EP1196608A2 - Nouveaux antimycotiques et fongicides, procede permettant de les preparer et leur utilisation - Google Patents

Nouveaux antimycotiques et fongicides, procede permettant de les preparer et leur utilisation

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Publication number
EP1196608A2
EP1196608A2 EP00945695A EP00945695A EP1196608A2 EP 1196608 A2 EP1196608 A2 EP 1196608A2 EP 00945695 A EP00945695 A EP 00945695A EP 00945695 A EP00945695 A EP 00945695A EP 1196608 A2 EP1196608 A2 EP 1196608A2
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EP
European Patent Office
Prior art keywords
nucleic acid
yeast
toxin
seq
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00945695A
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German (de)
English (en)
Inventor
Klaus Rehfeldt
Simone Theisen
Frank Weiler
Manfred Schmitt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aventis Research and Technologies GmbH and Co KG
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Aventis Research and Technologies GmbH and Co KG
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Publication of EP1196608A2 publication Critical patent/EP1196608A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to new antimycotics and fungicides obtainable from yeast, processes for their preparation and use.
  • the antimycotics currently used to treat such infections have considerable side effects, since they destroy the structural integrity of the eukaryotic cytoplasmic membrane and thereby also damage the infected host organism [Hector, 1993].
  • the application of conventional antimycotics has also led in a short time to a rapid increase in resistance to fluconazole, which spread rapidly among the human-pathogenic microorganisms and represent 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].
  • a target of selective antimycotics are the ß-1, 3-D-glucans of the yeast cell wall, which are essential for the mechanical and osmotic stability of the cell, but which are not found in higher eukaryotes and are therefore used as "Achilles'verses" in the fight against pathogenic yeasts could [Roemer et al., 1994].
  • substances that selectively intervene in the cell wall structure of yeasts and fungi are of great interest, no antibiotic-like inhibitors have so far been used to treat mycoses.
  • Yeast persist stably and in high copy number without recognizable damage to the eukaryotic host cell [Tipper & Schmitt, 1991].
  • the three hitherto known killer toxins (K1, K2, K28) of the yeast S. cerevisiae are non-glycosylated ⁇ / ß heterodimers, which are translated by the infected cell as higher molecular weight preprotoxins and by intracellular secretion through complex modifications are processed into the biologically active killer proteins [Hanes et al., 1986; Dignard et al., 1991; Schmitt & Tipper, 1995].
  • cerevisiae toxins are based either on the destruction of membrane integrity (toxins K1, K2) or (as in the case of killer toxin K28) on a cell cycle arrest with targeted inhibition of DNA synthesis [Bussey, 1991; Schmitt &
  • killer toxins of classes K1, K2 and K28 differ significantly in their modes of action and physicochemical properties, they have in common that they have narrow spectrum of activity and kill predominantly sensitive yeasts of closely related species. This restricted spectrum of action is based on the fact that the previously characterized
  • Baker's yeast killer toxins must interact with different receptor populations at the yeast cell wall and cytoplasmic membrane levels in order to kill a sensitive target cell.
  • yeast zeilwand is either highly branched ⁇ -1,6-D-glucans or the outer mannotriose side chains of a cell wall mannoprotein [Bussey, 1991; Schmitt & Radler 1987, 1988].
  • killer strains have also been described in the genera Debaryomyces, Hansenula, Cryptococcus, Rhodotorula, Trichosporon, Pichia, Kluyveromyces, Torulopsis and Williopsis [McCracken et al., 1994; Park et al., 1996; Schmitt s Neuhausen, 1994; Walker er al., 1995].
  • the genetic basis of the killer phenomenon in these yeasts is not based on viral genomes, but rather either on linear dsDNA plasmids or on chromosomal yeast genes [Schründer et al., 1994].
  • Yeast is widespread and represents a potential that should not be underestimated in the development of selective antimycotics [Walker et al., 1995; Hodgson et al., 1995; Polonelli et al., 1986; Schmitt & Neuhausen, 1994; Neuhausen & Schmitt, 1996; Schmitt et al., 1997], however, such protein toxins have not yet been provided.
  • the highly effectively produced and secreted killer toxin WICALTIN also protein toxin
  • DSM 12865 wild type yeast Williopsis californica strain 3/57
  • ZYGOCIN also protein toxin
  • DSM 12864 are particularly suitable Control of yeasts and / or fungi that are pathogenic to humans and plants.
  • toxin genes are suitably cloned and sequenced within the scope of this invention and thus a method for the genetic engineering production and overexpression in culture of WICALTIN and ZYGOCIN is established.
  • One object of the invention therefore relates to protein toxins obtainable from Williopsis californica, particularly preferably the strain DSM 12865 and Zygosaccharomyces bailii, particularly preferably the strain DSM 12864.
  • DSM 12864 and DSM 12865 in particular secrete biologically highly effective protein toxins which, due to their broad spectrum of action (see Examples 4 and 7), also kill numerous human and phytopathogenic pathogens.
  • the invention therefore also relates to selective antimycotics or fungicides, in the sense that the protein toxins - and the polypeptides according to the invention below and their coding nucleic acids according to the invention, in particular in the functional unit of a toxin gene - are potential bio-pharmaceuticals which due to their specific, receptor-mediated effect, kill only yeasts and / or fungi and are completely harmless for higher eukaryotes - and thus also for humans and mammalian cells - as well as plants, preferably cultivated plants [cf. Pfeiffer et al., 1988].
  • yeast Saccharomyces cerevisiae, Candida albicans, Candida krusei, Candida glabrata, Candida vinii, Hanseniaspora uvarum, Kl ⁇ yveromyces marxian ⁇ s, Methschnikowia pulcherrima, Ustilago maydis, Debaryomyces hansenii, Pichia jomadiaiaiaxiaaciaxiaciaiaiiaxiaacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia, Pichia anomalyacia.
  • Candida albicans Candida glabrata, Candida tropicalis, Debaryomyces hansenii, Kluyveromyces lactis, Metschnikowia pulcherrima, Pichia anomala, Pichia jadinii, Saccharomyces cerevisiae, Sporthrix spec, Torulaspora delbrueckomycesiaisailiaisiazomycysacialisiazolysporia, liposaccharides, Torulasporia delia
  • the particularly strong activity of the wicaltin-producing yeast strain DSM 12865 is probably due to its pronounced secretion efficiency, which is significantly more pronounced in comparison to other strains of the same type of yeast.
  • the 'killer' property of the zygocin-producing yeast strain DSM 12864 is based on an infection with toxin-coding double-stranded RNA viruses (Mz t rdsRNA), which persist in the cytoplasm in a stable and high copy number and the relevant yeast (strain DSM 12864) for production and enable secretion of zygocin [Cf. Schmitt & Neuhausen, 1994].
  • Other strains of the same species showed no toxin production because they do not harbor toxin-encoding dsRNA viruses in the cytoplasm and can therefore be classified phenotypically as 'non-killers'.
  • the present invention therefore furthermore relates to nucleic acids coding for a protein toxin - with an amino acid sequence according to SEQ ID No 1 and No 2 and a glucanase activity - or a functional variant thereof, and
  • nucleic acid (s) Parts thereof with at least 8 nucleotides, preferably with at least 15 or 20 nucleotides, in particular with at least 100 nucleotides, especially with at least 300 nucleotides (hereinafter referred to as "nucleic acid (s) according to the invention").
  • the complete nucleic acids code for protein toxins, which after intracellular
  • Processing and secretion have a size of 309 amino acids and a molecular mass of 34 kDa (SEQ ID No 1) or of 99 amino acids and a molecular mass of 10 kDa (SEQ ID No 2).
  • the expression of the nucleic acid according to SEQ ID No 1 in the yeast S. cerevisiae results in a recombinant WICALTIN, which as a glycosylated protein with significant ß-1, 3-D-glucanase activity in the
  • nucleic acids according to the invention are nucleic acids which, in the case of SEQ ID No 1 for encode a protein toxin with glucanase activity and, in the case of SEQ ID No 2, for an in vivo presumably O-glycosylated protein toxin called ZYGOCIN.
  • the nucleic acids according to the invention are obtainable from DSM 12865 (SEQ ID No 1) and DSM 12864 (SEQ ID No 2).
  • the nucleic acids according to the invention are a DNA or RNA, preferably a double-stranded DNA, and in particular a DNA with a nucleic acid sequence according to SEQ ID No 1 from item 1 to item 947 and according to SEQ ID No 2 from item 1 to item . 713.
  • the two positions determine the start and the end of the coding region, i.e. the first and last amino acid of the respective reading frame.
  • the term “functional variant” means a nucleic acid which is functionally related to the nucleic acids according to the invention.
  • related nucleic acids are nucleic acids from different yeast cells or strains and cultures or allelic variants.
  • the present invention also encompasses variants of nucleic acids which can come from various yeast / yeast strains or other infectious agents such as dermatophytes and molds (according to the DHS system).
  • variants means nucleic acids which have a homology, in particular a sequence identity of approximately 60%, preferably approximately 75%, in particular approximately 90% and above all approximately 95 % exhibit.
  • the parts of the nucleic acid according to the invention can be used, for example, for the production of individual epitopes, as probes for identifying further functional variants or as antisense nucleic acids.
  • 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 the PCR
  • nucleic acid from at least approx. 20 nucleotides for the identification of further variants and a nucleic acid from at least approx. 100 nucleotides as a probe.
  • the nucleic acid according to the invention contains one or more non-coding sequences and / or a poly (A) sequence, one or more (for intracellular pro-protein processing) Kex2p endopeptidase recognition sequences and one or more potential N -Glykosyl michsstellen.
  • the non-coding sequences are regulatory sequences, such as promoter or enhancer sequences, for the controlled expression of the coding toxin gene containing the nucleic acids according to the invention.
  • the nucleic acid according to the invention is therefore contained in a vector, preferably in an expression vector or vector which is active in gene therapy.
  • the expression vectors can, for example in the case of the nucleic acid according to SEQ ID No 2, prokaryotic and / or eukaryotic expression vectors or in
  • nucleic acid according to SEQ ID No 1 is exclusively eukaryotic expression vectors. Expression of the toxin-coding nucleic acid according to SEQ ID No 1 in Escherichia coli is not possible because the heterologously expressed protein toxin in question is toxic to the bacterial cell. A cloning of the WICALTIN-encoding nucleic acid according to SEQ ID No 1 is only possible in E. coli with plasmids that do not carry a promoter (eg with the aid of derivatives of the plasmid pBR322).
  • prokaryotic vector that allows heterologous expression of the ZYGOCIN-coding nucleic acid according to SEQ ID No 2 is the commercially available vector pGEX-4T-1, which in E. coli expresses a gluthathione-S-transferase-ZYGOCIN- Fusion protein allowed.
  • Another vector for the expression of ZYGOCIN in E. coli is, for example, the T7 expression vector pGM10 (Martin, 1996), which codes for an N-terminal Met-Ala-His6 tag, which advantageously cleans the expressed protein via Ni 2 + -NTA column enables.
  • suitable eukaryotic expression vectors for expression in Saccharomyces cerevisiae are the vectors p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl.
  • the expression vectors also contain suitable regulatory sequences for the host cell, such as, for example, the trp promoter for expression in E. coli (see, for example, EP-B1-0154133), the ADH-2 promoter for expression in yeasts (Radorel et al. (1983), J. Biol. Chem. 258, 2674), the baculovirus polyhedrin promoter for expression in insect cells (see, for example, EP-B1-0127839) or the early SV40
  • suitable regulatory sequences for the host cell such as, for example, the trp promoter for expression in E. coli (see, for example, EP-B1-0154133), the ADH-2 promoter for expression in yeasts (Radorel et al. (1983), J. Biol. Chem. 258, 2674), the baculovirus polyhedrin promoter for expression in insect cells (see, for example, EP-B1-0127839) or the early SV40
  • Promoter or LTR promoters e.g. by MMTV (Mouse Mammary Tumor Virus; Lee et al. (1981) Nature, 214, 228).
  • MMTV Mammary Tumor Virus
  • virus vectors preferably adenovirus vectors, in particular replication-deficient adenovirus vectors, or adeno-associated virus vectors, e.g. an adeno-associated virus vector consisting exclusively of two inserted terminal repeat sequences (ITR).
  • ITR inserted terminal repeat sequences
  • Suitable adenovirus vectors are described, for example, in McGrory, W.J. et al. (1988)
  • Suitable adeno-associated virus vectors are described, for example, 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 with gene therapy effects can also be obtained by complexing the nucleic acid according to the invention with liposomes.
  • Lipid mixtures such as those of Feigner, PL et al. (1987) Proc. Natl. Acad. Sei, USA 84, 7413; Behr, JP et al. (1989) Proc. Natl. Acad. Be. USA 86, 6982; Feigner, JH et al. (1994) J. Biol. Chem. 269, 2550 or Gao, X. & Huang, L. (1991) Biochim. Biophys. Acta 1189, 195.
  • the DNA is bound ionically on the surface of the liposomes in such a ratio that a positive net charge remains and the DNA is completely complexed by the liposomes.
  • the nucleic acids according to the invention are therefore contained in a vector, preferably in an expression vector for the production of transgenic plants. Since the killer toxins WICALTIN and ZYGOCIN described have a broad spectrum of activity and also kill plant-pathogenic yeasts and fungi, it is possible to provide transgenic plants which, for example, are resistant to infection with the maize pathogen Ustilago maydis. Similar experiments have already been carried out on tobacco plants which, by heterologous expression of the naturally virally coded killer toxin KP4 from U.
  • the nucleic acids according to the invention also represented in the toxin genes WCT and ZBT, can be cloned into so-called bidirectional pBI vectors (from CLONTECH) and used for Production of transgenic plants can be used.
  • the relevant toxin genes WCT and ZBT are brought under transcriptional control of the strong cauliflower mosaic virus promoter (CaMV-P). The more precise structure of the vectors to be constructed is shown schematically in Example 9.
  • nucleic acids according to the invention can, for example, be chemically disclosed using the sequences disclosed in SEQ ID No 1 and No 2 or using the peptide sequences disclosed in SEQ ID No 1 and No 2 using the genetic code, e.g. can be synthesized according to the phosphotriester method (see e.g. Uhlman, E.
  • Another way of getting hold of the nucleic acid according to the invention is to isolate it from a suitable gene bank using a suitable probe (see, for example, Sambrook, J. et al. (1989) Moleeular Cloning. A laboratory manual. 2nd Edition, Cold Spring Harbor, New York). For example, single-stranded DNA
  • the present invention further relates to the polypeptides as such with an amino acid sequence according to SEQ ID No 1 and No 2 or a functional variant thereof, and parts thereof with at least six amino acids, preferably with at least 12 amino acids, in particular with at least 65 amino acids. and especially with 309 amino acids (SEQ ID No 1) and with 99 amino acids
  • an approximately 6-12, preferably approximately 8 amino acid long polypeptide can contain an epitope which, after coupling to a support, is used to produce specific poly- or monoclonal antibodies (see, for example, US Pat. No. 5,656,435).
  • Polypeptides with a length of at least approx. 65 amino acids can also be used directly without a carrier for the production of poly- or monoclonal antibodies.
  • the term "functional variant” in the sense of the present invention means polypeptides which are functionally related to the peptide according to the invention, i.e. have glucanase activity. Variants are also understood to mean allelic variants or polypeptides which can come from various yeast / yeast strains or other infectious agents such as dermatophytes, molds (according to the DHS system).
  • this also includes polypeptides which have a sequence homology, in particular a sequence identity of approximately 70%, preferably approximately 80%, in particular approximately 90%, in particular approximately 95%, of the polypeptide with the amino acid sequence Figure 2 have. Furthermore, this also includes deletion of the polypeptide in the range from about 1 to 60, preferably from about 1 to 30, in particular from about 1 to 15, especially from about 1 to 5, amino acids. For example, the first amino acid methionine may be absent without significantly changing the function of the polypeptide. In addition, this also includes fusion proteins which contain the polypeptides according to the invention described above, the fusion proteins themselves already having the function of a glucanase or being able to acquire the specific function only after the fusion portion has been split off.
  • this includes fusion proteins with a portion of, in particular, non-human sequences from about 1 - 200, preferably about 1 - 150, in particular about 1 - 100, especially about 1 - 50 amino acids.
  • non-human peptide sequences are prokaryotic peptide sequences, for example from the galactosidase from E. coli or a so-called histidine tag, for example a Met-Ala-His 6 tag.
  • a fusion protein with a so-called histidine tag is particularly advantageously suitable for purifying the expressed protein on columns containing metal ions, for example on a Ni 2+ NTA column.
  • NTA stands for the chelator "nitrilothacetic acid” (Qiagen GmbH, Hilden).
  • the invention also includes those polypeptides according to the invention which are masked, in the sense of a proprotein or in the broadest sense as a pre-drug.
  • the parts of the polypeptides according to the invention represent, for example, epitopes that can be specifically recognized by antibodies.
  • polypeptides according to the invention are produced, for example, by expression of the nucleic acid according to the invention in a suitable expression system, as already described above, using methods which are generally known to the person skilled in the art.
  • a suitable expression system as already described above.
  • Only eukaryotic organisms are suitable as host cells for the production of correctly processed and thus biologically active protein toxins, preferably the sprout yeast Saccharomyces cerevisiae and the split yeast Schizosaccharomyces pombe.
  • the parts of the polypeptide mentioned can also be synthesized using classic peptide synthesis (Merrifield technique). They are particularly suitable for obtaining antisera, with the aid of which suitable gene expression banks can be searched in order to arrive at further functional variants of the polypeptide according to the invention.
  • Another object of the present invention therefore relates to a
  • the split yeast Schizosaccharomyces pombe is very particularly preferred, since this yeast behaves naturally WICALTIN and ZYGOCIN resistant and has already been used successfully several times for heterologous expression of foreign proteins [Giga-Hama & Kumagai (1997), in "Foreign Gene Expression in Fission Yeast : Schizosaccharomyces pombe ", Springer Verlag].
  • the toxin-coding nucleic acids according to SEQ ID No 1 and SEQ ID No 2 can be cloned, for example, into the S. pombe vector pREP1 [Maundrell (1990), J. Biol. Chem.
  • pombe we have already constructed an expression / secretion vector [vector pTZ ⁇ / ⁇ ; see Example 11], which contains the secretion and processing signal of the viral K28 preprotoxin gene [Schmitt & Tipper, 1995] and thereby enables an effective secretion of the foreign protein which is connected 'in-frame'.
  • Another object of the present invention also relates to antibodies which react specifically with the polypeptide according to the invention, the above-mentioned parts of the polypeptide either being immunogenic themselves or by
  • suitable carriers e.g. bovine serum albumin
  • suitable carriers e.g. bovine serum albumin
  • suitable carriers e.g. bovine serum albumin
  • the antibodies are either polyclonal or monoclonal.
  • the preparation which is also an object of the present invention, is carried out, for example, by generally known methods by immunizing a mammal, for example a rabbit, with the polypeptide according to the invention or the parts thereof, optionally in the presence of, for example, Freund's adjuvant and / or aluminum hydroxide gels (see, for example, Diamond, BA et al. (1981) The New England Journal of Medicine, 1344).
  • the polyclonal antibodies produced in the animal as a result of an immunological reaction can then be easily isolated from the blood by generally known methods and purified, for example, by column chromatography.
  • affinity purification of the antibodies in which for example, the respective antigen (ZYGOCIN or WICALTIN) is covalently coupled to a generally available CnBr-activated Sepharose matrix and used to purify the respective toxin-specific antibodies.
  • the respective antigen ZYGOCIN or WICALTIN
  • Monoclonal antibodies can for example by the known method of
  • Another object of the present invention is also a medicament containing the nucleic acids according to the invention or the polypeptides according to the invention
  • a medicament for the treatment of mycoses such as superficial, cutaneous and subcutaneous dermatomycoses, mucous membrane and systemic mycoses, particularly preferred Candida mycoses, in which an invented nucleic acid according to the invention or a polypeptide according to the invention is formulated with pharmaceutically acceptable additives and / or auxiliaries.
  • toxin WICALTIN produced and purified by strain DSM 12865
  • strain DSM 12865 has a significantly higher toxicity on yeast than the topical antimycotics clotrimazole and miconazole which have been tested in comparison and are frequently used for the treatment of mycoses.
  • the invention therefore also relates to a medicament in the above sense containing an antimycotic or a protein toxin obtainable from DSM 12864 and / or DSM 12865 and / or polypeptides according to the invention having an antimycotic effect.
  • a drug is particularly suitable for gene therapy use in humans which contains the nucleic acid according to the invention in naked form or in the form of one of the above-described gene therapy vectors or in a form complexed with liposomes.
  • Suitable additives and / or auxiliary substances are, for example, a physiological saline solution, stabilizers, proteinase inhibitors, nuclease inhibitors, etc.
  • Another object of the present invention is also a diagnostic agent containing a nucleic acid according to the invention, a polypeptide according to the invention or antibodies according to the invention and optionally suitable additives and / or auxiliaries and a method for producing a diagnostic agent for diagnosing mycoses, such as superficial, cutaneous and subcutaneous dermatomycoses, Mucosal and systemic mycoses, particularly preferred Candida mycoses, in which a nucleic acid according to the invention, a polypeptide according to the invention or antibodies according to the invention are mixed with suitable additives and / or auxiliary substances.
  • a diagnostic agent based on the polymerase chain reaction PCR diagnostics, for example in accordance with EP-0200362
  • a Northern and / or Southern blot as described in more detail in Example 13
  • PCR diagnostics for example in accordance with EP-0200362
  • a Northern and / or Southern blot as described in more detail in Example 13
  • These tests are based on the specific hybridization of the nucleic acid according to the invention with the complementary counter strand, usually the corresponding mRNA.
  • the nucleic acid according to the invention can also be modified here, as described for example in EP0063879.
  • a DNA fragment according to the invention is preferably labeled using suitable reagents, for example radioactive with ⁇ -P 32 -dATP or non-radioactive with biotin, according to generally known methods and with isolated RNA, which was preferably bound to suitable membranes made of cellulose or nylon, for example. incubated. It is also advantageous to separate the isolated RNA prior to hybridization and binding to a membrane, for example by means of agarose gel electrophoresis. With the same amount of RNA examined from each tissue sample, the amount of mRNA that was specifically labeled by the probe can thus be determined.
  • suitable reagents for example radioactive with ⁇ -P 32 -dATP or non-radioactive with biotin
  • Another diagnostic agent contains the polypeptide according to the invention or the immunogenic parts thereof described in more detail above.
  • the polypeptide or parts thereof which are preferably bound to a solid phase, for example made of nitrocellulose or nylon, can be brought into contact with the body fluid to be examined, for example blood, in vitro, in order to be able to react with antibodies, for example.
  • the antibody-peptide complex can then be detected, for example, using labeled anti-human IgG or anti-human IgM antibodies become.
  • the label is, for example, an enzyme, such as peroxidase, that catalyzes a color reaction. The presence and the amount of autoimmune antibodies present can thus be easily and quickly detected via the color reaction.
  • Another diagnostic agent contains the antibodies according to the invention themselves. With the aid of these antibodies, for example, a tissue sample from humans can be easily and quickly examined to determine whether the polypeptide in question is present.
  • the antibodies according to the invention are labeled, for example, with an enzyme, as already described above.
  • the specific antibody is labeled, for example, with an enzyme, as already described above.
  • Peptide complex can thus be detected easily and just as quickly via an enzymatic color reaction.
  • the invention further relates to a fungicide which comprises the nucleic acids and / or the polypeptides according to the invention (individually or in
  • Combination and optionally suitable additives or auxiliaries and a method for producing a fungicide for controlling harmful yeasts and harmful fungi, in which a nucleic acid or a polypeptide according to the invention is formulated with agriculturally acceptable additives and / or auxiliaries.
  • a transgenic plant is produced which expresses the protein toxin according to the invention.
  • the invention therefore also relates to plant cells and inherently to the transgenic plant as such containing the polypeptides and / or protein toxins according to the invention.
  • Another object of the present invention also relates to a test for the identification of functional interactors, such as e.g. Inhibitors or stimulators containing a nucleic acid according to the invention, a polypeptide according to the invention or the antibodies according to the invention and, if appropriate, suitable additives and / or auxiliaries.
  • functional interactors such as e.g. Inhibitors or stimulators containing a nucleic acid according to the invention, a polypeptide according to the invention or the antibodies according to the invention and, if appropriate, suitable additives and / or auxiliaries.
  • a suitable test for the identification of functional interactors is for example the so-called “two-hybrid system” (Fields, S. & Sternglanz, R. (1994) Trends in Genetics, 10, 286).
  • a cell for example a yeast cell, is transformed or transfected with one or more expression vectors which express a fusion protein which contains the polypeptide according to the invention and a DNA binding domain of a known protein, for example Gal4 or LexA from E.
  • coli contains and / or express a fusion protein containing an unknown polypeptide and a transcription activation domain, for example from Gal4, herpes virus VP16 or B42.
  • the cell contains a reporter gene, for example the LacZ gene from E. coli, "Green Fluorescence Protein” or the amino acid biosynthesis genes of the yeast His3 or Leu2, which is regulated by regulatory sequences such as the lexA promoter / operator or by a so-called "Upstream Activation Sequence" (UAS) of the yeast is controlled.
  • the unknown polypeptide is encoded, for example, by a DNA fragment that comes from a gene bank, for example from a human gene bank.
  • a cDNA library is immediately produced in yeast using the expression vectors described, so that the test can be carried out immediately thereafter.
  • the nucleic acid according to the invention is encoded in a functional unit on the nucleic acid for the LexA DNA
  • cDNA fragments from a cDNA library are cloned in a functional unit to the nucleic acid coding for the Gal4 transcription activation domain, so that a fusion protein from an unknown polypeptide and the Gal4 transcription activation domain in the transformed yeast is expressed.
  • the yeast transformed with both expression vectors which is for example Leu2 " , additionally contains a nucleic acid which codes for Leu2 and is controlled by the LexA promoter / operator.
  • the Gal4 binds -Transcription activation domain via the LexA DNA binding domain to the LexA promoter / operator, whereby this is activated and the Leu2 gene is expressed.
  • the Leu2 " yeast can grow on minimal medium which does not contain leucine .
  • the LacZ or "Green Fluorescence Protein” reporter gene instead of an amino acid biosynthesis gene, the activation of the transcription can be demonstrated by the fact that blue or green fluorescent colonies form.
  • the blue or green fluorescence staining can also be easily quantified in the spectrophotometer, for example at 585 nm in the case of a blue staining.
  • expression gene banks can be easily and quickly searched for polypeptides that interact with the polypeptide according to the invention.
  • the new polypeptides found can then be isolated and further characterized.
  • Another possible application of the "two-hybrid system” is to influence the interaction between the polypeptide according to the invention and a known or unknown polypeptide by other substances, such as e.g. chemical compounds. In this way it is also easy to find new valuable chemically synthesizable active ingredients that can be used as therapeutic agents.
  • the present invention is therefore not only intended for a method for finding polypeptide-like interactors, but also extends for a method for finding substances which can interact with the protein-protein complex described above.
  • Such peptide-like as well as chemical interactors are therefore referred to in the sense of the present invention as functional interactors which can have an inhibiting or a stimulating effect.
  • the invention further relates to a method for producing the protein toxins by culturing and secreting the protein toxins in a medium which is a synthetic culture medium (BAVC medium) which is used for chromatographic purification of the secreted toxins, for example by means of ultrafiltration and cation exchange chromatography and / or affinity chromatography on laminarin-Sepharose and / or mannoprotein-Sepharose, much easier [Cf. Example 1 and Appendix to Examples].
  • BAVC medium synthetic culture medium
  • Strain DSM 12865 produced and secreted WICALTINS can achieve a further increase in toxin production if the medium by adding the vegetable (and generally available) ß-1, 3-D-glucans laminarin in one Final concentration of 1% is supplemented. As explained in Example 14, the addition of laminarin to the culture medium leads to an induction of the WICALTIN production, which could be attributed to an induction of the transcription by Northern analyzes.
  • Synthetic B medium can be used to produce the toxin ZYGOCIN secreted by DSM 12864 [cf. Radler et al., 1993].
  • killer yeast W. californica strain 3/57 shows maximum toxin production when cultivated in BAVC medium (pH 4.7).
  • the killer yeast was first incubated for 24 h in 5 ml of YEPD medium at 30 ° C. with shaking, then completely transferred to 200 ml of BAVC medium and again cultured on the shaker (140 rpm) at 20 ° C. for 48 h.
  • the dialyzed preparation was sterile filtered through a 0.2 ⁇ m membrane and frozen in 1 ml aliquots at -20 ° C. Detection and calibration of toxin activity was carried out in the agar diffusion test on methylene blue agar (MBA; pH 4.7) against the sensitive indicator yeast Saccharomyces phenomenon visiae 192.2d. For this purpose, logarithmic dilution levels were prepared from the toxin concentrate in 0.1 M citrate-phosphate buffer (pH 4.7) and 100 ⁇ l each in previously punched holes (hole diameter 9 mm) of an MBA plate inoculated with the sensitive indicator yeast (2x10 5 cells / ml) pipetted in.
  • the concentrated WICALTIN was purified either by cation exchange chromatography on Bioscale-S (FPLC) or by affinity chromatography on an epoxy-activated Sepharose 6B matrix (from Pharmacia), to which the vegetable ⁇ -1,6-D- Glucan Pustulan has been coupled.
  • the toxin preparation enriched 625-fold in this way in its specific activity (Table 1) was gel-electrophoretically pure and showed SDS-PAGE (10-22%)
  • WICALTIN has an N-glycosidically linked carbohydrate content of about 3 kDa, which in this size also indicates a single N-glycosylation site in the protein toxin in yeast. Since the deglycosylated WICALTIN has a clearly limited toxicity, it can be concluded that the carbohydrate portion of WICALTIN is presumably necessary for binding to the sensitive target cell and thereby indirectly influences the biological activity of the toxin.
  • the first ten amino acids were determined by N-terminal amino acid sequencing of the purified killer toxin.
  • the N-terminus of WICALTIN has a significant homology to the amino terminus of the endo- ⁇ -1,3-glucanase encoded by the BGL2 gene of the yeast Saccharomyces cerevisiae.
  • glucanase activity can be detected in the unpurified toxin concentrate and in the purified toxin preparation. Both in the enzymatic test with the ß-1, 3-D-glucan laminarin as a substrate and in the fluorescence test with 4-methyl-umbelliferyl-ß-D-glucoside (MUC) as a substrate, a clear ß-1 was found in the WICALTIN preparations , 3-D glucanase activity can be detected; the ß-1, 6-D-glucan pustulan also tested was not hydrolyzed by WICALTIN.
  • MUC 4-methyl-umbelliferyl-ß-D-glucoside
  • Table 2 Spectrum of activity of WICALTIN on pathogenic and non-pathogenic yeasts of different types. All strains were tested in an agar diffusion test (MBA; pH
  • the toxin activity applied was 1x10 6 U / ml in all cases.
  • the C. tropicalis strain (patient number 541965) came from the Institute for Medical Microbiology and Hygiene at the University Clinic Mainz.
  • WICALTIN is a glycoprotein which is extremely toxic to yeasts and whose primary" target "is the cell wall ⁇ -1,3-glucans found in yeasts. Its selective Toxicity to yeasts and fungi is based on the fact that WICALTIN in the sensitive target cell destroys the structure and / or integrity of the cell wall and thus attacks yeast at its most sensitive point and ultimately kills it.
  • the virus-encoded killer toxin ZYGOCIN of the yeast Z. bailii strain 412 was according to the method of Radler et al. (1993) isolated method from the culture supernatant of killer yeast, concentrated by ultrafiltration and finally purified by affinity chromatography.
  • the purification of ZYGOCIN developed in this study in just one step uses the natural affinity of the toxin
  • Yeast cell wall mannoproteins The isolated and partially isolated method from S. cerevisiae strain 192.2d described by Schmitt & Radler (1997) Purified mannoprotein was covalently coupled to an epoxy-activated Sepharose-6B matrix (Pharmacia) and used for FPLC for column chromatographic toxin purification. According to SDS-PAGE, the biologically highly active ZYGOCIN purified in this way showed a single protein band with an apparent molecular weight of about 10 kDa (FIG. 4).
  • the spectrum of activity of the viral ZYGOCIN of the yeast Z. bailii 412 (DSM 12864) determined in the agar diffusion test includes pathogenic and non-pathogenic yeast genera, of which Candida albicans and Sporothnx schenkii are pathogens in humans and animals, and Ustilago maydis and Debaryomyces hansenii and harmful yeasts in agriculture are feared in the food sector (Tab. 3).
  • Table 3 Spectrum of action of ZYGOCIN on pathogenic and non-pathogenic yeasts of different types. All strains were tested in an agar diffusion test (MBA; pH 4.5) against a ZYGOCIN preparation with an activity of 1x10 4 U / ml.
  • ZBT ZYGOCIN-encoding ZBT gene
  • the cDNA synthesis of the toxin-coding double-stranded RNA genome of the killer yeast Z. bailii 412 was carried out in accordance with the method described by Schmitt (1995) with purified M-dsRNA denatured by methylmercury hydroxide as a template and various hexanucleotides as 'primers'. After ligation in the EcoRI-restricted vector pUC18, transformation in E. coli and isolation of the recombinant plasmids, several cDNA clones could be identified and sequenced.
  • the cDNA sequence of the ZYGOCIN-coding reading frame contains the genetic information for a precursor protein (pro-toxin) from 238 amino acids, which carries a potential Kex2-endopeptidase cleavage site in amino acid position RR 139 .
  • the biologically active ZYGOCIN, its molecular weight (10 kDa; 99 amino acids) and N-terminals result from the Kex2-mediated pro-ZYGOCIN processing that takes place in vivo in the late Golgi stage
  • Amino acid sequence exactly match the values determined for the purified ZYGOCIN.
  • heterologous expression of the ZßT cDNA in the yeast S. cerevisiae resulted in the transformed yeasts being killed by their own toxin.
  • heterologous ZYGOCIN expression in the toxin-resistant fission yeast Schizosaccharomyces pombe will be sought, since, as with the example of the viral K28 toxin, it has already been shown that the fission yeast is particularly suitable for the expression and secretion of foreign proteins.
  • the toxin genes WCT and ZBT we cloned can be cloned into so-called bidirectional pBI vectors (from CLONTECH) and used to produce transgenic plants .
  • the relevant toxin genes WCT and ZBT are brought under the transcriptional control of the strong cauliflower mosaic virus promoter (CaMV-P).
  • CaMV-P cauliflower mosaic virus promoter
  • the WICALTIN-encoding WCT gene was cloned as a 930 bp EcoRI / Smal fragment into the generally available 2 ⁇ vector pYX242.
  • the resulting vector pSTH2 (FIG. 6) contains the toxin gene under transcriptional control of the yeast's own triose phosphate isomerase promoter (TPI) and thereby enables a constitutive expression of WICALTIN after transformation in yeast (S. cerevisiae).
  • the split yeast behaves resistant to WICALTIN and ZYGOCIN both as an intact cell and as a cell wall-free spheroplast, it is suitable as a host for the heterologous expression of the toxins in question.
  • a vector was constructed (pTZ ⁇ / ⁇ ; Figure 7), the carries a secretion and processing signal (S / P) which is functional in S. pombe and which originates from the cDNA of the viral K28 preprotox gene of the yeast S. cerevisiae [cf.
  • the secretion and processing signal ensures that the 'in-frame' downstream protein in the split yeast is imported into the lumen of the endoplasmic reticulum and thus introduced into the secretion pathway of the yeast. Due to the Kex2p cleavage site at the C-terminus of the S / P region, the desired foreign protein in a late Golgi compartment is split off from its intracellular transport vehicle by the yeast's own Kex2p endopeptidase and can finally be used as a biologically active protein ( ZYGOCIN and / or WICALTIN) are secreted into the external medium.
  • a biologically active protein ZYGOCIN and / or WICALTIN
  • WICALTIN Since purified WICALTIN has a broad spectrum of activity and also effectively kills human-pathogenic yeasts and / or fungi, it is important as a potential antimycotic. Therefore, comparative studies were carried out with WICALTIN on the topical antimycotics clotrimazole and microconazole that are currently used very frequently. First, the toxic effects of clotrimazole and
  • clotrimazole was dissolved in a concentration of 10 mg / ml in ethanol (96%); this stock solution was diluted with H 2 0 i des t and used in concentrations of 0.1 to 10 mg / ml of 100 ⁇ l each in the MBA test.
  • the inhibition zone diameter was between 12 and 32 mm when an amount of 10-50 ⁇ g clotrimazole was used.
  • a stock solution of 100 ⁇ g / ml in DMSO (100%) was prepared from miconazole, which in the same way as clotrimazole in the MBA test for biological activity against Sporothrix spec. was investigated.
  • the use of 0.08-0.3 ⁇ g miconazole led to inhibition zones between 22 and 36 mm in the 'bioassay'.
  • the biological activities of 10 ⁇ g clotrimazole and 0.08 ⁇ g miconazole correspond to that
  • Example 13 Detection of the WICALTIN-coding WCT gene of the yeast W. californica 3/57
  • nucleic acid according to SEQ ID No. 1 can be used to produce a WICALTIN-specific DNA probe for a subsequent Southern hybridization
  • a DIG-labeled, 930 bp long DNA probe was used to detect the WCT gene cloned into the vector pSTH1.
  • the vector pSTH1 constructed represents a derivative of the commonly available prokaryotic cloning vector pBR322.
  • the agarose gel electrophoresis shown in FIG. 9 and the corresponding Southern blot show beyond any doubt that the WICALTIN-encoding WCT gene can be detected with the nucleic acid probe produced.
  • the yeast strain DSM 12865 was added in 300 ml BAVC medium or in BAVC medium with the addition of 0.03% of the vegetable ⁇ -1,3 -D-Glucans Laminarin cultivated for 48 h at 20 ° C with gentle shaking (60 rpm) and used after different times to prepare the total RNA. All samples (10 ml) were pre-RNA
  • Isolation set to an equal cell number of 1.8 x 10 8 cells / ml and electrophoresed in denaturing agarose-formaldehyde gels.
  • a size of 1,100 bases could be detected for the WCT transcript both under non-inducing conditions (BAVC medium without additive) and in the laminarin-supplemented BAVC medium.
  • BAVC medium without additive BAVC medium without additive
  • a maximum WCT expression was reached towards the end of the exponential growth phase (after 19 h); the hybridization signals, which become significantly weaker in the stationary growth phase, indicate a weakened transmission out there.
  • the WCT transcript Under inducing culture conditions (in the presence of laminarin) the WCT transcript showed a significantly higher intensity after 10 h than in the non-induced culture, so that it can be concluded that the transcription of the WICALTIN-encoding WCT gene by adding ß- 1, 3-D glucans can be induced.
  • Biotin in 5 g KH 2 PO. ⁇ / 50 ml aqua dest. to solve.
  • Folic acid in 50 ml distilled water. dissolve with the addition of a few drops of dilute NaOH.
  • Riboflavin in 500 ml distilled water. and dissolve a few drops of HCI while heating. The remaining vitamins are in a little aqua dest. soluble.
  • the pH of the BAVC medium was adjusted to 4.7 by adding KOH.
  • Glucose and stock solutions were sterilized separately.
  • Amino acid, vitamin and trace element stock solutions were sterilized in flowing steam at 100 ° C for 20 minutes and then added to the autoclaved BAVC medium.
  • SEQ ID No. 1 DNA and derived amino acid sequence of the WCT-encoded protein toxin WICALTIN of the yeast Williopsis californica strain 3/57.
  • SEQ ID No. 2 cDNA and deduced amino acid sequence of the ZßT-encoded protein toxin ZYGOCIN of the yeast Z. bailii
  • Figure 1 N-terminal amino acid sequences of the W californica toxin WICALTIN and the endo- ⁇ -1,3-glucanase Bgl2 of the yeast S. cerevisiae. The only deviation of the otherwise identical partial sequences is shown in bold (Bgl2p sequence according to Klebl & Tanner, 1989)
  • FIG. 2 Kinetics of WICALTIN-treated cells of the sensitive yeast S. cerevisiae 192.2d in the presence (2a) and absence (2b) of the ⁇ -D-glucans laminarin (L) and pustulan (P).
  • the toxin used had a total activity of 4.0 x 10 5
  • Figure 3 (a, b, c, d): Agar diffusion test to detect a WICALTIN sensitivity / resistance in Kre1 + and Kre1 " strains of the yeast S. cerevisiae. By transforming the WICALTIN-resistant krel zero mutant S. cerevisiae
  • FIG. 4 (A) Gel electrophoretic analysis (SDS-PAGE) of the ZYGOCINS produced and secreted by the yeast Z. bailii strain 412 (DSM 12864) according to affinity
  • Figure 5 Schematic structure of a ZBT or WCT-bearing expression vector for the production of transgenic plants.
  • RB, LB right and left border sequences of the natural Ti plasmid from Agrobacterium tumefaciens
  • CaMV-P 35S promoter of the cauliflower mosaic virus
  • NOS-P, NOS-T transcription promoter and terminator of nopaline synthase
  • kan R Kanamycin resistance gene from Streptococcus for selection in E. coli
  • NPT-II Neomycin phosphotransferase gene from the transposon Tn5 for selection in plants.
  • FIG. 6 (A) Partial restriction map of the episomal vector pSTH2 for the heterologous expression of the WICALTIN-encoding toxin gene WCT in the yeast Saccharomyces cerevisiae.
  • Vector pSTH2 is a constructed plasmid based on the commercially available 2 ⁇ multi-copy vector pYX242, into which the WCT gene from strain DSM 12865 was cloned as a 930 bp EcoRI / Smal fragment.
  • the toxin in question is under the transcriptional control of the yeast's own TPI
  • Figure 7 Scheme of the structural structure of the vector pTZ ⁇ / ⁇ for the heterologous expression and secretion of foreign proteins (in particular WICALTIN and ZYGOCIN) in the fission yeast Schizosaccharomyces pombe.
  • P nm t ⁇ , nmt ⁇ transcription promoter and terminator of the thiamine-regulated nmtl gene of the split yeast S. pombe
  • S / P secretion and processing sequence of the viral K28 preprotoxin of the shoot yeast S. cerevisiae
  • arsl autonomously replicating sequence from chromosome 1 of the fission yeast
  • Ieu2 marker gene for the selection of leucine-prototrophic transformants from S. pombe
  • Figure 8 Comparative biological activities of purified WICALTIN, clotrimazole and miconazole; the specified molar amounts are generated in a 'bioassay' (agar diffusion test) against the sensitive indicator yeast Sporothrix spec. an inhibitor diameter of 12 mm.
  • FIG. 9 Detection of the WICALTIN-encoding WCT gene of the yeast W. californica 3/57 (DSM 12865) encoded in pSTHI (pBR322 derivative) by agarose gel electrophoresis (A) and Southern hybridization with a DIG-labeled WCT probe (B).
  • A agarose gel electrophoresis
  • B Southern hybridization with a DIG-labeled WCT probe
  • Figure 10 Northem analysis for transcriptional induction of the WICALTIN-encoding WCT gene of the yeast W californica 3/57 (DSM 12865) under non-inducing culture conditions in BAVC medium (A) and under inducing conditions in BAVC medium with additive of 0.03% laminarin (B).
  • the total RNA isolated from strain DSM 12865 was electrophoresed in a denaturing agarose / formaldehyde gel at constant voltage (7 V / cm). The RNA was hybridized on a nylon membrane against a WICALTIN-specific, DIG-labeled DNA probe (630 bp) and detected by chemiluminescence.
  • Lanes 1-8 correspond to the time of sampling to isolate the total RNA: lane 1, 10 h; Lane 2, 15 h; Lane 3, 19 h; Lane 4, 24 h; Lane 5, 33 h; Lane 6, 38 h; Lane 7, 43 h; Lane 8, 48 h]
  • microorganisms used in the context of the present invention were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Maschenroder Weg 1, 38124 Braunschweig, Federal Republic of Germany, recognized as an international depository, in accordance with the requirements of the Budapest Treaty for the international recognition of the deposit of microorganisms deposited for the purpose of patenting (filing number; filing date):
  • K28 a new double-stranded RNA killer virus of Saccharomyces cerevisiae. Mol. Cell. Biol. 10: 4807-4815.
  • Schmitt, MJ & F. Radler (1996). dsRNA viruses encode killer toxins in the yeast Saccharomyces. BioEngineering 2: 30-34. Schmitt, MJ & G. Schernikau (1997). Construction of a cDNA-based K1 / K2 / K28 triple killer strain of Saccharomyces cerevisiae. Food Technol. Biotechnol. 35: 281-285. Schmitt, MJ & P. Compain (1995). Killer toxin resistant kre12 mutants of Saccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor. Arch. Microbiol. 164: 435-443.

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Abstract

L'invention concerne la préparation par technique génétique, de toxines protéiques à base de levures (dites levures tueuses), pour lutter contre des levures et/ou des moisissures pathogènes chez l'homme et les végétaux, qui sont tuées de manière sélective. La grande spécificité permet d'utiliser les toxines protéiques comme antimycotique et/ou comme fongicide. Ces toxines protéiques peuvent également s'utiliser dans le domaine phytosanitaire.
EP00945695A 1999-07-05 2000-05-31 Nouveaux antimycotiques et fongicides, procede permettant de les preparer et leur utilisation Withdrawn EP1196608A2 (fr)

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