamination of food and animal feed. Mycosis has particularly important consequences in immunosuppressed patients where cellular and body defense systems are maintained at a level where they are not fully functional (Anaissie, 1992; Meunier et al., 1992; Wingard, 1995]. Patients who are extremely endangered by mycosis are those who are infected with HIV-1 (AIDS), which frequently die at the end of opportunistic infections caused by hospital-acquired fungi and / or yeast to humans (Levy, 1993). Antifungal drugs currently used to treat such infections (such as amphotericin B, fluconazole, itraconazole, and ketoconazole) cause significant side effects because they destroy the structural integrity of the eukaryotic membrane and damage the infected host organism : Hector, 1993]. In addition, when conventional antifungal drugs are applied, they quickly increase fluconazole resistance rapidly diffusing in microorganisms that are pathogenic to humans and increase conventional problems (Cameron et al., 1993; Chavenet et al., 1994; Maenza et al., 1996; Pfaller et al., 1994; Rex et al., 1995; Troillet et al., 1993]. Thus, it is becoming important to develop antifungal drugs that are distinguished by high selectivity, such as bacterial antibiotics, and possibly attacking only hospital-acquired fungi and yeast in humans. However, the development of " specific antifungal antibiotics " has so far failed because most of the cellular processes in higher organisms are dominated by gene products that exhibit a high degree of functional homology in eukaryotic cells (Kurz, 1998; Komiyama et al., 1998].

The target of the selective antifungal drug is β-1,3-D-glucan of yeast cell wall which is essential for the mechanical stability and osmotic stability of the cell but does not occur in the higher eukaryotic cell and constitutes the "Achilles tendon" (Roemer et al., 1994). Materials that are selectively involved in the cell wall structure of yeast and fungi are of great interest, but no antibiotic-like inhibitor has been used to control mycosis. Bacterial antibiotic producers were first discovered early in the 20th century, but similar effects in yeast were observed in the early 1960s by identifying so-called killer yeast (Bevan & Makower, 1963), bacillus subtilis The toxin-producing killer strain of Saccharomyces cerevisiae produces and secretes a protein named " killer toxin " that destroys the susceptible yeast in a receptor-dependent process (Bussey, 1991; Tipper & Schmitt, 1991]. In the case of Saccharomyces cerevisiae, the ability of the toxin to produce is based on the infection by a virus of the double-stranded double-stranded RNA virus, which stably maintains a high copy number in the yeast cytoplasm without significantly impairing eukaryotic host cells (Tipper & Schmitt, 1991). The three killer toxins (K1, K2 and K28) of yeast Saccharomyces cerevisiae, which have been known to date, are detoxified by the infected cells as high molecular weight pre-pro-toxins and processed by complex transformation during the intracellular secretory pathway Glycosylated < RTI ID = 0.0 > a / -heteroamer < / RTI > (Hanes et al., 1986; Dignard et al., 1991; Schmitt & Tipper, 1995]. The toxic effect of toxin on Saccharomyces cerevisiae is based on disruption of membrane integrity (toxins K1 and K2) or on the interruption of the cell cycle (as in the case of killer toxin K28) due to direct inhibition of DNA synthesis [ See Bussey, 1991; Schmitt & Compain, 1995; Schmitt et al., 1996]. Although the killer toxins of the K1, K2 and K28 classes are significantly different from each other in terms of their mode of action and physical properties, they share the property of significantly narrowing susceptible yeast species closely related to their narrow range of action. This limited range of action is based on the fact that the thus characterized brewer's yeast killer toxin must interact with different receptor populations at yeast cell wall and cytoplasmic membrane levels to destroy sensitive target cells. The major toxin receptor on the yeast cell wall is the highly branched β-l, 6-D-glycan or the external mannose side chain of the cell wall mannoprotein (Bussey, 1991; Schmitt & Radler 1987, 1988].

Apart from the virus protein toxins of Hanseniaspora uvarum, Zygosaccharomyces bailii and Ustilago maydis in Saccharomyces cerevisiae, which is a yeast, Killer strains may be selected from the group consisting of Debaryomyces, Hansenula, Cryptococcus, Rhodotorula, Trichosporon, Pichia, Kluyveromyces spp., Torulopsis spp. And Williopsis spp. (McCracken et al., 1994; Park et al., 1996; Schmitt & Neuhausen, 1994; Walker et al., 1995]. However, in these yeasts, the gene base of the killer phenomenon is not a viral genome, but a linear dsDNA plasmid or a chromosomal yeast gene (Schrunder et al., 1994).

An intensive study of the molecular biology of the "killer yeast" that produces various toxins has revealed that the secretion of toxic proteins ('killer toxins') constitutes the development potential of selective antifungal drugs that should not be underestimated and spread in yeast Walker et al., 1995; Hodgson et al., 1995; Polonelli et al., 1986; Schmitt & Neuhausen, 1994; Neuhausen & Schmitt, 1996; Schmitt et al., 1997], so far it has not been possible to provide such a protein toxin.

Accordingly, it is an object of the present invention to provide a suitable antifungal or fungicide protein toxin for the treatment of hospitalized yeast and / or fungi against humans and plants.

Surprisingly, the killer toxin WICALTIN (also a protein toxin) from the wild-type yeast Willoopsis californica strain 3/57 (DSM 12865), which is produced and secreted in a highly efficient manner, and the yeast Gosaka saccharomyces bailei (DSM 12864 ZYGOCIN (also a protein toxin) from E. coli was found to be particularly suitable for controlling hospitalized yeasts and / or fungi for humans and plants. It can also destroy dangerous fungi and harmful yeasts in the food and animal feed sector. Thus, the above two protein toxins can be used as antifungal drugs and / or fungicides for controlling yeast and / or fungal infections, especially mycosis. Such attempts are evidenced in the present invention as a study of the mode of action. The toxin gene is cloned and sequenced in a manner suitable for the purposes of the present invention, thus establishing recombinant production and overexpression of gastricin and gigantin in the culture.

Accordingly, the subject of the present invention relates to a protein toxin obtainable from Willy opsys californica, particularly preferably strain DSM 12865, and Gigus saccharomyces bailei, particularly preferably strain DSM 12864. These two strains were deposited in the Deutsche Jahrung von Mikro Organism Management Challenge Tunnel German Beha (DSMZ) (www.dsmz.de), Maheshlöderbeck 1, Braunschweig, 38124, Germany, under the Budapest Treaty, on June 9, 1999 Was deposited.

In particular, for the purposes of the present invention, DSM 12864 (see Examples 4 and 7), which secretes a biologically potent protein toxin that destroys a large number of yeast and fungi in hospitals for humans and plants, And DSM 12865. Thus, the present invention relates to selective antifungal drugs or fungicides in terms of protein toxins, particularly in the functional units of toxin genes, the polypeptides according to the invention and their encoded nucleic acids according to the invention, Is an efficacious herb that entirely destroys yeast and / or fungi due to mediator production and is harmless to higher eukaryotes (and also to humans and mammalian cells) and plants, preferably crops (Pfeiffer et al. 1988].

Non-pathogenic or hospital-grown yeast and / or fungi may then be selectively destroyed for humans and plants:

Candida albicans, Candida krusei, Candida glabrata, Candida vinii, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Hanseniaspora uvarum, Kluyveromyces marxianus, Methschnikowia pulcherrima, Ustilago maydis, Debaryomyces hansenii, and the like. , Pichia anomala, Pichia jadinii, Pichia membranefaciens, Yarrowia lipolytica and Zygosaccharomyces rouxii (Zygosaccharomyces rouxii) ).

Candida tropicalis, Candida tropicalis, Kluyveromyces lactis, Methisnycopia cherima, Pichia, Candida albicans, Candida albicans, Candida albicans, Candida tropicalis, Anthomolas, Pichia jadini, Saccharomyces cerevisiae, Sporthrix spec., Torulaspora delbrueckii, Torulaspora pretoriensis, Yarorospora spp. Wai Lipotrica and Gigi Sakaro Maisse Bailey.

The particularly high activity of the gastrin-producing yeast strain DSM 12865 appears to be based on a more pronounced secretion efficacy compared to other strains of the same yeast species. 'Killer' property of being new generation yeast DSM 12864 strain was toxin that allows getting persists stably in the cytoplasm by the number of high copy of the art yeasts (DSM 12864 strain) can be secreted by creating put encryption double-stranded RNA virus (M _Zb -dsRNA) (Schmitt & Neuhausen, 1994). Other strains of the same species exhibit non-toxic production as they do not contain the toxin-encoding dsRNA virus in the cytoplasm and the phenotype is classified as a 'non-killer'.

Thus, another subject of the present invention is a nucleic acid encoding a protein toxin, or a functional variant thereof, comprising an amino acid sequence and a glucanase activity according to SEQ ID NO: 1 and SEQ ID NO: 2, and a nucleic acid having a nucleotide of 8 or more, 15 or 20 or more, particularly preferably 100 or more, more particularly preferably 300 or more (hereinafter referred to as " nucleic acid according to the present invention ").

The complete nucleic acid encoding the protein toxin has, after intracellular processing and secretion, a 309 amino acid size and a molecular weight of 34 kDa (SEQ ID NO: 1) or a 99 amino acid size and a molecular weight of 10 kDa (SEQ ID NO: 2). Expression of the nucleic acid according to SEQ ID NO: 1 in yeast Saccharomyces cerevisiae produces a recombinant gastricin, which is a glycosylated protein with marked? -1,3-D-glucanase activity, Lt; / RTI > (Example 10). A further experiment according to the present invention is that the nucleic acid according to the invention is a nucleic acid encoding a protein toxin having glucanase activity in the case of SEQ ID NO: 1 and in the case of SEQ ID NO: 2 it is presumably O-glycosylated in vivo Is a nucleic acid that encodes a protein toxin called < RTI ID = 0.0 > Nucleic acids according to the present invention can be obtained from DSM 12865 (SEQ ID NO: 1) and DSM 12864 (SEQ ID NO: 2).

In a preferred embodiment, the nucleic acid according to the invention comprises a DNA or RNA, preferably a double-stranded DNA, and in particular a nucleic acid sequence according to 1 to 947 of SEQ ID NO: 1 and a nucleic acid sequence according to 1 to 713 of SEQ ID NO: Lt; / RTI > According to the invention, the two positions determine the starting and terminating amino acids of the coding region, in each case the first and last amino acids of the corresponding reading frame.

In the present invention, the term " functional variant " should be understood to mean a nucleic acid functionally associated with a nucleic acid according to the present invention. Examples of related nucleic acids are nucleic acids from different yeast cells or strains and cultures or allelic variants. The present invention also encompasses nucleic acid variants that can be derived from a variety of yeast / yeast strains or other pathogens (e.g., dermatophytes and fungi) (according to the DHS system).

Furthermore, the term " variant " according to the invention refers to a nucleic acid which exhibits homology, in particular about 60%, preferably about 75%, particularly preferably about 90% and more particularly about 95% . A fragment of a nucleic acid according to the present invention can be used, for example, as a probe for further identifying a functional variant or as an antisense nucleic acid to produce an individual epitope. For example, nucleic acids having about eight nucleotides or more are suitable as antisense nucleic acids, nucleic acids having about 15 nucleotides or more are suitable as primers in the PCR method, and nucleic acids having about 20 or more nucleotides are suitable for identifying additional variants And nucleic acids having about 100 or more nucleotides are suitable as probes.

In a further preferred embodiment, the nucleic acid according to the invention comprises at least one non-coding sequence and / or a poly (A) -sequence, at least one Kex2p endopeptidase recognition sequence (required for intracellular protein processing) N-glycosylation site. An unencrypted sequence is a promoter or enhancer sequence for regulating the expression of a regulatory sequence, e. G., A cognitive toxin gene containing a nucleic acid according to the invention.

In a further aspect, the nucleic acid according to the invention is contained in a vector, preferably an expression vector, or a vector effective for gene therapy.

An example of an expression vector may be a prokaryotic and / or eukaryotic expression vector in the case of a nucleic acid according to SEQ ID NO: 2, and a eukaryotic expression vector in the case of a nucleic acid according to SEQ ID NO: 1. The expression of the toxin-encoding nucleic acid according to SEQ ID NO: 1 in Escherichia coli is not possible because the heterologously expressed protein toxin is toxic to bacterial cells. this. In E. coli, the cloning of the chiculotin encoding nucleic acid according to SEQ ID NO: 1 is possible only by plasmids that do not contain a promoter (e. G., Using a derivative of the plasmid pBR322). An example of a prokaryotic cell vector that heterologously expresses the luciferin-encoding nucleic acid according to SEQ ID NO: 2 is glutathione S transferase / Lt; RTI ID = 0.0 > pGEX-4T-1 < / RTI > this. For example, the T7 expression vector pGM10 (Martin, 1996), which encodes an N-terminal Met-Ala-His6 tag and is expressed through a Ni2 ⁺ -NTA column The protein is advantageously purified. An example of a suitable eukaryotic expression vector for expression in Saccharomyces cerevisiae is the vector p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767] and examples of expression vectors suitable for expression in insect cells are baculovirus vectors such as those described in EP 0 127 839 or EP 0 549 721, An example of an expression vector suitable for expression in a cell is the SV40 vector, which is freely available.

In general, the expression vector may also contain a regulatory sequence suitable for the host cell, e. A trp promoter for expression in E. coli (e. G., EP 0 154 133), an ADH-2 promoter for expression in yeast (Russel et al., 1983, J. Biol. Chem. 258, 2674), baculovirus polyhedrin (e.g., EP 0 127 839) for expression in insect cells, or early SV40 promoter or LTR promoter such as the promoter of MMTV (see Mouse Mammary Tumor Virus; Lee et al., 1981, Nature, 214, 228).

An example of a vector suitable for gene therapy is a viral vector, preferably an adenoviral vector, particularly preferably a replication deficient adenoviral vector, or an adeno-associated viral vector, for example, with only two inserted terminal repeat sequences (ITRs) Lt; / RTI > associated viral vector.

Suitable adenoviral vectors are described in McGrory, 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 WO95 / 00655.

Examples of suitable adeno-associated viral vectors are described in Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol. 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.

Vectors effective for gene therapy can also be obtained by complexing nucleic acids according to the invention with liposomes. Lipid mixtures suitable for this purpose can be found 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. & Huang, L. (1991) Biochim. Biophys. Acta < / RTI > 1189,195. When making liposomes, the DNA is bound by ions on the surface of the liposome at a rate at which the total positive charge remains and the DNA is fully complexed by the liposomes.

In a further embodiment, the nucleic acid according to the invention is contained in a vector, preferably an expression vector, to produce a transgenic plant. The above-mentioned killer toxin gastric and gigasin have broad action and destroys hospitalized yeast and fungi for the plant. Therefore, the transgenic plants that are resistant to infection by the host adult pathogens, Plants can be provided. Similar experiments have already been performed on tobacco plants, which are due to the heterotypic expression of the naturally encoded maize kills and the maize killer toxin KP4 by the virus, which can secrete the protein toxin, Increased specific protection from infection by strains (Park et al., 1996; Kinal et al., 1995; Bevan, 1984]. Starting from a commercial transformation system based on a modified derivative of the native Agrobacterium tumefaciens Ti plasmid, the nucleic acid according to the invention, described as the toxin genes WCT and ZBT, is usually an anisotropic pBl vector (CLONTECH ) To be used in the production of transgenic plants. To do this, the respective toxin genes WCT and ZBT are placed under the transcriptional control of the strong cauliflower mosaic virus promoter (CaMV-P). A more detailed structure of the vector to be constructed is schematically shown in Example 9.

For example, the nucleic acid according to the present invention can be obtained by using the sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 or the peptide sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 with reference to the genetic code, (Uhlman, E. & Peyman, A. (1990) Chemical Reviews, 90, 543, p. 4]. Another possibility of obtaining a nucleic acid according to the present invention is to isolate a suitable gene bank using a suitable probe (Sambrook, J. et al. (1989) Molecular Cloning. A laboratory manual. 2nd Edition, Cold Spring Harbor, New York]. Suitable probes include, for example, single stranded DNA fragments of about 100 to 1000 nucleotides in length, preferably about 200 to 500 nucleotides in length, particularly preferably about 300 to 400 nucleotides in length, wherein the sequence is SEQ ID NO: 1 and SEQ ID NO: 2 Can be deduced from the nucleic acid sequence according to < RTI ID = 0.0 >

Another object of the present invention is a polypeptide or a functional variant thereof such as an amino acid sequence according to SEQ ID NO: 1 and SEQ ID NO: 2, and an amino acid sequence having 6 or more, preferably 12 or more, particularly preferably 65 or more Preferably at least 309 (SEQ ID NO: 1) and 99 (SEQ ID NO: 2) (hereinafter referred to as "polypeptides according to the present invention"). For example, a polypeptide having an amino acid length of about 6 to 12, preferably about 8, contains an epitope that is used to generate a specific polyclonal or monoclonal antibody after binding to a support [ See U.S. Patent No. 5,656,435). Polypeptides having an amino acid length of about 65 or more can also be used directly in the manufacture of polyclonal or monoclonal antibodies without support.

For purposes of the present invention, the term " functional variant " should be understood to mean a polypeptide functionally associated with the peptide of the present invention, i.e., exhibiting glucanase activity. In addition, variants are understood to refer to allelic variants or polypeptides (depending on the DHS system) that can be derived from various yeast / yeast strains or other infectious agents, for example, dermatophytes or fungi.

In a broader aspect, they have a homology of about 70%, preferably about 80%, particularly preferably about 90%, more preferably about 90% sequence homology with a polypeptide having the amino acid sequence shown in Figure 2 ≪ / RTI > is about 95%. The term also includes polypeptides in which about 1 to 60, preferably about 1 to 30, particularly preferably about 1 to 15, more particularly about 1 to 5 amino acids have been deleted in the region do. For example, the first amino acid methionine can be absent without significantly altering the function of the polypeptide. In addition, it also comprises a fusion protein containing the above-described polypeptide according to the invention, which fusion protein itself has glucanase function or can only acquire a specific function after the fusion protein has been separated off have. In particular, it includes fusion proteins, which include in particular about 1 to 200, preferably about 1 to 150, particularly preferably about 1 to 100, more particularly about 1 to 50 non-human amino acids Lt; / RTI > Examples of non-human peptide sequences include, for example, Coli galactosidase or a so-called histidine tag, for example, a prokaryotic peptide sequence from the Met-Ala-His ₆ tag. Fusion proteins with so-called histidine tags are particularly advantageous for the purification of expressed proteins via metal ion containing columns, for example, Ni ²⁺ -NTA columns. &Quot; NTA " means the chelator nitrilotriacetic acid [Qiagen GmbH, Hilden]. In this regard, the invention also encompasses polypeptides according to the invention which are blocked as free-drugs in terms of pro-protein, or in a broader sense.

A portion of the polypeptide according to the present invention means, for example, an epitope that the antibody specifically recognizes.

The polypeptides according to the invention are prepared by methods generally known to those skilled in the art, for example by expressing the nucleic acid according to the invention in a suitable expression system as generally described above. Suitable host cells suitable for the production of precisely processed and viable protein toxins are exclusively eukaryotic organisms, preferably germinated Saccharomyces cerevisiae and Schizosaccharomyces pombe fission yeast.

Particularly, a portion of the above-mentioned polypeptides can also be synthesized using the conventional peptide synthesis method (Merrifield technique). They are particularly suitable for obtaining antisera that can be screened with suitable gene expression libraries to reach additional functional variants of the polypeptides according to the invention.

Accordingly, a further object of the present invention relates to a method for the expression of a nucleic acid according to the invention in a suitable host cell and, optionally, isolation to produce a polypeptide according to the invention.

The fission enzyme skiing investigation is particularly desirable since carmyeses formovetes have been successfully and successfully used for the expression of heterologous exogenous proteins and exogenous proteins and gastric and nociceptive naturally occurring yeast (Giga-Hama & Kumagai 1997), "Foreign Gene Expression in Fission Yeast: Schizosaccharomyces pombe", Springer Verlag]. As exemplified in Example 11, the toxin-encoding nucleic acid according to SEQ ID NO: 1 and SEQ ID NO: 2 is present, for example, under the transcriptional control of the thiamine-regulated nmt1 promoter of the cleavage yeast [nmt = 'no message by thiamine' The ski survey was conducted using the caromaieth pombe vector pREP1 (Maundrell (1990), J. Biol. Chem. 265: 10857-10864]. The yeast transformed with this vector expresses the foreign gene as a function of the respective thiamine concentration in the yeast culture medium. In some cases, it separates the yeast growth phase from the phase from which the foreign protein is produced, so that in principle it is capable of expressing a protein that is toxic to the yeast. In order to secrete toxin gastric carnitine and isozymes which are heterologously expressed in carmy-maths pombe, the inventors of the present invention have found that secretion and processing signals of the virus K28 pre-propoxin gene (Schmitt & Tipper, 1995) Secretion vector [vector pTZa / gamma; < / RTI >; < / RTI > See Example 11].

Still another object of the present invention relates to an antibody that specifically reacts with a polypeptide according to the present invention, which may make said portion of the polypeptide itself immunogenic or immunogenic, For example, they may be coupled to suitable carriers, such as bovine serum albumin, to enhance their immunogenicity.

The antibody is a polyclonal or monoclonal antibody. The preparations constituting the subject of the present invention can be prepared by immunizing a mammal, for example a rabbit, with a polypeptide according to the invention or a part thereof as described above, optionally in the presence of a Freund ' s adjuvant and / (See Diamond, BA < RTI ID = 0.0 > et al. (1981) The New England Journal of Medicine, 1344]. The polyclonal antibody formed in the animal due to the immune response can subsequently be easily separated from the blood in a conventional manner and purified, for example, by column chromatography. For example, when the antigen is covalently bound to a freely obtainable CnBr-activated sepharose matrix (IgG or wicalin), the antibody is preferably affinity purified when it is used for the purification of a toxin specific antibody.

The monoclonal antibody can be produced, for example, by a known method (Winter, G. & Milstein, C. (1991) Nature, 349, 293).

Another object of the present invention is a pharmaceutical composition comprising a nucleic acid according to the present invention or a polypeptide according to the present invention (individually or in combination) and, optionally, a drug comprising a suitable additive or adjuvant, Mucous membrane fungus and systemic mycosis, particularly preferably a method for producing a drug for treating candida mycosis, wherein the nucleic acid according to the present invention or the polypeptide according to the present invention is a pharmaceutically acceptable additive and / ≪ / RTI >

Example 12 demonstrates that the toxin gastrin produced and purified by the strain DSM 12865 is significantly more potent against yeast than the topical antifungal drugs clotrimazole and myconazole tested for comparison and frequently used in the treatment of mycosis Illustrate.

Accordingly, the present invention also relates to the aforementioned medicament comprising antifungal or protein toxins obtainable from DSM 12864 and / or DSM 12865 and / or antifungal active polypeptides according to the invention.

Drugs containing the nucleic acid according to the invention in pure form or in any of the abovementioned vectors effective for gene therapy or in complex form with liposomes are particularly suitable for use in human gene therapy.

Examples of suitable additives and / or adjuvants are physiological saline, stabilizers, protease inhibitors, nocrease inhibitors and the like.

Another subject of the invention is the use of a nucleic acid according to the invention, a polypeptide according to the invention or an antibody according to the invention and, optionally, diagnostic agents comprising suitable additives and / or adjuvants, A method for producing a diagnostic agent for diagnosis of Candida mycosis, wherein the nucleic acid according to the present invention, the polypeptide according to the present invention or the antibody according to the present invention is administered in combination with a suitable additive And / or formulated with adjuvants).

For example, a polymerase chain reaction (e. G., PCR diagnostics according to European Patent Publication No. 0 200 362) or Northern blotting and / or Southern blotting, as described in more detail in Example 13, Diagnostic agents based on the present invention can be prepared according to the present invention using nucleic acids according to the present invention. These tests are based on the specific hybridization of a nucleic acid according to the invention with a complementary strand, usually the corresponding mRNA. The nucleic acid according to the present invention can be modified, for example, as described in European Patent Publication No. 0 063 879. Preferably, the DNA fragment according to the invention is labeled with suitable reagents by conventional methods (for example, by providing a radiolabeled or non-radioactive biotin label using? -P ³² -dATP), and preferably Is incubated with separate RNA bound to a suitable membrane (e.g., cellulose or nylon). It is also advantageous to separate the separated RNAs prior to hybridization and membrane binding, for example, by cloning by agarose gel electrophoresis. If the amount of test RNA from each tissue sample is the same, the amount of mRNA specifically labeled by the probe can be determined.

Another diagnostic agent comprises a polypeptide according to the invention or an immunogenic part thereof as described in more detail above. For example, a polypeptide, or a portion thereof, conjugated to a solid phase such as nitrocellulose or nylon is preferably contacted in vitro, for example, with a body fluid (e.g., blood) to be tested, . The antibody / peptide complex can then be detected using, for example, labeled anti-human-IgG or anti-human-IgM antibodies. The label is, for example, an enzyme that catalyzes the color reaction (e.g., peroxidase). Thus, the presence and amount of an autoimmune antibody can be detected easily and quickly through a color reaction.

Another diagnostic agent comprises the antibody itself according to the invention. These antibodies allow for the easy and rapid examination of human tissue samples, for example, against the presence of the polypeptide in question. In this case, the antibody according to the invention is labeled, for example, with an enzyme as already described above. Thus, specific antibody / peptide complexes can be readily and rapidly detected through enzyme color reaction.

Another object of the present invention is to provide a pharmaceutical composition comprising a nucleic acid according to the present invention and / or a polypeptide according to the present invention, alone or in combination, and optionally containing a fungicide containing a suitable additive or adjuvant and a harmful yeast and a harmful fungus Wherein the nucleic acids according to the invention or the polypeptides according to the invention are formulated together with agriculturally acceptable additives and / or adjuvants.

As already described, transgenic plants are produced in a preferred embodiment to express the protein toxin according to the invention. Accordingly, the present invention relates to plant cells and transgenic plants themselves, including polypeptides and / or protein toxins according to the present invention.

Another subject of the invention is a pharmaceutical composition comprising a functional interaction reagent, for example a nucleic acid according to the invention, a polypeptide according to the invention or an antibody according to the invention and, if appropriate, suitable additives and / or adjuvants Inhibitors or stimulants for use in the treatment of cancer.

Suitable test substances for identifying functional interactions, especially those that interact with the protein toxin receptor according to SEQ ID NO: 2 in susceptible yeast cells are, for example, two hybrid systems (see Fields, S. & Sternglanz, R. (1994) Trends in Genetics, 10, 286]. In such assays, a cell, e. G., A yeast cell expresses and / or expresses a fusion protein comprising a polypeptide according to the invention and a DNA binding domain of a known protein (e. G. Ga14 or LexA from E. coli) Or is transfected with or transfected with one or more expression vectors expressing a fusion protein comprising an unknown polypeptide and a transcription activation domain (e.g., Gal4, herpes virus VP16 or B42), or the cell may be a reporter gene, The E. coli LacZ gene, the green fluorescent protein or the yeast amino acid biosynthesis gene His3 or Leu2, wherein the reporter antibody is regulated by a regulatory sequence such as lexA promoter / activator or yeast upper activation sequence (UAS) Unknown polypeptides include, but are not limited to, a gene library, for example, DNA originating from a human gene library In general, the cDNA gene library is first generated in yeast using the expression vector described above so that subsequent assays can be performed immediately.

In the yeast expression vector, for example, the nucleic acid according to the present invention can be prepared by introducing a fusion protein of the polypeptide according to the present invention and the LexA DNA binding domain into a nucleic acid encoding a LexA DNA binding domain Cloned in sex units. In another yeast expression vector, the cDNA fragment of the cDNA gene library contains a functional group for a nucleic acid encoding a Gal4 transcription activation domain such that a fusion protein of an unknown polypeptide and a Gal4 transcription activation domain is expressed in the transfection yeast Lt; / RTI > The transformed yeast into two expression vectors (for example: Leu2 ^-) is, encrypts the Leu2 further comprises a nucleic acid that is controlled by the LexA promoter / operator factor. In the case of a functional interaction between a polypeptide according to the present invention and an unknown polypeptide, the Gal4 transcription activation domain binds to the LexA promoter / operative factor through the LexA DNA binding domain to activate the LexA promoter / Lt; / RTI > As a result, Leu2 ^- yeast can grow on minimal media that does not contain leucine.

When LacZ or a green fluorescent protein reporter gene is used instead of an amino acid biosynthesis gene, transcription activation can be detected by the formation of a colony that emits blue or green light. However, blue or green fluorescent staining can also be easily quantified at 585 nm for spectrometers, e.g., blue staining.

In this method, an expression gene library can be screened easily and rapidly for polypeptides that interact with the polypeptides according to the invention. The novel polypeptides that are found can be subsequently further characterized and further characterized. Another possible use of the two hybrid systems is in the influence of other substances (e.g., chemicals) on the interaction between the polypeptides according to the invention and the known or unknown polypeptides. It also allows the discovery of new useful active ingredients that can be chemically synthesized and used as therapeutic agents. Accordingly, the present invention extends to a method for searching for a substance capable of interacting with the above-described protein / protein complex, as well as a method for searching for a polypeptide-like interactive agent. Thus, such peptide-like, and chemical, interacting agents are termed functional interactions that may have inhibitory or stimulatory effects for the purposes of the present invention.

Another object of the present invention is to provide a process for the production of protein toxins by culturing protein toxins and treating the protein toxins with synthetic culture media (BAVC medium), such as ultrafiltration and cation exchange on laminarin-sepharose and / or mannoprotein- Which significantly promotes the chromatographic purification of the toxin secreted by chromatography and / or affinity chromatography (see, for example, Example 1 and Examples). The. For wikartin produced and secreted by the strain DSM 12865, toxin production can be further enhanced by supplementing the plant with a plant-derived (and readily available)? -1,3-D-glucanamininin to a final concentration of 1% Can be further increased.

As illustrated in Example 14, addition of laminarin to the culture medium induces gastric kallin production, which, according to Northern analysis, contributes to the induction of transcription. Using synthetic B medium, it is possible to produce toxin rich in secreted by DSM 12864 (Radler et al., 1993).

The following examples are intended to illustrate the invention, but the invention is not limited to these examples.

Example 1

Concentration and purification of the anti-Candida toxin chitin from the culture supernatant of the killer yeast W. californica 3/57 strain (DSM 12865)

In the Agar diffusion test on methylene blue agar to the susceptible yeast, the killer toxin gastricin secreted by the killer yeast Wilheloxis californica 3/57 showed optimal inhibition at pH 4.7 and 20 ° C. In the synthetic liquid medium, killer yeast Willysys californica strain 3/57 shows maximum toxin production when grown in BAVC medium (pH 4.7). For toxin enrichment, the killer yeast is first incubated for 24 hours in 5 ml of YEPD medium with shaking for 24 hours, then transferred to 200 ml of BAVC medium and incubated again at 20 DEG C for 48 hours on a shaker (140 rpm) . Four primary cultures of 2.5 L BAVC medium (pH 4.7 in 5 L Elenmeyer flask) are each inoculated with secondary pre-culture (1% inoculum) and incubated at 20 ° C for 5 days with gentle shaking (60 rpm) . To concentrate the secreted killer toxin, the cell-free culture supernatant was ultrafiltered on a polysulphonic membrane (EasyFlow) (Fa Sartorius; exclusion limit 10 kDa) at a pressure of 1 bar at + 4 ° C Concentrate 200 times in 50 ml volume. To remove low molecular weight compounds and desalt the resulting concentrate, the toxin is 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 product is filter-sterilized through a 0.2 [mu] m membrane and frozen in 1 ml aliquots at -20 [deg.] C.

The toxin activity is detected and standardized in a susceptibility indicator yeast Saccharomyces cerevisiae by agar diffusion test on methylene blue agar (MBA; pH 4.7) to 192.2d. To this end, the index ever toxin concentrate of step is diluted and prepared in 0.1M citrate / phosphate buffer (pH 4.7), sensitive indicator yeast (2 × 10 ⁵ cells / ml) wells pre-punched with a MBA plate inoculated with (Well diameter 9 mm). The plate is incubated at 20 ° C for 3 days, and then the inhibition area that can be observed clearly is measured. There was a linear relationship between the inhibition zone diameter and the toxin concentration index. The random toxin activity of 1 x 10 ⁴ units / ml is designated as the inhibition zone diameter of 20 mm (corrected to the well diameter).

Concentrated wicallin is purified by cation exchange chromatography on a bioscale-S (FPLC) or by an epoxy-activated sepharose-6B matrix (Pharmacia (< RTI ID = 0.0 > Pharmacia). ≪ / RTI > The toxin preparation (Table 1), whose specific activity was enriched 625 times (Table 1), was purified by gel electrophoresis and showed a single band at about 37 kDa only after SDS-PAGE (10-22% gradient gel) Coomassie Blue (protein stain) and peridoxic acid-Schiff stain (PAS; carbohydrate stain). Positive PAS staining suggests strong N-glycosylation of the anti-Candida toxin cholines. The treatment of the purified toxin with endoglycosidase-H confirms that gastricin has an approximately 3 kDa carbohydrate moiety linked by N-glycosidation, and its size in the yeast is a single N-glycosylation site in the protein toxin . Since the deglycosylated gastrocartin exhibits a markedly limited toxicity, it can be inferred that the carbohydrate moiety of gastrocartin is probably required for binding to the susceptible target cell, thus directly affecting the viability of the toxin.

Concentration of gastricin from the culture supernatant of killer yeast Willoopsis californica [UF, ultrafiltration] Formulation Volume (ml) Total protein (mg) Total toxin activity (E) Specific toxin activity (E / mg) Active yield (%) Refining factor Culture supernatant 10,000 24,600 7.9 × 10 ⁵ 3.2 × 10 ¹ 100 One UF Holder 50 162 6.3 × 10 ⁵ 3.9 × 10 ³ 80 122 Lyophilized dialysate 25 45.8 3.1 × 10 ⁵ 6.8 × 10 ³ 39 213 Bio-scale S (cation exchange) 64 1.28 2.5 x 10 ⁴ 2.0 x 10 ⁴ 3.2 625

Example 2

NH of Weikartin ₂ - Determination of terminal amino acid sequence and detection of enzyme? -1,3-glucanase activity

The first 10 amino acids are determined by sequencing the N-terminal amino acid of the purified killer toxin. As can be seen from Fig. 1, the N-terminus of gastricin is markedly homologous to the amino terminus of endo-beta-1,3-glucanase encoded by the BGL2 gene in yeast Saccharomyces cerevisiae .

Since homology of gastricin and Bgl2 has been determined, the detectability of glucanase activity in untreated toxin concentrates and purified toxin concentrates is investigated. In the wikartin preparation, an enzyme assay using β-1,3-D-glucanaminarin as a substrate and a fluorescence assay using 4-methyl-umbelliferyl-β-D-glucoside (MUC) β-1,3-D-glucanase activity is detected, and the further tested β-1,6-D-glucan fructulan is not hydrolyzed by wicallin.

Example 3

Survival of gastricin treated yeast with and without cell wall glucan: competition analysis

The susceptible yeast cells of Saccharomyces cerevisiae 192.2d strain grown in a YEPD liquid medium (pH 4.7) at 20 ° C in the presence of purified gastricin 1 × 10 ⁵ U / ml showed the lethal dynamics shown in FIG. 2 . The addition of plant-derived β-1,6-D-glucan fusTulen significantly increases the survival rate of toxin-treated yeast cells, and when added at a concentration of 10 mg / ml, the toxicity of gastrocartin is completely reversed. Contrary to fructoolan, β-1,3-D-glucanaminamin could not increase the survival rate of toxin-treated yeast (FIG. 2).

Therefore, this finding can be concluded that the action of gastricin requires binding with? -1,6-D-glucan to act as a major blocking site (a toxin receptor) in yeast cell walls. Consistent with this finding, the yeast deleted at the chromosome KRE1 gene position exhibited toxin resistance, but was found to regain toxin susceptibility when re-transformed into an episome vector containing KRE1 (FIG. 3). The toxin resistance in the kre1 mutant is based on the significantly reduced? -1,6-D-glucan content and thus a reduction in toxin binding to the yeast cell surface is required for lethal action.

Example 4

The range of action and mortality of gastricin

In the agar diffusion test, purified Willy opsys californica toxin gastricin exhibits significant toxicity to the yeast shown in Table 2. All 22 clinical patient isolates tested and all other control strains of Candida species hospitalized for humans, except for three strains of yeast Candida krusei, were destroyed by gastrocartin in a very efficient manner. For 14 toxin susceptible yeast species from all 10 different species, wicallin exhibits a particularly broad range of action against killer toxins.

The range of action of gastrocartin on pathogenic and nonpathogenic yeasts of different species. All strains are tested by the Agar diffusion test (MBA; pH 4.7) on purified gastrocartin. The toxin activity applied is 1 × 10 ⁶ U / ml. The Candida tropicallis (patient number 541965) strain is obtained from The Department of Medical Microbiology and High Grade of the University Hospital Mainz. Yeast strain Phenotype Diameter of inhibition zone (mm) Candida albicans ATCC 10231 S 11 Candida Glabrata NCYC 388 S 12 Candida Cruisei 185 R 0 Candida tropicallis patient number 541965 S 11 Devari Ohmesse Sansei 223 S 16 Han Senia Spra Uba Room ATCC 64295 R 0 Hansega Waja asaponica variant Versace tillis 191 R 0 Cluye veromycetes lactis CBS 2359/152 S 22 Clu Beromayces Marcianus C 8,1 R 0 MetShunnico Via Pool Cherryma K / 31 B6 S 8 Pikia Anomala 245 S 17 Pichia Parisa 258 R 0 Piquiadzini 251 S 6 Pichia clueberry ATCC 64301 R 0 Pikia Membrane Epiences NCYC 333 R 0 Sakaromayses Serabiziae 192.2d S 30 381 S 23 ATCC 42017 (K1 super killer) S 19 NCYC 738 (K2 killer) S 14 452 (= NCYC 1006) S 16 Sakaro Maiko des Ruud Crisis 240 R 0 Ski investigation Karomaisse Pombe CBS 1042 R 0 Sporotrix species 1129 S 11 Torulospora del Brucci 208 S 18 Torulosfora pretoriais 186 S 10 Yarowia Repositica 271 S 8 Jisakaro Maisse Bailey 412 S 23

Example 5

Cloning, Sequencing and Molecular Characterization of the Wycholin-Encoding WCT Gene of Yeast Wiley-opsy californica strain 3/57 (DSM 12865)

Starting with the N-terminal amino acid sequence of gastricin, it produces a specific DNA oligonucleotide, which leads to the identification, cloning and characterization of the molecular biology of the toxin gene WCT located on the chromosome. The DNA sequence of WCT (SEQ ID NO: 1) encodes a strongly N-glycosylated 109 amino acid protein and represents a single open reading frame with a calculated molecular weight of 34,017 Da. Studies of the action of the WCT-encoded killer toxin showed that it was a glycoprotein that was highly toxic to yeast and its major target was the cell wall β-1,3-D-glucan found in yeast. Its selective toxicity to yeast and fungi is based on wicallin, which destroys cell wall structure and / or integrity in susceptible target cells, and eventually attacks them to the most sensitive yeast.

Example 6

Killer yeast Concentrate and purify the viral toxin from the culture supernatant of Gigasacolomyces Bailey strain 412 (DSM 12864)

The virus-encoded killer toxin lysine of the yeast Gigasacolomyces Bailey 412 strain was isolated from the culture supernatant of killer yeast by a known method (Radler et al., 1993), concentrated by ultrafiltration, Purification by flash chromatography. The first step purification of Joshin developed in this study develops the natural affinity of the toxin for the cell wall mannoprotein of the susceptible yeast. The partially purified mannoprotein isolated from the strain 192.2d in Saccharomyces cerevisiae was treated with the epoxy-activated sepharose-6B matrix (Pharmacia) by the method described in Schmitt & Radler (1997) And used as an FPLC for purifying toxins by column chromatography. After performing SDS-PAGE, the highly purified liposomes purified by this method exhibit a single protein band with an apparent molecular weight of about 10 kDa (FIG. 4).

Example 7

Range of action and range of death

The range of action of the viral overgrowth on the yeast Gigasaccharomyces bailei 412 (DSM 12864) as measured by the Aga diffusion test includes pathogenic and non-pathogenic yeast, among which Candida albicans and Sporotrichs senki are used for the treatment of humans and animals It is an important pathogen, and osteil, maddis and debarrionisseus hanseini are important harmful yeasts in the agriculture and food sector (Table 3).

Scope of action of overexpressed genes on pathogenic yeast and non - pathogenic yeast of different genus. All strains are tested with an agar diffusion test (MBA; pH 4.5) on a lysine preparation with an activity of 1 x 10 ⁴ U / ml. Lysosomesensitive yeast Relative Sensitivity Sakaromayses Serabiziae ++ Candida albicans + Candida Cruise ++ Candida Glavata ++ Candida binii + Han Senia Spra Uba Room ++ Cluj Beromayes Marcianus + Metz Shnico Via Pool Cherryma + Woo Sutilla ++ Debari Omaiseth Hanseini ++ Pikia Anomala ++ Pikia Zadini + Pikia Membrane Feathians + Jarowiapolitica + Jisakaromaisaisurookushi ++

Example 8

Cloning and Sequencing of the ZigBee ZBT Gene (ZBT) of Yeast Gigusaccharomyces cerevisiae 412 strain (DSM 12864)

Killer Yeast The cDNA of the double-stranded RNA genome of the Gigasaccharomyces bailey 412 was amplified using purified M-dsRAN modified with methyl mercurial hydroxide as a template and a hexanucleotide as a primer, using Schmidt (1995)]. The EcoRI restriction vector pUC18 was ligated and ligated. After isolating recombinant plasmids transformed and identified in E. coli, several cDNA clones are isolated and sequenced. The cDNA sequence (SEQ ID NO: 2) of the amplified read frame contains genetic information for the 238 amino acid precursor protein (pro-toxin), which contains a strong Kex2-endopeptidase cleavage site at amino acid position RR ¹³⁹ . The viability of precisely matched data for the molecular weight (10 kDa; 99 amino acids) and N-terminal amino acid sequence purified from the lysine is formed by Kex2 mediated pro-lobe processing, which occurs during the late Golgi phase It occurs in vivo.

Due to the toxicity of Joshin Shin, the heterologous expression of ZBT-cDNA in yeast Saccharomyces cerevisiae causes the death of yeast transformed by their own toxins. As already proven in the example of the virus K28 toxin, since the cleavage yeast is particularly suited for the expression or secretion of foreign proteins, a further object is to express heterologous and exogenous genes in the toxin resistant fission yeast scout caroiomyces pombe.

Example 9

Expression of toxin genes WCT and ZBT in transgenic plants

Since the killer toxins gastric and gastrointestinal tracts described above have a wide range of action and destroy phytopathogenic yeasts and fungi, for example, transgenic plants which are resistant to infection by corn pathogens osteil and maize can be constructed . Similar experiments have already been carried out on tobacco plants and have been naturally encoded by viruses. Due to the heterologous expression of the maize killer toxin KP4, the tobacco plants can secrete the killer toxin and can specifically protect against infection by certain phytopathogenic fungi and Maize's strain (Part et al. , 1996; Kinal et al., 1995; Bevan, 1984). Starting with a commercially available transformation system based on a modified derivative of the native Agrobacterium tumefaciens Ti-plasmid, the toxin genes WCT and ZBT cloned by the present inventors were cloned into a so-called anisotropic pBl vector (CLONTECH) They can be used for the production of transgenic plants. To this end, the toxin genes, WCT and ZBT, are placed under the transcriptional control of the strong cauliflower mosaic virus promoter (CaMV-P). The structure of the vector to be constructed is schematically shown in Fig.

Example 10

Expression of heterozygosity of Wycardin-encoding WCT gene of yeast Willoopsis californica 3/57 (DSM 12865) in Saccharomyces cerevisiae

To heterologously express the WCT gene in yeast Saccharomyces cerevisiae, the Wicollin-encoding WCT gene is cloned as a 930 bp EcoRI / SmaI fragment into a generally available 2μ vector pYX242. The resulting vector pSTH2 (Figure 6) contains the toxin gene under the transcriptional control of the yeast triose phosphate isomerase promoter (TPI) and is thus transformed into yeast (Saccharomyces cerevisiae) Lt; / RTI > Analysis of the culture supernatant of the thus-obtained yeast transformant by gel electrophoresis showed that the recombinant gastricin was secreted into the external medium and the β-1,3-D-glucanase activity corresponding to the activity of the homologous gastrocartin (From wild-type strain DSM 12865) (Fig. 6).

Example 11

Experiments on heterologous expression of gastrocartin and oligosin in Karomayses pombe

Since cleavage yeast is resistant to gastrocartin and oligosaccharide, it is suitable both as a pure cell and as a cell wall free spiroplast, and as a host for heterologous expression of the toxin. (S / P), which is functional in the ski-inspired caromyces pombe, to secrete not only the recombinant toxin as a cleavage yeast but also to the intracellular secretory pathway and secrete it into the external medium, A vector derived from the cDNA of the virus K28-preprotoxin gene of Mysis cerevisiae is constructed (pTZ? / ?; Fig. 7) (Schmitt, 1995; Schmitt & Tipper, 1995]. The secretion and processing signals allow exogenous proteins aligned downward in the frame to intervene in the lumen of the endoplasmic reticulum in the fission yeast to feed the yeast's secretory pathway. The Kex2p cleavage site at the C-terminus of the S / P region is cleaved by yeast Kex2p-endopeptidase to cleave the desired foreign protein from its intracellular transport vehicle in the late Golgi compartment, Can be secreted into the outer medium (oversampling and / or gastricin).

Example 12

Comparison of purified gastric and topical antifungal drugs chlorothymazole and myconazole

Purified gastrocartin is important as a candidate antifungal drug because it has a broad range of action and is effective in killing hospitalized yeast and / or fungi against humans. Therefore, a comparative study of chitrimazoles and myconazole and wicallin, which are currently widely used topical antifungal drugs, is conducted. First, toxic effects of clotrimazole and myconazole on sporotrichus species as indicator yeast are tested by the MBA agar diffusion test. To this end, clotrimazole is dissolved in ethanol (96%) at a concentration of 10 mg / ml and the stock solution is diluted with ddH ₂ O and used for MBA testing at a concentration of 0.1 to 10 mg / ml per 100 μl. When clotrimazole is used in an amount of 10 to 50 μg, the inhibition region diameter is 12 to 32 mm. 100 mg / ml stock solution in DMSO (100%) was prepared using myconeazole and tested in the same manner as clotrimazole in the MBA test for the viability of sporotrichoids. In the biopsy method, when the amount of myconazole is used in an amount of 0.08 to 0.3 μg, the inhibition area is 22 to 36 mm. Thus, the viability of 10 μg of clotrimazole and 0.08 μg of myconazole corresponds to the toxicity of 2 μg of purified gastricin. As a result of comparison based on the molecular weight of the three test compounds, wicallin is a very potent antifungal drug (Fig. 8) since it exhibits the same activity as 0.2 pmol of myconazole and 29 pmol of clotrimazole at a concentration of 0.07 pmol of gastricin.

Example 13

Detection of Wycardin-encoding WCT gene of yeast Wilheloxis californica 3/57 (DSM 12865) by Southern hybridization with a gene-specific DNA probe

To demonstrate that a nucleic acid according to SEQ ID NO: 1 can be used to prepare a gastricin-specific DNA probe for subsequent Southern hybridization, a DIG labeled 930 bp DNA probe is used to detect the WCT gene cloned into the vector pSTH1. The constructed vector pSTH1 is a derivative of the generally available prokaryotic cloning vector pBR322.

The agarose gel electrophoresis and the corresponding Southern blotting shown in Figure 9 indicate that the nucleic acid probe can be used for the detection of the Wicollin encoded WCT gene.

Example 14

Northern Blot Analysis for Detecting the Transcription Induction of Wycardine-Encoding WCT Gene in Yeast Wiley-opsys californica 3/57 (DSM 12865) by β-1,3-D-glucan

To detect β-1,3-D-glucan induced WCT transcription, yeast strain DSM 12865 was cultivated in BAVC medium supplemented with 300 ml of BAVC medium or 0.03% of β-1,3-D-glucan laminarin from plant Grown at 20 캜 for 48 hours with gentle shaking (60 rpm), and used for the preparation of total RNA after different time intervals. Prior to RNA isolation, all samples (10 ml) are adjusted to the same cell density of 1.8 x 10 ⁸ cells / ml and separated by electrophoresis on denaturing agarose formaldehyde gel. As can be seen in FIG. 10, 1100 base sizes were detected for WCT transcripts in both non-in vitro conditions (non-supplemented BAVC medium) and in laminarin supplemented BAVC medium. Without addition of glucan, maximal WCT expression is achieved at the end of the exponential growth phase (after 19 hours), and the hybridization signal, which is significantly attenuated in the stationary growth phase, suggests a decrease in transcription. Under the induction of culture conditions (in the presence of laminarin), WCT transcripts exhibit higher intensity after 10 hours than in non-inoculated cultures, suggesting that transcription of the Wicollin-encoding WCT gene results in the formation of &bgr; -1,3-D-glucan ≪ / RTI >

Supplements to the Examples:

The medium and solution used in the examples:

a) BAVC medium

Glucose 50g / L D, L-Malate 20g / L Trisodium citrate 0.5 g / L (NH _{4) 2} SO ₄ 1.5 g / L MgSO ₄ 1.0 g / L CaCl ₂ 0.5 g / L Mio-Inositol 0.04 g / L Amino acid stock solution (10x) 200ml / L Trace element stock solution (100x) 10ml / L Vitamin stock solution (100x) 20ml / 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 3g / L Histidinium monochloride 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 solution (100x)

Boric acid 200mg / L FeCl ₃ 6H ₂ O 200mg / L ZnSO ₄ x 7H ₂ O 200mg / L AlCl ₃ 200mg / L CuSO ₄ × 5H ₂ O 100 mg / L Na ₂ MoO ₄ x 2H ₂ O 100 mg / L Li ₂ SO ₄ x H ₂ O 100 mg / L KI 100 mg / L Potassium hydrogen tartrate 2 g / L

d) Vitamin stock solution (100x)

4-aminobenzoic acid 20mg / L Biotin 2 mg / L Folic acid 2 mg / L Nicotinic acid 100 mg / L Pyridoxine hydrochloride 100 mg / L Riboflavin 50 mg / L Thiamineethyl dichloride 50 mg / L Calcium D-Pantothenate 100 mg / L

It is dissolved in a KH ₂ PO ₄ 5g / 50ml distilled water: biotin.

Folate: Dissolve in 50 ml of distilled water with a few drops of dilute NaOH.

Riboflavin: dissolve in 500 ml of distilled water and a few drops of HCl while heating.

The remaining vitamins can be dissolved in a small amount of distilled water.

The pH of the BAVC medium is adjusted to pH 4.7 by the addition of KOH. The glucose and stock solutions are sterilized separately. Amino acid, vitamin and trace element stock solutions are sterilized at 100 ° C for 20 minutes with valve opening and then added to autoclave BAVC medium.

Drawings and most important sequences

Sequence 1 is the DNA sequence and the deduced amino acid sequence of the WCT-encoded protein toxin gastric fibrinolytic gene of yeast Wilheloxys californicus 3/57 strain.

Sequence 2 is the ZBT-encoded protein toxin lordosis cDNA sequence and the deduced amino acid sequence of the yeast gigasikomosaeth bailey.

1 shows the N-terminal amino acid sequence of Willy opsys californica toxin gastricin and the N-terminal amino acid sequence of endo-β-1,3-glucanase Bgl2 in yeast saccharomyces cerevisiae. Other sequences are identical, and only the subsequences differed in bold (Bgl2p sequence after Klebl & Tanner, 1989).

Figure 2 is a graph showing the effect of the presence of 192.2d of soluble < RTI ID = 0.0 > (SEQ ID NO: 2) < / RTI > on susceptible yeast saccharomyces cerevisiae in the presence of? -D- This shows the lethal dynamics of gastrocartin-treated cells. The toxin used had a total activity of 4.0 × 10 ⁵ U / ml at a specific activity of 4.2 × 10 ⁵ U / protein mg.

Figures 3 (a, b, c and d) show the Agar diffusion test for detecting gastricin sensitivity / resistance in Kre1 ⁺ and Kre1 ^- strains to yeast saccharomyces cerevisiae. The transformation of SEY6210 [DELTA kre1] into the gastrin-kallin-resistant kre1 0-mutant Saccharomyces cerevisiae using the KRE1-containing vector pPGK [KRE1] completely restores gastricin sensitivity.

Figure 4a shows gel electrophoresis analysis (SDS-PAGE) of bovine kidney produced and secreted by yeast Gigasaccharomyces bailei 412 strain (DSM 12864) after affinity chromatography on mannoprotein-Sepharose. FIG. 4B shows an agar diffusion test for detecting the viability of a purified killer toxin.

Figure 5 shows the schematic structure of a ZBT or WCT-containing expression vector for producing transgenic plants. [Abbreviations: RB, LB: right and left border sequences of the natural Ti plasmid of Agrobacterium tumefaciens; CaMV-P: Cauliflower Mosaic Virus 35S promoter; NOS-P, NOS-T: Nosalin synthase transcription promoter and terminator; kan ^R : This. Streptococcus kanamycin resistance gene for screening in E. coli; NPT-II: neomycin phosphotransferase gene from transposon Tn5 for selection in plants].

Figure 6a is a partial restriction map of the episomal vector pSTH2 for the heterologous expression of the ictalcine coding toxin gene WCT in yeast Saccharomyces cerevisiae. The vector pSTH2 is a constructed plasmid based on the commercially available 2 mu multiplex replication vector pYX242, in which the WCT gene from strain DSM 12865 is cloned as a 930 bp EcoRI / SmaI fragment. The toxin gene is under the transcriptional control of the TPI promoter of yeast, and thus strongly transforms the wikartin by structurally expressing Saccharomyces cerevisiae.

FIG. 6b shows the gel electrophoresis analysis (SDS-PAGE) of the concentrated culture supernatant of Saccharomyces cerevisiae after transformation using the constructed gastricin expression vector pSTH2 (lane 1) and the basic vector pYX242 (lane 2) PAGE; 10 to 22.5% gradient gel). The gastrocartin heterologously expressed in Saccharomyces cerevisiae is indicated by an arrow.

FIG. 6C shows the transformation of yeast Saccharomyces cerevisiae using the gastrin cell expression yeast vector pSTH2 and the detection of extracellular &bgr; -1,3-D-glucanase activity. To measure exo-beta-1,3-D-glucanase activity, yeast colonies grown on leucine-free SC agar were inoculated with 0.04% 4-methylumbelliferyl- Spray β-D-glucoside (MUG). After incubation at 37 ° C for 30 minutes, the agar plate is irradiated with UV light (wavelength: 254 nm). Glucanase activity is detected by fluorescence due to MUG hydrolysis [Abbreviations: 1 and 4: Saccharomyces cerevisiae transformed with a vector (pEP-WCT) expressing the Wycholin-encoding WCT gene under its own promoter To Jia; 2: wild-type yeast Willysys californica 3/57 (DSM 12865); 3: wild-type yeast Willysys californica 3/111; 5: Saccharomyces cerevisiae after transformation with gastricin expression vector pYX-WCT; 6: Saccharomyces cerevisiae transformed with the basic vector pYX242 (no toxin gene)].

Figure 7 diagrammatically shows the structure of the vector pTZa / gamma for heterologous expression and secretion of foreign proteins (in particular, gastricin and gigantin) in the fission yeast skiing caroiomyces pombe [Abbreviations: P _nmt1 , T _nmt1 : A transcriptional promoter and a transcription terminator of the thiamin-regulated nmt1 gene of the carnomyces pombe; S / P: Secretion sequence and processing sequence of the virus K28 preprotoxin from the yeast Saccharomyces cerevisiae; ars1: self-replication sequence from chromosome 1 of cleavage yeast; leu2: a leucine-2 marker gene for screening carnomyces pombe transformants for a leucine-type nutritional bacterium skiing investigation].

Figure 8 compares the viability of purified gastric, clotrimazole and myconazole. Susceptibility indicator In the biopsy (agar diffusion test) for yeast sporotrichus species, the indicated molar concentration produces a suppression area diameter of 12 mm.

Figure 9 shows the WCALT encoding WCT gene of yeast Willy opsys californica 3/57 (DSM 12865) cloned in pSTHl (pBR322 derivative) into agarose gel electrophoresis (Figure 9a) and Southern blot with DIG labeled WCT probe (FIG. 9B). [Abbreviation: M: Standard of DNA size of DIG labeled DNA II; Lane 1: pSTH1 restricted to EcoRI and SalI; Lane 2: "smart ladder" DNA marker.

FIG. 10 shows the results of the cell culture of yeast Wilheloxs californicus 3/57 (DSM 12865) on the BAVC medium under non-inoculum culture conditions (FIG. 10a) and on BAVC medium supplemented with 0.03% Lt; RTI ID = 0.0 > WCT < / RTI > gene. Total RNA isolated from strain DSM 12865 is separated by electrophoresis on denaturing agarose / formaldehyde gel at a constant voltage (7 V / cm). The RNA is hybridized on a nylon membrane against a gastrin-specific, DIG-labeled DNA probe (630 bp) and detected by chemiluminescence. [Abbreviation: M: DIG labeled RNA size standard I: lanes 1-8 Corresponds to the sampling time for isolating total RNA; Lane 1: 10 hours; Lane 2:15 hours; Lane 3: 19 hours; Lane 4: 24 hours; Lane 5: 33 hours; Lane 6: 38 hours; Lane 7: 43 hours; Lane 8: 48 hours].

Abbreviations used in the specification:

WCT Willoopsis californica toxin ( W illiopsis C alifornica T oxin) ZBT Is My process Bailey toxin as Saccharomyces (Z ygosaccharomyces B ailii T oxin) Joshin Shin Toxin isolated from DSM 12864 Weikartin Toxin isolated from DSM 12865

Microbial deposit

The following microorganisms used for the purposes of the present invention were deposited with the Deutsche Jahrung Phone Microorganism Mentullum Turengelm GmbH (DSMZ), Germany, 38124, Braunschweig, Germany, Shall be recognized as an international deposit under the provisions of the Budapest Treaty with respect to ratification:

Accession number Date of deposit Willy opsys californica strain 3/57 DSM 12865 1999. 06. 09 Jisakaro Maisse Bailey strain 412 DSM 12864 1999. 06. 09

references

Claims (26)

Protein toxins obtainable from Williopsis californica and / or Zygosaccharomyces bailii. The protein toxin according to claim 1, obtainable from DSM 12864 and / or DSM 12865. The protein toxin according to claim 1 or 2, which has an antifungal activity and / or a fungicidal action. 4. The protein toxin according to any one of claims 1 to 3 having glucanase activity. The protein toxin according to claim 4, which binds to? -1,6-D-glucan and has? -1,3-D-glucanase and / or? -1,3-glucanosyltransferase activity. 6. The nucleic acid molecule according to any one of claims 1 to 5, which comprises an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2, wherein SEQ ID NO: 1 or SEQ ID NO: 2 is a part of the claim or a functional variant thereof and a portion thereof having 8 or more nucleotides And / or a nucleic acid encoding a glucanase. The nucleic acid according to claim 6, which is DNA or RNA, preferably double stranded DNA. The method according to claim 6 or 7, wherein the DNA comprises a nucleotide sequence according to base position 1 to 951 of SEQ ID NO: 1 or base position 1 to 717 of SEQ ID NO: 2, wherein SEQ ID NO: 1 or SEQ ID NO: ). ≪ / RTI > 9. The method of claim 8, further comprising one or more regulatory regions (promoter, enhancer or terminator), a 3'-terminal poly-A sequence, a Kex2p endopeptidase cleavage site required for intracellular proteolytic processing and / - a nucleic acid containing a glycosylation site. 10. A nucleic acid according to claim 8 or 9, obtainable from DSM 12864 and / or DSM 12865. 11. A nucleic acid according to any one of claims 6 to 10, which is contained in a vector, preferably an expression vector or a vector effective for gene therapy. 10. A method for producing a nucleic acid according to any one of claims 6 to 10, which comprises chemically synthesizing a nucleic acid or separating it from a gene library using a probe. A polypeptide comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 or a functional variant thereof and a portion thereof having 6 or more amino acids. 14. A method for producing a polypeptide according to any one of claims 1 to 5 and 13, comprising expressing a nucleic acid according to any one of claims 6 to 11 in a suitable host cell. 17. An antibody against a polypeptide according to any one of claims 1 to 5 and 13. 16. A method for the manufacture of an antibody according to claim 15, comprising immunizing a mammal with the polypeptide according to claim 7, optionally separating the formed antibody. A nucleic acid according to any one of claims 6 to 10, a polypeptide according to any one of claims 1 to 5 or 13 or an antibody according to claim 15 and, optionally, Drugs containing acceptable additives and / or adjuvants. Use of a nucleic acid according to any one of claims 6 to 10, a polypeptide according to any one of claims 1 to 5 or 13 or an antibody according to claim 15 in the form of a pharmaceutical acceptable excipient and / Fungi and systemic mycorrhizae, particularly preferably Candida fungi, which comprises a formulation together with an adjuvant or an adjuvant. 10. A nucleic acid according to any one of claims 6 to 10, a polypeptide according to any one of claims 1 to 5 or 13 or an antibody according to claim 15 and, if appropriate, / RTI > and / or adjuvants. 11. A nucleic acid according to any one of claims 6 to 10, a polypeptide according to any one of claims 1 to 5 or 13 or an antibody according to claim 15 in combination with a pharmaceutically acceptable carrier Fungal diseases including mucous membranous fungi and systemic mycoses, particularly preferably candidiasis mycotoxins, including superficial, skin and subcutaneous skin fungi. 10. A nucleic acid according to any one of claims 6 to 10, a polypeptide according to any one of claims 1 to 5 or 13 or an antibody according to claim 15 and, if appropriate, / RTI > and / or an adjuvant. 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 the identification of a functional indicator. A nucleic acid according to any one of claims 6 to 10 for detecting a variant, comprising screening the gene library with a nucleic acid according to any one of claims 6 to 10 and isolating the detected mutant Usage. Use of a polypeptide according to any one of claims 1 to 5 and 13 for the control of harmful yeasts and fungi in food and animal feeds. DSM 12864 and DSM 12865 in a synthetic B and / or BAVC medium. Use of a nucleic acid according to any one of claims 6 to 11 for the production of transgenic plants and plant cells.