WO2005095975A2 - Anti-fungal target genes - Google Patents

Anti-fungal target genes Download PDF

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Publication number
WO2005095975A2
WO2005095975A2 PCT/GB2005/001289 GB2005001289W WO2005095975A2 WO 2005095975 A2 WO2005095975 A2 WO 2005095975A2 GB 2005001289 W GB2005001289 W GB 2005001289W WO 2005095975 A2 WO2005095975 A2 WO 2005095975A2
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WO
WIPO (PCT)
Prior art keywords
protein
polynucleotide
sequence
fungal
dna
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Application number
PCT/GB2005/001289
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French (fr)
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WO2005095975A3 (en
Inventor
Daniel Scott Tuckwell
Michael John Bromley
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F2G Ltd
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Publication date
Priority claimed from GB0407613A external-priority patent/GB0407613D0/en
Priority claimed from GB0418895A external-priority patent/GB0418895D0/en
Application filed by F2G Ltd filed Critical F2G Ltd
Publication of WO2005095975A2 publication Critical patent/WO2005095975A2/en
Publication of WO2005095975A3 publication Critical patent/WO2005095975A3/en

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    • 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
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus

Definitions

  • the present invention relates to a method of screening for an anti-fungal agent, to fungal genes, and to diagnosis and therapy of fungal infections.
  • polyenes e.g., amphotericin B
  • azoles e.g., ketoconazole or itraconazole
  • echinocandins e.g., caspofungin
  • flucytosine e.g., flucytosine
  • amphotericin B interacts with membrane sterols to produce pores allowing leakage of cytoplasmic components and subsequent cell death.
  • Azoles work by inhibition of the 14 ⁇ -demethylase via a cytochrome P450 dependent mechanism. This leads to a depletion of the membrane sterol ergosterol and the accumulation of sterol precursors resulting in a plasma membrane with altered fluidity and structure.
  • Echinocandins work by the inhibition of the cell wall synthetic enzyme ⁇ -glucan synthase. This leads to abnormal cell wall formation, osmotic sensitivity and cell lysis.
  • Flucytosine is a pyrimidine analogue interfering with cellular pyrimidine metabolism as well DNA, RNA and protein synthesis.
  • widespread resistance to flucyotosine limits its therapeutic use.
  • the currently available antifungal agents act primarily against only two cellular targets; membrane sterols (ployenes and azoles) and ⁇ -glucan synthase (echinocandins). Resistance to both azoles and polyenes has been widely reported leaving only the recently introduced echinocandins to combat invasive fungal infections. As the use of echinocandins increases resistance by fungi will inevitably occur.
  • the inventors have found a set of twenty two genes which are essential for the viability of fungal cells. This finding allows the identification of anti-fungal agents based on their ability to target these genes.
  • the invention provides a set of twenty two proteins which can be used to screen for anti-fungal agents.
  • a set of twenty two proteins from Aspergillus fumigatus (see Table I) is provided.
  • the invention provides the following: - a method of identifying an anti-fungal agent which targets an essential protein or gene of a fungus comprising contacting a candidate substance with (i) a protein which comprises the sequence shown by SEQ LD NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90 (ii) a protein which has 50% identity with (i), (iii) a protein comprising a fragment of (i) or (ii) which fragment has a length of at least 50 amino acids, (iv) a polynucleotide that comprises sequence which encodes (i), (ii) or (iii), (v) a polynucleotide comprising sequence which has at least 70% identity with the coding sequence of (iv), and determining whether the candidate substance binds or modulates (i), (ii), (iii), (iv) or (v), wherein binding or
  • an isolated protein or polynucleotide of the invention - an organism which is transgenic for a polynucleotide of the invention, - an organism which has been genetically engineered to render a polynucleotide or protein of the invention non-functional or inhibited.
  • an antibody which is specific for a protein of the invention - a method for preventing or treating a fungal infection comprising administering an anti-fungal agent identified by the screening method of the invention, and a fungus which has been killed, or whose growth has been impaired, by inhibition of the expression or activity of a protein or polynucleotide of the invention.
  • proteins of the invention and polynucleotide sequences (termed “proteins of the invention” and “polynucleotides of the invention” herein), including homologues and/or fragments of the fungal proteins and polynucleotides, to identify anti-fungal agents.
  • An essential fungal gene may be defined as one which, when disrupted genetically (for example when not expressed) in a fungus, prevents survival or significantly retards growth of the cell on minimal or defined medium, or in guinnea pigs, mice, rabbits or rats infected with the fungus.
  • the protein of the invention is able to complement such an effect of the genetic disruption. Thus the protein may cause survival (viability) of a fungal cell which does not express its native protein.
  • a protein or polynucleotide of the invention may be defined by similarity in sequence to a another member of the family. As mentioned above this similarity may be based on percentage identity (for example to the sequences shown in the sequence listing).
  • the protein or polynucleotide of the invention may align with other proteins or polynucleotides of the invention (as shown in SEQ LD Nos. 1-3, 5-7, 9-11, 13-15, 17- 19, 21-23, 25-27, 29-31, 33-35, 37-19, 41-43, 45-17, 49-51, 53-55, 57-59, 61-63, 65- 67, 72-74, 76-78, 80-82, 84-86, 88-90).
  • the protein or polynucleotide of the invention may be in isolated form (such as non- cellular form), for example when used in the method of the invention.
  • the polynucleotide may comprise native, synthetic or recombinant polynucleotide, and the protein may comprise native, synthetic or recombinant protein.
  • the polynucleotide or protein may comprise combinations of native, synthetic or recombinant polynucleotide or protein, respectively.
  • the polynucleotides and proteins of the invention may have a sequence which is the same as, or different from, naturally occurring polynucleotides and proteins.
  • references to polynucleotides and proteins being “isolated from” a particular organism include polynucleotides and proteins which were prepared by means other than obtaining them from the organism, such as synthetically or recombinantly.
  • the polynucleotide or protein is isolated from a fungus, more preferably a filamentous fungus, even more preferably an Ascomycete.
  • the polynucleotide or protein is isolated from an organism selected from Aspergillus; Blumeria; Candida; Colletotrichium; Cryptococcus; Encephalitozoon; Fusarium; Histoplasma; Leptosphaeria; Magnaporthe; Mycosphaerella; Neurospora, Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Saccharomyces; Schizosaccharomyces, Trichophyton; and Ustilago.
  • an organism selected from Aspergillus; Blumeria; Candida; Colletotrichium; Cryptococcus; Encephalitozoon; Fusarium; Histoplasma; Leptosphaeria; Magnaporthe; Mycosphaerella; Neurospora, Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia
  • the polynucleotide or protein is isolated from Aspergillus.
  • the polynucleotide or protein is isolated from an organism selected from the species Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans;
  • Mycosphaerella graminicola Neurospora crassa; Phytophthora capsici;
  • Phytophthora infestans Plasmopara viticola; Pneumocystis jiroveci; Puccinia coronata; Puccinia graminis; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Saccharomyces cerevisiae; Schizosaccharomyces pombe; Trichophyton inter digitale; Trichophyton rubrum; and Ustilago maydis.
  • the polynucleotide or protein is isolated from an organism selected from Aspergillus fumigatus;.
  • the polynucleotide, and preferably the protein, may be isolated from A. fumigatus AF293. Variants of the above mentioned polynucleotides and proteins are also provided, and are discussed below.
  • RNA sequences are given in the sequence listing with Thymidine (T), although it is understood that in vivo Undine (U) would be present.
  • T Thymidine
  • U in vivo Undine
  • the protein of the invention may comprise an amino acid sequence substantially as set out and independently selected from any of SEQ ID Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90, or variants thereof.
  • the polynucleotide of the invention may comprise DNA, such as genomic DNA.
  • the polynucleotide may comprise a sequence substantially as set out and independently selected from any of SEQ ID Nos. 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 72, 76, 80, 84 or 88, or complements, or variants thereof.
  • the polynucleotide may comprise RNA, preferably mRNA, preferably spliced mRNA.
  • the polynucleotide comprises substantially the sequence shown as SEQ ID Nos: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 73, 77, 81, 85 or 89, or a complement, or a variant thereof.
  • the protein is encoded by the regions of sequences SEQ LD Nos. 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 72, 76, 80, 84 or 88, as described in the column "gDNA” in Table I, or a complement, or a variant thereof.
  • the polynucleotide encodes a protein which comprises substantially the amino acid sequences SEQ TD Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90, or a variant thereof.
  • amino acid/polynucleotide/protein an amino acid, polynucleotide or protein produced naturally from biological sources either in vivo or in vitro.
  • synthetic amino acid/polynucleotide/protein is meant an amino acid, polynucleotide or protein which has been produced artificially or de novo using a DNA or protein synthesis machine known in the art.
  • recombinant amino acid/polynucleotide /protein is meant an amino acid, polynucleotide or protein which has been produced using recombinant DNA or protein technology or methodologies which are known to the skilled technician.
  • variant and the terms “substantially the amino acid/polynucleotide/protein sequence” are used herein to refer to related sequences. As discussed below such related sequences are typically homologous to (share percentage identity with) a given sequence, for example over the entire length of the sequence or over a portion of a given length.
  • the related sequence may also be a fragment of the sequence or of a homologous sequence.
  • a variant protein may be encoded by a variant polynucleotide.
  • variant and the terms “substantially the amino acid/polynucleotide/ protein sequence” we mean that the sequence has at least 30%, preferably 40%, more preferably 50%, and even more preferably, 60% sequence identity with the amino acid/polynucleotide/protein sequences of any one of the sequences referred to.
  • Calculation of percentage identities between different amino acid/polynucleotide/ protein sequences may be carried out as follows.
  • a multiple alignment is first generated by the ClustalX program (pairwise parameters: gap opeining 10.0, gap extension 0.1, protein matrix Gonnet 250, DNA matrix IUB; multiple parameters: gap opening 10.0, gap extension 0.2, delay divergent sequences 30%>, DNA transition weight 0.5, negative matrix off, protein matrix gonnet series, DNA weight IUB; Protein gap parameters, residue-specific penalties on, hydrophilic penalties on, hydrophilic residues GPSNDQERK, gap separation distance 4, end gap separation off).
  • the percentage identity is then calculated from the multiple alignment as (N/T)*100, where N is the number of positions at which the two sequences share an identical residue, and T is the total number of positions compared.
  • percentage identity can be calculated as (N/S)*100 where S is the length of the shorter sequence being compared.
  • the amino acid/polynucleotide/protein seqences may be synthesised de novo, or may be native amino acid/polynucleotide/protein sequence, or a derivative thereof. An amino acid/polynucleotide/protein sequence with a greater identity than 65% to any of the sequences referred to is also envisaged.
  • amino acid/polynucleotide/ protein sequence with a greater identity than 70% to any of the sequences referred to is also envisaged.
  • An amino acid/polynucleotide/protein sequence with a greater identity than 75% to any of the sequences referred to is also envisaged.
  • An amino acid/polynucleotide/protein sequence with a greater identity than 80% to any of the sequences referred to is also envisaged.
  • the amino acid polynucleotide/protein sequence has 85% identity with any of the sequences referred to, more preferably 90% identity, even more preferably 92% identity, even more preferably 95% identity, even more preferably 97% identity, even more preferably 98% identity and, most preferably, 99% identity with any of the referred to sequences.
  • percentage identities may be measured over the entire length of the original sequence or over a region of 15, 20, 50 or 100 amino acids/bases of the original sequence.
  • percentage identity is measured with reference to SEQ LD Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90.
  • the variant protein has at least 40% identity, such as at least 60% or at least 80% identity with SEQ ID Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90, or a portion of SEQ LD Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90.
  • a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID Nos. 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 72, 73, 76, 77, 80, 81, 84, 85, 88 or 89, or their complements under stringent conditions.
  • a substantially similar protein may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90. Such differences may each be additions, deletions or substitutions.
  • nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • suitable variants are those having homologous nucleotide sequences " but comprising all, or portions of, sequence which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and metliionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Certain organisms, including Candida are known to use non-standard codons compared to those used in the majox ⁇ ty of eukaryotes.
  • mitocliondrial sequences may use the TGA codon for tryptophan rather than as a stop codon. Any comparisons of polynucleotides and proteins from such organisms and/or organelles with the sequences given here should take these differences into account.
  • Align program Use of the Align program is also preferred (http://www.gwdg.de/ ⁇ dhepper/download/; Hepperle, D., 2001: Multicolor Sequence Alignment Editor. Institute of Freshwater Ecology and Inland Fisheries, 16775 Stechlin, Germany), although others, such as JalView or Cinema are also suitable.
  • variants also include a fragment of the relevant polynucleotide or protein sequences, including a fragment of the homologous sequences (which have percentage identity to a specified sequence) referred to above.
  • a polynucleotide fragment will typically comprise at least 10 bases, such as at least 20, 30, 50, 100, 200, 500 or 1000 bases.
  • a protein fragment will typically comprise at least 10 amino acids, such as at least 20, 30, 50, 80, 100, 150, 200, 300, 400 or 500 amino acids.
  • the fragments may lack at least 3 amino acids, such as at least 10, 20 or 30 amino acids of the amino acids from either end of the protein.
  • the invention provides a method of screening which may be used to identify modulators of the proteins or polynucleotides of the invention, such as inhibitors of expression or activity of the proteins or polynxicleotides of the invention.
  • a candidate substance is contacted with a protein or polynucleotide of the invention and whether or not the candidate substance binds or modulates the protein or polynucleotide is detennined.
  • the modulator may promote (agonise) or inhibit (antagonise) the activity of the protein.
  • a therapeutic modulator (against fungal infection) will inhibit the expression or activity of protein or polynucleotide of the invemtion.
  • the method may be carried out in vitro (inside or outside a cell) or in vivo.
  • the method is carried out on a cell, c ell culture cell extract.
  • the cell may or may not be a cell in which the polynucleotide or protein is naturally present.
  • the cell may or may not be a fungal cell, or may or may not be a cell of any of the fungi mentioned herein.
  • the protein or polynucleotide may be present in a non-cellular form in the method, thus the protein may be in the form of a recombinant protein purified from a cell.
  • Methods which determine whether a candidate substance is able to bind the protein or polynucleotide may comprise providing the protein or polynucleotide to a candidate substance and determining whether binding occurs, for example by meas ⁇ iring the amount of the candidate substance which binds the protein or polynucleotide.
  • the binding may be determined by measuring a characteristic of the protein or polynucleotide that changes upon binding, such as spectroscopic changes.
  • the assay format may be a 'band shift' system. This involves determining whether a test candidate advances or retards the protein or polynucleotide on gel electrophoresis relative to the absence of the compound.
  • the method may be a competitive binding method. This determines whether the candidate is able to inhibit the binding of the protein or polynucleotide to an agent which is known to bind to the protein or polynucleotide, such as an antibody specific for the protein.
  • Whether or not a candidate substance modulates the activity of the protein may be determined by providing the candidate substance to the protein under conditions that permit activity of the protein, and determining whether the candidate substance is able to modulate the activity of the product.
  • the activity which is measured may be any of the activities of the proteins of the invention mentioned herein, including dehydrogenase, galactosidase, glycophosphatidyl inositol derivitisation, kinase, helicase, maxim-m acidification, nucleotide polymerase, oxidoreductase, transferase or vesicle motility activities.
  • the screening method comprises carrying out a reaction in the presence and absence of the candidate substance to determine whether the candidate substance inhibits the activity of the protein of the invention.
  • a candidate substance is contacted with a cell heterozygous for an underexpressed, mutated, disrupted or deleted copy of the gene, and the extent to which the candidate substance inhibits growth of the cell is determined by any suitable means and compared to the effects of the candidate substance on cells homozygous for unaltered copies of the gene.
  • the heterozygous cell will show greater sensitivity to substances that inhibit the gene or its ene product.
  • Suitable candidate substances which can tested in the above methods include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies). Furthermore, combinatorial libraries, defined chemical identities, peptide and peptide numerics, oligonucleotides and natural product libraries , and display libraries (e.g. phage display libraries) may also be tested.
  • the candidate substances may be chemical compounds. Batches of the candidate substances may be used in an initial screen of, for example, ten substances per reaction, and the substances from batches which show inhibition tested individually.
  • a polynucleotide or protein of the invention for use as a medicament or in diagnosis.
  • the polynucleotide or protein may be modified prior to use, preferably to produce a derivative or variant thereof.
  • the polynucleotide or protein may be derivatised.
  • the protein may be modified by epitope tagging, addition of fusion partners or purification tags such as glutathione S-transferase, multiple histidines or maltose binding protein, addition of green fluorescent protein, covalent attachment of molecules including biotin or fluorescent tags, incorporation of selenomethionine, inclusion or attachment of radioisotopes or fluorescent/non-fuorescent lanthanide chelates.
  • the polynucleotide may be modified by methylation or attachment of digoxygenin (DIG) or by addition of sequence encoding the above tags, proteins or epitopes.
  • DIG digoxygenin
  • the medicament is adapted to retard or prevent a fungal infection.
  • the fungal infection may be in human, animal or plant.
  • the polynucleotide or protein may be used for the development of a drug.
  • the polynucleotide or protein may be used in, or for the generation of, a molecular model of said polynucleotide or said protein.
  • a polynucleotide or protein of the invention for the preparation of a medicament for the treatment of a fungal infection.
  • the polynucleotide or protein may be modified prior to use, preferably to produce a derivative or variant thereof.
  • the polynucleotide or protein may be derivatised.
  • the polynucleotide or protein may not be modified or derivatised.
  • the medicament is adapted to retard or prevent a fungal infection.
  • the treatment may comprise retarding or preventing fungal infection.
  • the drug and/or medicament comprises an inhibitor.
  • the drug or medicament is adapted to inhibit expression and/or activity of the polynucleotide of the invention-, or a fragment thereof, and/or the function of the protein of the invention or a fragpnent thereof.
  • the fungal infection comprises an infection by a fungus, more preferably an Ascomycete, and even more preferably, an organism selected from the genera Aspergillus; Blumeria; Candida; Colletotrichium; Cryptococcus; Encephalitozoon; Fusarium; Histoplasma; Leptosphaeria; Magnaporthe; Mycosphaerella; Neurospora, Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Trichophyton; and Ustilago.
  • a fungus more preferably an Ascomycete, and even more preferably, an organism selected from the genera Aspergillus; Blumeria; Candida; Colletotrichium; Cryptococcus; Encephalitozoon; Fusarium; Histoplasma; Leptosphaeria; Magnaporthe; Mycosphaerella; Neurospora, Phytophthora; Pla
  • the fungal infection comprises an infection by an organism of the genus Aspergillus.
  • the fungal infection comprises an infection by an organism selected from the species Aspergillus flavus; Aspergillus fumigatus; Aspergillus ni ⁇ vdans;
  • Histoplasma capsulata Leptosphaeria nodorum; Magnaporthe gr ⁇ isea;
  • the fungal infection comprises an infection by Aspergillus fumigatus.
  • a method of detecting the presence of a fungal infection in an individual comprising ⁇ ng:- (i) obtaining a sample from an organism; and (ii) detecting in the said sample the presence of a polynucleotide or protein of the invention.
  • the individual may be a person (human) or animal (such as a mammal or bird) or a plant.
  • the fungal infection may arise from infection with an organism s. elected from the genera Aspergillus; Blumeria; Candida; Colletotrichium; Cryptococcus; Encephalitozoon; Fusarium; Histoplasma; Leptosphaeria; dagnaporthe; Mycosphaerella; Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Trichophyton; and Ustilago
  • the fungal infection may arise from infection with an organism selected from the species Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans - Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria gramirzis; Candida albicans; Candida cruzei; Candida glabrata; Candida parapsiloszs; Candida tropicalis; Colletotrichium trifolii; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium graminarium; Fusarium solani; Fusarium sporotrichoides; Histoplasma capsulata; Leptosphaeria nodorum; Magnaporthe grisea; Mycosphaerella graminicola; Phytophthora capsici; Phytophthorcz infestans; Plasmopara viticola; Pneumocystis jirov
  • the sample comprises a biological sample which, preferably, comprises nucleic acid and/or protein.
  • the nucleic acid or protein is purified (at least partially) from the sample before the detection is performed.
  • the sample may comprise sputum, bronchoalveloar lavage, urine, respiratory specimens, endotracheal aspirates, sterile specimens obtained by an invasrve procedure such as vitreous tap, tympanocentesis, brain biopsy or aspiration, na_sal or sinus specimens, blood, tissue or autopsy.
  • the sample may comprise rice leaf or rice stem.
  • said detecting of the presence in the said sample of a polynucleoticLe as defined by the invention comprises use of at least one oligonucleotide pair adapted to be used for amplification of DNA, preferably genomic, more preferably, fu-ngal genomic DNA.
  • the amplification may be PCR amplification.
  • said detecting comprises subjecting the amplified DNA to size analysis, preferably, electrophoresis and, preferably, comparing the results to a positive control and, preferably, a negative control.
  • Said detecting may also comprise sequencing of the amplified DNA to demonstrate the correct sequence.
  • said detecting of the presence in the said sample of a protein comprises use of a monoclonal or polyclonal antibody directed to part or all of the protein of the invention.
  • a recombiznant DNA molecule or vector comprising a polynucleotide of the invention.
  • the recombinant DNA molecule or vector may comprise an expression cassette.
  • the recombinant DNA molecule or vector comprises an expression vector.
  • the polynucleotide sequence is operatively linked to an expression control sequence.
  • a suitable control sequence may comprise a promoter, an enhancer etc.
  • a cell containing a polynucleotide, recombinant DNA molecule or vector of the invention a polynucleotide, recombinant DNA molecule or vector of the invention.
  • the cell may be transformed or transfected with the polynucleotide, recombinant DNA molecule or vector by suitable means.
  • the cell produces a recombinant protein of the invention.
  • the invention also provides an organism which is transgenic for the polynucleotide of the invention (whose cells may be the same as the cells of the invention mentioned herein).
  • Such an organism is typically a fungus, such as any genera or species of fungus mentioned herein.
  • the organism may be microorganism, such as a bacterium, virus or yeast.
  • the organism may be a plant, animal (including birds and mammals), such as any of the animals mentioned herein.
  • the organism may be produced by introduction of the polynucleotide of the invention into a cell of the organism, and in the case of a multicellular organism allowing the cell to grow into a whole organism.
  • a cell in which a native polynucleotide or protein of the invention protein is non-functional and/or inhibited is non-functional and/or inhibited.
  • the cell may be of, or present in, a multicellular organism.
  • the cell may be a mutant cell.
  • the cell is typically a fungal cell, such as of any genera or species of fungus mentioned herein.
  • a preferred means of generating the cell is to modify the polynucleotide of the invention, such that the polynucleotide is non-functional. This modification may be to cause a mutation, which disrupts the expression or function of a gene product. Such mutations may be to the nucleic acid sequences that act as 5' or 3' regulatory sequences for the polynucleotide, or may be a mutation introduced into the coding sequence of the polynucleotide. Functional deletion of the polynucleotide may be, for example, by mutation of the polynucleotide in the form of nucleotide substitution, addition or, preferably, nucleotide deletion.
  • the polynucleotide may be made non- functional and/or inhibited by: (i) shifting the reading frame of the coding sequence of the polynucleotide; (ii) adding, substituting or deleting amino acids in the protein encoded by the polynucleotide; or (iii) partially or entirely deleting the DNA coding for the polynucleotide and/or the upstream and or downstream regulatory sequences associated with the polynucleotide. (iv) inserting DNA into the coding or non-coding regions.
  • a preferred means of introducing a mutation into a polynucleotide is to utilize molecular biology techniques specifically to target the polynucleotide which is to be mutated. Mutations may be induced using a DNA molecule.
  • a most prefened means of introducing a mutation is to use a DNA molecule that has been especially prepared such that homologous recombination occurs between the target polynucleotide and the DNA molecule. When this is the case, the DNA molecule, which may be double stranded, may contain base sequences similar or identical to the target polynucleotide to allow the DNA molecule to hybridize to (and subsequently recombine with) the target.
  • inhibitors include agents that prevent transcription of the polynucleotide, or prevent translation, expression or disrupt post-translational modification.
  • the inhibitor may be an agent that increases degradation of the gene product (e.g. a specific proteolytic enzyme).
  • the inhibitor may be an agent which prevents the polynucleotide product from functioning, such as neutralizing antibodies.
  • the inhibitor may also be an antisense oligonucleotide, or any synthetic chemical capable of inhibiting expression of the gene or the stability and/or function of the protein.
  • the inhibitor may also be a protein which interacts with a protein of the invention to prevent its function.
  • the inhibitor may also be an RNA molecule which causes inhibition by RNA interference.
  • the antisense polynucleotide or RNA molecule which causes RNA interference is an example of a polynucleotide of the invention.
  • an antibody exhibiting immunospecificity for a protein of the invention may be used as a diagnostic reagent.
  • the antibody may be monoclonal or polyclonal, and may be raised in mouse, rat, rabbit, chicken, turkey, horse, goat or donkey.
  • the antibody may be raised against one or all of the proteins together, or may be raised against proteolytic or recombinant fragments.
  • the term "antibody”, unless specified to the contrary, includes fragments which bind a protein of the invention. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR- grafted antibodies or humanised antibodies.
  • any of the therapeutic substances e.g. proteins, polynucleotides or modulators
  • Any such substance may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The substance may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.
  • the substance is formulated for use with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, com starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g.
  • Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
  • Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymefhylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • a therapeutically effective non-toxic amount of substance is administered.
  • the dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical daily dose is from about 0.1 to 50 mg per kg, preferably from about O.lmg/kg to lOmg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • daily dosage levels are examples of daily dosage levels.
  • Modulators identified by the method of the invention may be administered to plants in order to prevent or treat fungal infections.
  • the modulators are normally applied in the form of compositions together with one or more agriculturally acceptable carriers or diluents and can be applied to the crop area or plant to be treated, simultaneously or in succession 'with further compounds.
  • the modulators of the invention can be applied together with carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation.
  • Suitable carriers and diluents coreespond to substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
  • a prefened method of applying the modulators of the present invention or an agrochemical composition which contains them is leaf application.
  • the number of applications and the rate of application depend on the intensity of infection by the fungus.
  • the active ingredients can also penetrate the plant through the roots via the soil (systemic action) by impregnating the locus of the plant with a liquid composition, or by applying the compounds in solid form to the soil, e.g. in granular fomi (soil application).
  • the active ingredients may also be applied to seeds (coating) by impregnating the seeds either with a liquid formulation containing active ingredients, or coating them with a solid formulation. In special cases, further types of application are also possible, for example, selective treatment of the plant stems or buds.
  • the active ingredients are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances.
  • the methods of application such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.
  • Advantageous rates of application are normally from 50g to 5kg of active ingredient (a.i.) per hectare ("ha", approximately 2.471 acres), preferably from lOOg to 2kg a.i./ha, most preferably from 200g to 500g a.i./ha.
  • the formulations, compositions or preparations containing the active ingredients and, where appropriate, a solid or liquid adjuvant are prepared in known manner, for example by homogeneously mixing and/or grinding active ingredients with extenders, for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).
  • Suitable solvents include aromatic hydrocarbons, preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol, monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl formamide, as well as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil; or water.
  • aromatic hydrocarbons preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthal
  • the solid carriers used e.g. for dusts and dispersible powders are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite.
  • Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand, hi addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.
  • suitable surface-active compounds are nonionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties.
  • surfactants will also be understood as comprising mixtures of surfactants.
  • Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds.
  • Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon atoms), for example the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained for example from coconut oil or tallow oil.
  • the fatty acid methyltaurin salts may also be used.
  • fatty sulfonates especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.
  • the fatty sulfonates or sulfates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammoniums salts and have a 8 to 22 carbon alkyl radical which also includes the alkyl moiety of alkyl radicals, for example, the sodium or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture of fatty alcohol sulfates obtained from natural fatty acids.
  • These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts.
  • the sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 8 to 22 carbon atoms.
  • alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product.
  • corresponding phosphates e.g. salts of the phosphoric acid ester of an adduct of p- nonylphenol with 4 to 14 moles of ethylene oxide.
  • Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 3O glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.
  • non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit.
  • non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol.
  • Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.
  • Cationic surfactants are preferably quaternary ammonium salts which have, as N- substituent, at least one C 8 -C 22 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals.
  • the salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium bromide.
  • the agrochemical compositions usually contain from about 0.1 to about 99% preferably about 0.1 to about 95%, and most preferably from about 3 to about 90% of the active ingredient, from about 1 to about 99.9%, preferably from about 1 to 99%, and most preferably from about 5 to about 95%> of a solid or liquid adjuvant, and from about 0 to about 25%, preferably about 0.1 to about 25%>, and most preferably from about 0.1 to about 20% of a surfactant.
  • a solid or liquid adjuvant preferably about 0 to about 25%, preferably about 0.1 to about 25%>, and most preferably from about 0.1 to about 20% of a surfactant.
  • Example 1 Identification of essential genes in. Aspergillus fumigatus Essential regions of the A. fumigatus genome were identified using the mycobank technology as described in patent WO00177295 Al with the following modifications:
  • P24 lines 11-18 Conidia ⁇ A. fumigatus) were collected from a stable diploid transformant colony and approximately 3xlO 4 spores were used to inoculate 1 ml of SAB broth containing lmg/ l FPA. This culture was incubated with shaking (200 rpm) at 37°C for 20 hours. lOO ⁇ l of the culture was spread onto complete media containing 0.2 mg/ml FPA and incubated at 37 °C for 3 days or until rapidly growing sectors emerged. Conidia were collected from each sector and plated onto nitrate, nitrite and hypoxanthine media and the nitrogen utilisation profiles of the resulting conidia assessed.
  • Colonies with the nitrogen utilisation profiles of the parental strains indicated breakdown of the diploid to a haploid. Multiple haploid sectors were isolated. None of the haploids were hygromycin resistant indicating the insertion of the hph gene into a portion of the genome required for function.
  • P25 line 9 Plasmid pAN7-l linearised with Hindlll was used as the transforming vector. PAN7-1 carries the hph gene which confers hygromycin resistance. P25 lines 17-20: 1 ml of cold YED was added to the cuvette and incubated at 37 °C for 1 h. Aliquots were spread on selective agar (complete media with 250 ⁇ g/ml hygromycin). Colonies growing on selective media were deemed putative transformants.
  • the points of insertion were identified using the plasmid rescue method outlined on page 31 lines 5-17.
  • the insertion sites were confirmed by employing PCR: Using the sequence obtained from plasmid rescue data, complementary primers were designed within the predicted sequence near the point of insertion. A primer corresponding to the sequence of pAN7-l was also used. Genomic DNA samples isolated from diploids of experiments 1745, 1775, 1784, 1859, 1871, 2008, 2009, 2022, 2083, 2139, 2610, 2905, 2975, 2994, 2998, 3026, 3034, 3336, 3345, 3508, 3524 and 3640 were used as templates. The resulting sequences are given as SEQ J-D Nos. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 75, 79, 83, 87 and 91 respectively. The relationship between the individual experiments and the SEQ LD numbers is given in Table I.
  • Example 2 Characterisation of the essential genes 2.1 Genome analysis
  • the A. fumigatus database www.TIGR.org or www.sanger.ac.uk
  • E. coli Select96 cells (Promega) are used in accordance with manufacturers' instructions.
  • A. fumigatus clinical isolate AF293 (ref. No. NCPF7367; available to the public from the NCPF repository; Bristol, U.K.); the CBS repository (Belgium) or from Dr. David Denning' s clinical isolate culture collection, Hope Hospital, Salford. U.K.) is the prefened strain according to the present invention.
  • AF293 was isolated in 1993 from the lung biopsy of a patient with invasive aspergillosis and aplastic anaemia. It was donated by Shrewsbury PHLS.
  • A. fumigatus genomic DNA To obtain mycelial material for genomic DNA isolation, approximately IO 7 A. fumigatus conidia are inoculated in 50 ml of Vogel's minimal medium and incubated with shaking at 200 rpm until late exponential phase (18-24 h) at 37°C. Mycelium is dried down onto Whatmann 54 paper using a Buchner funnel and a side-arm flask attached to a vacuum pump and washed with PBS/Tween. At this point, the mycelium can be freeze-dried for extraction at a later date.
  • the mycelium (fresh or freeze dried) is ground to a powder using liquid nitrogen in a - 20°C cooled mortar.
  • the ground biomass is transfened to 50 ml tubes on ice up to the 10 ml mark.
  • An equal volume of extraction buffer (0.7 M NaCl; 0.1 M Na 2 SO 3 ; 0.1 M Tris-HCl pH 7.5; 0.05 M EDTA; l%(w/v) SDS; pre-warmed to 65°C) is then added to each tube, mixed thoroughly with a pipette tip and incubated at 65°C for 20 minutes in a water bath.
  • a volume of chloroform/isoamyl alcohol (24:1) equivalent to the volume of the original biomass is then added to each tube, tubes are mixed thoroughly and incubated on ice for 30 min. Tubes are then centrifuged at 3,500 x g for 30 min and the aqueous phase carefully transferred to fresh 50 ml tubes without disturbing the interface.
  • the pellet is suspended in 2 ml sterile water. 1 ml of 7.5 M ammonium acetate is added, mixed and incubated on ice for 1 hour. Tubes are centrifuged at 12,000 x g for 30 min, the supernatants transfened to a fresh tube and 0.54 volumes of isopropanol are added, mixed and incubated at room temperature for at least 15 minutes. Tubes are then centrifuged at 5,930 x g for IO min, the supernatant is removed and the pellet washed in 1 ml of 70%> ethanol. Tub>es are centrifuged at 5,930 x g for 10 min and all the ethanol is removed.
  • the pellet is air dried for 20-30 minutes at room temperature and suspended in 0.5-1.0 ml of TE (L 0 mM Tris-HCl pH 7.5; lmM EDTA) Finally, the DNA is treated with RNase A (5 ⁇ l of 1 mg/ml stock).
  • Primers pairs are designed to the upstream and downstream regions of the A. fumigatus AF293 genes: The 200 base regions flanking the predicted gene of interest are used as input sequence for Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3 www. cgi) to provide a primer pair that spans the gene. If the gene is particularly long, it may be necessary to design primer pairs with internal sequences and thus sequence the gene in parts.
  • the PCR product is then ligated into pGEM-Teasy (Promega) using the following ligation mixture: 2x Buffer 5 ⁇ l pGEM Teasy 1 ⁇ l
  • the reaction is incubated over-night at 4° C.
  • 2 ⁇ l of the ligation mix are then added to Select 96 cells (Promega) and incubated for 20 min on ice. Cells are then heat shocked at 42° C for 45 sees and placed back on ice. 250 ml of room temp. SOC medium is then added and the cells incubated for 1 hour at 37° C, with shaking at 220 rpm. 50 and 200 ⁇ l amounts are then plated on to LB agar plates containing ampicillin (100 ⁇ g/ml), 50 ⁇ l X-gal (4%) and 10 ⁇ l LPTG (100 mM) and incubated over night at 37° C.
  • Plasmid DNA is extracted using Qiagen miniprep kit according to the manufacturers instructions. 1 ⁇ l of plasmid DNA is digested with restriction enzymes for 1 hour at 37° C. Results are compared with the predicted sizes for constructs and clones showing the correct restriction digest pattern are sequenced at MWG Biotech UK Ltd, Waterside House, Peartree Bridge, Milton Keyn s, MK6 3BY.
  • Example 3 cDNA sequencing and RACE The internal sequences of the genes of interest are experimentally determined by cloning and sequencing cDNA, and the 5' and 3 ' ends of the genes are determined by RACE (Rapid Amplification of cDNA Ends).
  • RNA and cDN ⁇ are prepared as described in Example 2.2.2. Cultures are harvested by filtration, then washed twice with DEPC-treated water and transfened to a 50 ml Falcon tube. Samples are frozen in liquid nitrogen and stored at -80°C until required. To prepare RNA, fungal samples are ground to a fine powder under liquid nitrogen.
  • RNA is then extracted using the Qiagen RNeasy Plant Mini Kit following the protocol for isolation of total RNA from filamentous fungi in the RNeasy Mini Handbook (06/2001, Pages 75-78, http://www.qiagen.com/literature/ handbooks/ma/mamini/1016272HBRNY_062001WW.pdf).
  • step 3 RLC is used as the Lysis buffer of choice;
  • step 7 the Rneasy column is incubated for 5 min at room temperature after addition of RW1;
  • step 9a is canied out;
  • step 10 30 ⁇ l _RNase-free water is added, the samples incubated for 10 min at room temperature, and then centrifuged;
  • step 11 the elution step is repeated to give a total volume of 60 ⁇ l RNA.
  • DNA contamination is removed from the RNA by the addition of Dnase, using 2 ⁇ l DNase per ⁇ g RNA, in the presence of 10X DNase buffer and incubating at 37° C for 2h.
  • DNase-treated RNA is cleaned up using the RNeasy Plant Mini Kit following the RNeasy Mini Protocol for RNA Cleanup (RNeasy Mind Handbook 06/2001, pages 79- 81).
  • RNA-free RNA DNA-free RNA
  • 3 ⁇ l oligo (dT) 100 ng/ ⁇ l
  • DEPC-treated water a total volume of 42 ⁇ l.
  • Samples are incubated in a heat block at 65°C for 5 min after which they are allowed to cool slowly to room temperature.
  • 2 ⁇ l Ultrapure dNTPs, l ⁇ l reverse transcriptase (Stratascript) and 5 ⁇ l 10X reverse transcriptase reaction buffer (Stratascript) are added. Samples -ure incubated at 42°C for lh, denatured at 90°C for 5 min and then cooled on ice.
  • PCR is carried out using the cDNA above to generate cDNA fragments. Primers are designed based on the 5' and 3' ends of the predicted genes. PCR reactions are carried out using the following reagents and conditions: PCR Master Mix
  • Cycles 2-4 are run 40 times in total, bp.
  • the PCR products are purified using Qiagen' s QIAquick PCR Purification Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, Westshire, RH10 9AX, UK) according to the manufacturers instructions and run on agarose gels.
  • PCR products are ligated into pGEM-Teasy, used to transform Select 96 cells, and sequenced as described in 2.2.4 above.
  • A. fumigatus biomass is prepared as described in 2.2.2.
  • RNA is prepared using the FastRNA kit (QBIOgene) following the manufacturer's instructions (Revision 6030- 999-1J05) with the following amendments:
  • At step 1 40 mg of biomass is used per extraction;
  • samples are processed for 20 seconds at speed 5, incubated on ice for 3 minutes, and processed again for 20 seconds at speed 5;
  • At step 3 samples are centrifuged for 5 minutes;
  • 500 ⁇ l DLPS are added, mixed, and incubated- at room temperature for 2 minutes. Samples are mixed again and incubated for a furthex 2 minutes;
  • two washes in 250 ⁇ l SEWS are carried out;
  • the pellet is disolved in 50 ⁇ l SAFE buffer.
  • RNA 1 ⁇ g total RNA prepared as described above is de-phosphorylated in a 10 ⁇ l reaction using 10 units of calf intestinal phosphate (CIP), 1 ⁇ l 10X CIP buffer and 40U RNaseOutTM (made up to 10 ⁇ l in DEPC water) at 50°C for 1 hour. Samples are tnen made up to 100 ⁇ l with DEPC water and the RNA extracted with 100 ⁇ l (25:24 :1) phenohchloroform: isoamyl alcohol. RNA is then precipitated by the addition of 2 ⁇ l mussel glycogen (lOmg/ml), 10 ⁇ l 3M sodium acetate, pH 5.2 and 220 ⁇ l 95% ethanol and the sample frozen on dry ice for 10 minutes. RNA is pelleted by centrifugatiori- at 14,500 rpm for 20 minutes at 4°C, washed with 70% ethanol, air dried and ore- suspended in 8 ⁇ l DEPC water.
  • CIP calf intestinal
  • RNA De-phosphorylated RNA (7 ⁇ l) is de-capped in a 10 ⁇ l reaction with 0.5 U tobacco acid pyrophosphatase (TAP), 1 ⁇ l lOx TAP buffer and 40 U RnaseOutTM for 1 hour- at 37°C. RNA is extracted with phenohchloroform and precipitated as above, and then ire- suspended in 7 ⁇ l DEPC-treated water.
  • TAP tobacco acid pyrophosphatase
  • RNA Oligo (0.25 ⁇ g) and incubated at 65°C for 5 minutes. A 10 ⁇ l ligation reaction is then set up by the addition of 1 ⁇ l lOx ligase buffer, 1 ⁇ l 10 mM ATP, 40 U
  • First-strand cDNA is prepared by the addition of 1 ⁇ l GeneRacerTM Oligo dT primer and 1 ⁇ l dNTP mix (lOmM each) to 10 ⁇ l ligated RNA and incubated at 65°C for 5 minutes.
  • the following reagents are added to the 12 ⁇ l ligated RNA and primer mix; 4 ⁇ l 5x first strand buffer, 2 ⁇ l 0.1 M DTT, 1 ⁇ l RNaseOutTM and 1 ⁇ l SuperscriptTM II RT (200 U/ ⁇ l) and incubated first at 42°C for 50 minutes and then, to stop the reaction, at 70°C for 15 minutes. 2 U RNase H is added to the reaction mix and incubated at 37°C for 20 minutes.
  • a 50 ⁇ l PCR reaction is set up using 1 ⁇ l of the RACE- ready cDNA prepared above, 1 ⁇ l GeneRacerTM 5' primer, 1 ⁇ l reverse gene-specific primer (designed against the complementary strand of the coding sequence: 5 pmol/ ⁇ l stock), 1 ⁇ l dNTP solution (10 mM each), 2 ⁇ l 50 mM MgSO 4 , 5 ⁇ l High Fidelity PCR buffer, 0.5 ⁇ l Platinum® Taq DNA Polymerase High Fidelity (5 U/ ⁇ l) and 38.5 ⁇ l sterile water. Cycling parameters are given in Table II below.
  • a second, nested PCR stage may also be carried out. This is set up using 1 ⁇ l of the RACE cDNA from the first stage above, 1 ⁇ l Nested 5' primer (supplied with kit), 1 ⁇ l second reverse gene-specific primer (designed against the complementary strand of the coding sequence and nested with respect to the above primer: 5 pmol/ ⁇ l stock), 1 ⁇ l dNTP solution (10 mM each), 2 ⁇ l 50 mM MgSO 4 , 5 ⁇ l High Fidelity PCR buffer, 0.5 ⁇ l Platinum® Taq DNA Polymerase High Fidelity (5 U/ ⁇ l) and 38.5 ⁇ l sterile water. Cycling parameters are given in Table II below.
  • a 50 ⁇ l PCR reaction is set up using 1 ⁇ l of the RACE-ready cDNA prepared above, 1 ⁇ l GeneRacerTM 3' primer (10 ⁇ M), 1 ⁇ l forward gene-specific primer (designed against the coding strand of the coding sequence : 5 pmol/ ⁇ l stock), 1 ⁇ l dNTP solution (10 mM each), 2 ⁇ l 50 mM MgSO 4 , 5 ⁇ l High Fidelity PCR buffer, 0.5 ⁇ l Platinum® Taq DNA Polymerase High Fidelity (5 U/ ⁇ l) and 38.5 ⁇ l sterile water. Cycling parameters are given in Table II below: A second, nested PCR stage may also be carried out.
  • 5' and 3' RACE identify the 5' ATG and 3' stop codons as well as giving the 5' and 3' untranslated regions of the genes.
  • homologs of the proteins or polynucleotides of the invention are identified in other fungi by means of bioinformatics analysis. Sequences identified by bioinformatics can be used to design primers which in turn can be used in PCR to generate DNA coding for the homologs.
  • degenerate PCR can be used to obtain sequence, which can then be used to generate probes for screening cDNA or genomic libraries of the organism of interest to identify clones containing the homologs.
  • Southern blots using fragments of genes from one species as probes can be used to identify the presence of a homolog in the genome of a second species. The same probe can then be used to screen cDNA or genomic DNA libraries. Once clones conesponding to the novel genes have been identified they can be expressed for functional characterisation of the protein.
  • Homologs of the proteins and polynucleotides of the invention can be identified by searching locally held databases, as detailed in Table III, using BLAST with SEQ ID Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90 as the query sequence. Where necessary, matching contigs are down-loaded and genes predicted from genomic DNA by Genscan analysis, blast searches, alignment and visualisation with Artemis as described in Example 2.
  • This dataset contains ESTs from the following plant pathogen fungi: Blumeria graminis, Botryotinia, Cladosporium fulvum, Colletotrichum trifolii, Cryphonectria parasitica, Fusarium sporotrichioides, Gibberella zeae, Leptosphaeria maculans, Magnaporthe grisea, Mycosphaerella graminicola, Phytophthora infestans, Phytophthora sojae, Sclerotinia sclerotiorum, Ustilago maydis and Verticillium dahliae
  • Protein and nucleic acid multiple alignments are generated by means of programs such as ClustalX (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882;) and/or using manual alignment editors such as Align (http://www.gwdg.de/ ⁇ dhepper/ download/; Hepperle, D., 2001: Multicolor Sequence Alignment Editor. Institute of Freshwater Ecology and Inland Fisheries, 16775 Stechlin, Germany).
  • SEQ ID Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90, and hits identified from blast searches above can be clarified by phylogenetic analysis, for example using the PHYLLP suite of programs (Felsenstein, Felsenstein, J., 2002. PHYLIP (Phylogeny Inference Package) version 3.6a3. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle). A distance matrix is generated using PROTDIST with the Jones- Taylor-Thornton model and a tree infened using FITCH with global reanangements and 10 jumbles of input order.
  • Fungal cultures are prepared using methods suitable for particular species. For example, Aspergillus and Candida species, Cryptococcus neoformans, Fusarium solani and Trichophyton species are maintained on Sabouraud dextrose agar at 30- 35°C; Leptosphaeria nodorum on Malt agar medium (30 g/L malt extract; 15 g/L Bacto-agar, pH 5.5), 24.0°C; Magnaporthe grisea on Oatmeal agar (6.7 g/L agar, 53.3 g/L instant oatmeal) 25.0°C, or Cornmeal agar (Difco 0386), 26.0 C; Phytophthora capsici cultures were maintained on on V-8 agar at 24°C; Pyricularia oryzae cultures were maintained on rice polish agar at 24°C under white fluorescent lights (12 hr artificial day), and were subculrured every 7 - 14 days by the transfer of mycelial plugs to fresh plates; Pyth
  • Genomic DNA is prepared from cultures using standard methodologies, e.g. using the Qiagen DNeasy Plant Kit, or using methods described in Example 2.2. 4.2.2 PCR
  • Primers are designed to conespond to regions conserved between the gene of interest and its homologs (identified as described above). Those skilled in the art will appreciate that it may be necessary to try a range of primer pairs. PCR reactions using the primer pairs are set up as follows:
  • the PCR products are purified (to remove residual enzymes and nucleotides) using Qiagen' s QIAquick PCR Purification Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, Westshire, RHIO 9AX, UK) according to the manufacturers instructions and eluted into 40 ⁇ l of sterile water (BDH molecular biology grade/filter sterile).
  • the purified PCR products are examined on 1% agarose gels.
  • degenerate PCR may require variations in a number of parameters in the attempt to generate a product. These include primer concentration, template concentration, concentration of Mg 2+ ions, elongation and annealing times, and annealing temperature. Variations in temperature can be accomodated by the use of a gradient PCR machine.
  • the purified PCR products are cloned into pPEM-Teasy (Promega) and then transformed into XLIO-Gold ® Kan ultracompetent E. coli cells according to the manufacturers instructions.
  • the transformation reactions are then plated onto LB agar plates containing ampicillin (100 ⁇ g/ml), 50 ⁇ l X-gal (4%) and 10 ⁇ l LPTG (100 mM). Following overnight incubation at 37°C, individual white colonies from each transformation are sub-cultured into LB broth containing ampicillin (100 ⁇ g/ml). After overnight incubation at 37°C with shaking, plasmids are extracted using Qiagen spin mini plasmid extraction kits according to the manufacturers instructions and sent away for full-length sequencing.
  • Genomic DNA from the fungi of interest are digested with the appropriate restriction enzyme and run on 0.8 % agarose gel. The gel is then submerged in 250 mM HCl for no more than 10 mins, with shaking, at room temperature, after which the gel is rinsed with sterilised RO water.
  • Transfer of the DNA onto nylon membrane is carried out using 0.4 M NaOH. Transfer protocols and apparatus are well known and are described in e.g. Sambrook et al., (1989), Molecular Cloning, 2 nd Edition., Cold Spring Harbor Laboratory Press. After transfer, the DNA is fixed to the membrane by baking at 120°C for 30 min. The membrane can then be used immediately, or stored dry for future use.
  • Probes are generated either by restriction digests of DNA or by PCR of an appropriate region.
  • a suitable probe can be generated by PCR using a primer pair designed using Primer3(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_ www.cgi), A. fumigatus genomic DNA, and the methods give in 4.2.2.
  • 1 ⁇ g DNA template is diluted in molecular biology water to a total volume of 16 ⁇ l, denatured in a boiling water bath for 10 mins, and quickly chilled on ice.
  • the membrane is placed in a hybridisation tube containing 20 ml of prehybridisation solution (DIG Easy Hyb, Roche) per 100cm 2 of membrane surface area and prehybridised at 42°C for 2 hours in a hybridisation oven.
  • the DIG- labelled probe is denatured by heating in a boiling water bath for 10 min and then chilled directly on ice.
  • the probe is then diluted to -200 ng/mL in hybridisation solution (Easy Hyb, Roche; at least 5 mL of hybridisation solution is required per hybridisation).
  • the prehybridisation solution is discarded from the hybridization tube and the hybridisation solution containing the DIG-labelled probe added quickly.
  • the hybridisation then proceeds overnight at a 42°C in the hybridisation oven.
  • the optimum temperature is dependant on probe size and homology with target sequence and is determined empirically.
  • the membrane is washed twice at 42°C, 5 mins per wash, with 50 mL of stringency wash solution (3 x SSC, 0.1% SDS; where 20 x SSC buffer is 3 M NaCL, 300 mM sodium citrate, pH 7.0), followed by two washes at RT, 15 min per wash, in 50 mL stringency wash solution.
  • stringency wash solution 3 x SSC, 0.1% SDS; where 20 x SSC buffer is 3 M NaCL, 300 mM sodium citrate, pH 7.0
  • the stringency of these washes can be decreased by increasing the SSC concentration to 6 x SSC, 0.1% SDS and/or decreasing the wash temperatures.
  • the membrane is washed in 20 mL washing buffer (100 mM maleic acid, 150 mM NaCl; pH 7.5; 0.3% v/v Tween 20), and then incubated successively with the following; 20 mL blocking solution (1% w/v blocking reagent for nucleic acid hybridisation, Roche, dissolved in 100 mM maleic acid, 150 mM NaCl, pH 7), for 30 min at room temperature; Anti-DIG-alkaline phosphatase (Roche) diluted 1:5,000 in blocking buffer, 30 min at room temperature; Washing buffer, two washes each of 15 min at room temperature; Detection buffer (lOOmM Tris-Hcl, 100 mM NaCl; pH 9.5), 2 min at room temperature.
  • 20 mL washing buffer 100 mM maleic acid, 150 mM NaCl; pH 7.5; 0.3% v/v Tween 20
  • 20 mL blocking solution 1% w/v blocking reagent for nucleic
  • the membrane is then removed, placed on top of an acetate sheet, and ⁇ 0.5 ml (per 100cm 2 ) of CSPD or CDP-star added to the top of the membrane.
  • a second sheet of acetate is then placed over the surface of the membrane, the assembly incubated for 5 min at room temperature and then sealed in a plastic bag.
  • the assembly is then exposed to X-ray film for between 15 min and 1 hour. Optimal exposure time is determined empirically by increasing exposure time up to 24 hours.
  • the presence of a band on the gel is evidence of a gene in the genomic DNA of interest.
  • the molecular weight of the band depends on the size of the restriction fragment that contains the gene.
  • Example 5 Expression during infection of wax moth larvae (Galleria melonella) and mice infected with A. fumigatus
  • Wax moth larvae have been shown to be good model systems in which to study Candida infection (Cotter et al., 2000, FEMS Immunol Med Microbiol 27, 163-9; Brennan et al., 2002, FEMS Immunol Med Microbiol 34, 153-7). We have found that this insect system is also a good system in which to study Aspergillus infection (D. Law and J. Rooke, manuscript in preparation).
  • Wax moth larvae were purchased from Livefood UK, Somerset, UK (www.livefood.co.uk), and were maintained in the dark at room temperature in wood shavings prior to infection. Healthy larvae (250 mg +/- 50 mg) were selected and incubated at 4°C for 10 minutes immediately prior to infection to immobilise them. Larvae were then injected through the cuticle of the left last pro-leg with 10 ⁇ l spore suspension (lOOx stock), using a sterile Hamilton syringe. Larvae were then transfereed to a sterile Petri dish.
  • the following controls were also established: Larvae injected with 10 ⁇ l PBS/Tween only; larvae injected with 10 ⁇ l heat killed spores (killed by incubation for 20 min 100°C); larvae pierced but not injected; and untouched larvae. Larvae were incubated at 30°C and monitored at least twice daily. All treatments and controls were carried out on batches of 10 larvae. Larval deaths and general health condition was recorded every 24 hrs and dead or moribund larvae were removed from the test group.
  • RNA-free RNA from Aspergillus fumigatus-infected wax moth larvae was prepared from the following sources: Uninfected larvae; larvae after 48h infection with A. fumigatus (early infection); larvae after 72h infection with A. fumigatus (late infection); larvae infected with heat-killed A. fumigatus spores; and A. fumigatus grown in Sabaraud Dextrose agar broth for 16hr.
  • Frozen larvae were ground to a fine powder under liquid nitrogen in a mortar and pestle previously baked at 22°C overnight, treated with RNaseZAP, rinsed with DEPC- treated water (0.1% (v/v) DEPC, stured for lh and autoclaved for lh) and cooled with liquid nitrogen.
  • step 5a was carried out; At step 6, 30 ⁇ l RNase-free water was added, samples incubated for 10 min at room temperature and then centrifuged for 1 min at 14,000 RPM; At step 7, the eluate from step 6 was transfened onto the RNeasy column, incubated for 10 min at room temperature, and then centrifuged for 1 min at 14,000 RPM. A sample of the DNase-treated RNA was run on an agarose gel, quantified and stored at -80°C.
  • PCR was carried out using primers that amplify the ⁇ -tubulin gene (SEQ LD Nos. 69 and 70). In the absence of a reverse-transcription step, only gDNA will be detected and thus any gDNA contamination will be revealed.
  • the following reaction mixture was set up:
  • genomic DNA was present and the sample was DNase-treated again. If the PCR was negative, no DNA was present in the sample.
  • mice were infected with Aspergillus fumigatus and organs harvested as follows. Thirteen male CDl mice were injected with the immunosuppressant cyclophosphamide (0.025 g/ml; 200 mg/kg) LV via the tail vein. After 72 hours, twelve mice were injected with 0.15 ml Aspergillus fumigatus AF293 conidia (7.5 x 10 5 /ml). 11 hours after infection, four mice were sacrificed with an overdose of inhaled halothane. The brain, lungs, liver and kidney were removed, frozen by immersion in liquid nitrogen, and stored at -70°C. A further four mice were also sacrificed at 24 and 48 hours after infection.
  • RNA was prepared from mouse tissues as described for wax moth larvae above (5.1.2 and 5.1.3).
  • cDNA was prepared from DNA-free RNA using the Promega Reverse Transcription kit, following the protocol as supplied with the product (Technical Bulletin No. 099, http://www.promega.com/tbs/tb099/tb099.pdf). In a modification to the protocol, the cDNA synthesis reaction was incubated for 60 min at 42°C rather than for the suggested 15 min. Samples were stored in 5-1 O ⁇ l aliquots at -20°C.
  • the gDNA is omitted and the amount of water increased conespondingly.
  • 25 ⁇ l 1 is pipetted into 8 wells on a multiwell plate, and each well run at a different temperature (between 50 and 65°C) with the following conditions:
  • Steps 2-4 are run for 30 cycles
  • the PCR products are run on a 2% agarose gel.
  • the real-time primers designed above are further tested to ensure that mouse nucleic acid is not amplified using these primers.
  • Four reactions are set up, each containing the following: 12.5 ⁇ l Abgene Reddymix 1 ⁇ l each primer 9.5 ⁇ l H2O and either; 1 ⁇ l infected mouse kidney cDNA (50 ng/ul; experimental); 1 ⁇ l uninfected mouse kidney cDNA (50 ng/ul; uninfected control); 1 ⁇ l AF293 gDNA (10 ng/ ⁇ l; positive control); 1 ⁇ l water (negative control).
  • Stepl 95°C - 5 min
  • Step2 95°C - 1 min
  • Step6 8°C - hold Steps 2-4 were run 40 times
  • PCR products are run on a 2% agarose gel. If primers are species-specific, bands should be seen with A. fumigatus genomic DNA as template, but not with uninfected mouse cDNA as template.
  • reaction Four reactions are set up containing 72 ⁇ l of the above mix and either; 3 ⁇ l H 2 O; 3 ⁇ l uninfected larvae cDNA (50 ng/ ⁇ l ); 3 ⁇ l AF293 gDNA (5 ng/ ⁇ l); or 3 ⁇ l infected larvae cDNA (50 ng/ ⁇ l.
  • the four reactions contain 72 ⁇ l of the above SYBR mix and either; 3 ⁇ l uninfected mouse kidney (50 ng/ ⁇ l); 3 ⁇ l infected mouse kidney - 48h post-infection (50ng/ul); or 3 ⁇ l AT293 cDNA (5ng/ ⁇ l).
  • step 7 90 cycles of step 7 are carried out with the temperature increased by 0.5°C after each cycle starting with cycle 2. Melt curve data collection and analysis enabled.
  • Recombinant proteins or fragments are expressed to enable detailed study of function and for the development of an in vitro high-throughput screen for inhibitory compounds.
  • PCR is carried out using cDNA, prepared as described above, to generate polynucleotides encoding protein sequence essentially conesponding to SEQ LD Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90.
  • Primers are designed to encode the 5' and 3' ends of the coding sequences (SEQ LD Nos. 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 73, 77, 81, 85 or 89), with the addition of a 5' C in the forward primer and a 5' AA in the reverse primer. If the protein has an N-terminal leader peptide, this should be excluded. If the protein is made up of multiple domains, it may be desirable or necessary to express only a limited number of domains, or even a single domain. In these cases, primers are designed to conespond to domain boundaries.
  • PCR reactions are carried out using the following reaction mixture and conditions. All Reagents are present in the KOD kit (Novagen).
  • Step 1 94°C 2 min Step 2 94°C 15 sec
  • reaction mixture is incubated at 22°C for 15 mins, denatured at 75°C for 5 minutes, and then placed on ice for 2 mins. 1 ⁇ l T4 DNA ligase and 1 ⁇ l Blunt pETBlue-2 vector are then added and the ligation reaction carried out at 14°C overnight.
  • Ligation mixtures are then transformed into Nova Blue chemically competent E. coli cells, and plated on to a prewarmed LBAmp selection plate. After an overnight incubation at 37° C, ampicillin-resistant colonies are selected and grown up in ampicillin-containing LB medium. Plasmid DNA is isolated using the P*lasmid Mini Kit (Qiagen). Confirmation of the presence and conect orientation of the inserts is determined by restriction analysis and sequencing of the construct.
  • Purified plasmid DNA which has been confirmed to be of the conect sequence and orientation, is transformed into chemically competent Tuner(DE3)pLacI E. coli cells (Novagen) and grown overnight at 37°C. 500 ⁇ l of an over-night culture is used to inoculate 10 ml of LB, 1% glucose, 500 ⁇ g/mL ampicillin-, 34 ⁇ g/ml chloramphenicol, and the cultures incubated at 37° C, 220 rpm until the cell density reaches an optical density of 0.6 (approximately 3 hours). Expression of the recombinant protein is then induced with IPTG (ImM) for 21 hours.
  • IPTG IPTG
  • Samples are removed from the induction at 0, 1, 2, 3, 4, 5, and 21 hours and bacteria harvested by centrifugation at 1300 rpm for 5 minutes. Lysis buffer (1 ml Bugbuster (Novagen), 1 ⁇ l Benzonase (Novagen), 10 ⁇ l protease inhibitor cocktail (Novagen)) is added to each pellet; 50 ⁇ L for 1 OD 6 oo of culture, and samples incubated for 20 minutes at room temperature. Samples are then reduced b;y the addition of 1.5 ⁇ L lOx reducing mix (Invitrogen) and 3.5 ⁇ L sample loading buffer (Invitrogen) to a 10 ⁇ L sample, incubated at 70°C for 10 minutes, and then placed on ice. Reduced samples are run on an SDS-PAGE gel and stained with Coomassie. In this way the optimal duration of induction is determined. Larger cultures, e.g. 0.5 L final volume can then be prepared in the same way.
  • Recombinant proteins are purified from pellets as follows : Supernatant from the lysis stage above is added to prewashed Ni-Nta resin at a concentration of 5-10 mg protein per ml of resin and allowed to bind for 1 hour at 4° C.
  • Protein-resin mix is then poured into a column, washed twice with 4 ml of wash buffer (2.5 ml 1M phosphate buffer pH8 , 6.25 ml 4M NaCl, 1 ml 1M imidazole pH8, 0.5 ml 10% Tween 20; made up to 50 mis in n.H 2 O) and then eluted in 4x 0.5 ml fractions with elution buffer (250 ⁇ l 1M phosphate buffer pH8, 625 ⁇ l 4M NaCl, 1.25 ml 1M imidazole pH8, 50 ⁇ l 10% Tween 20, made up to 5 ml in n.H 2 O).
  • elution buffer 250 ⁇ l 1M phosphate buffer pH8, 625 ⁇ l 4M NaCl, 1.25 ml 1M imidazole pH8, 50 ⁇ l 10% Tween 20, made up to 5 ml in n.H 2 O.
  • Recombinant proteins can be assayed using an assay type specific for the particular protein.
  • the assay can then be used to screen for inhibitors. For example:
  • Galactosidases can be screened by incubating the galactosidase with fluorogenic substrates such as fluorescein digalactoside, methylumbelliferyl galactoside or fluorescent glycosphingolipid and monitoring the increase in fluorescence.
  • fluorogenic substrates such as fluorescein digalactoside, methylumbelliferyl galactoside or fluorescent glycosphingolipid
  • Candidate substances can be included and examined for an effect on- the increase in fluorescence.
  • Helicases can be screened by incubating the helicase with an oligonucleotide containing a fluorescent analog such as 2-aminopurine and monitoring the unwinding mediated by the helicase using fluorescence spectroscopy. The degree of inhibition by candidate compounds can be assessed.
  • a fluorescent analog such as 2-aminopurine
  • Kinases can be screened by incubating the kinase with [32PJ-ATP, substrate, and candidate substance, and measuring the decrease of 32P-label incorporation into the substrate due to the presence of the substance.
  • Suitable substrates may include myelin basic protein, glycogen synthase and enolase.
  • Oxidoreductases can be screened by carrying out a redox reaction and measuring the inhibition of the reaction by detecting the amount of NADH or NADPH oxidation, for example by measuring the generation of the oxidised forms of NADH and NADPH spectroscopically at 340nm.
  • a suitable colourimetric oxidoreductase substrate may be used to measure inhibition, such as methylene blue, phenazine methosulphate or 2, 6-dichlorophenolindophenol. 7.2 Genetic screen for the identification of inhibitors
  • the m- cobank mutants can be screened for inhibitors using a generic genetic screen. These mutants are heterozygotes, that is, they cany one normal and one disrupted version of a gene. In most cases this should result in less gene product being made by the hete-rozygote than the wild type diploid. If the gene is essential for growth then the hetexozygote should be more sensitive to a compound that targets the product of that gene. This phenomenon is called haploinsufficiency and has been demonstrated in yeast (Genomic profiling of drag sensitivities via induced haploinsufficiency. Giaever G, Shoemaker DD, Jones TW, Liang H, Winzeler EA, Astromoff A, Davis RW. Nat Genet. 1999 21:278-83.)
  • the primary screen for genes of unknown function involves monitoring the growth of the mycobank heterozygous mutant versus the growth of the wild type diploid strain of Aspergillus fumigatus, in the presence and absence of a panel of compounds. Spore suspensions of these strains are set up in RPMI 1640 medium in 96-well plates. lxlO 4 cfu/ml is the inoculum used. Potential inhibitors are added to give a final concentration of 32 ⁇ g/ml. The plates are then incubated at 37°C for 48h. The OD485 of the cultures is then measured using a plate reading spectrophotometer.
  • MIC Minimal Inhibitory Concentration
  • the heterozygote mycobank mutant and the wild type diploid are incubated in the presence of a range of concentrations of the chemical.
  • the lowest concentration of chemical that prevents growth of the organism (the Minimal Inhibitory Concentration, MIC) is calculated for both strains.
  • Doubling dilutions of the compound of interest are prepared in RPMI 1640 medium in 96-well plates starting at 50 ⁇ g/ml do ⁇ v to 0.1 ⁇ g/ml in duplicate. Each well is inoculated with either wild type or mutant ⁇ Aspergillus fumigatus and the plate incubated at 37°C for 24/48h prior to measuring the D485.
  • An inhibitor of the product of the gene of unknown function will have a lower MIC in the mutant strain than in the wild type strain, i.e., a 2-fold or more difference in MIC between the 2 strains.
  • This anti-fungal compound can then be used as the basis for chemistry approaches to improve the specificity, potency and other properties of the compound.
  • Example 8 Method for detecting fungal infection
  • Suiable primer pairs are designed using Primer3 (http://frodo.wi.init.edu/cgi- bin/primer3/primer3_www.cgi).
  • Appropriate controls include; (i) template DNA but no primers; primers but no template (negative controls); (ii) cDNA encoding the gene of interest or DNA from fungal cultures instead of patient DNA (positive control). PCR reactions are run as follows:
  • PCR products are examined on agarose gels.
  • the production of a band of the conect molecular weight is diagnostic of the presence of the particular fungus. It may be additionally necessary to carry out diagnostic restriction digests of the PCR products. If necessary, PCR products are subcloned into a vector, such as pGEM-Teasy (Promega), and sequenced to verify that the PCR products are from the appropriate fungus.
  • the presence of an infection with A. fumigatus, A. nidulans, C. albicans or M. grisea, C. trifolii, F. graminearum, F. sporotrichioides or M. graminicola, or other organisms is detected by means of antibodies raised against the fungal protein.
  • One suitable means is the use of a capture ELISA.
  • microtitre plates are coated with a monoclonal antibody raised against the fungal protein. Then the plates are incubated with diluted patient samples, or appropriate protein extracts of samples (particularly if the samples are biopsies or plant tissues). Plates are then incubated with a polyclonal antibody (again against the fungal protein). Finally, binding of the second antibody was detected by means of an enzyme-coupled or fluorescently-labelled antibody directed against the polyclonal.
  • two monoclonal or polyclonal antibodies or various combinations may be used.
  • Antibodies against the proteins of the invention will be of considerable use as diagnostic reagents (see example 8 above).
  • As an immunogen recombinant proteins are used (as described in Example 6).
  • synthetic proteins or polypeptides encoding regions either unique to the individual proteins, or likely to provide cross- reactivity within a set of homologs are used.
  • Peptides may need to be conjugated to carrier proteins before immunization.
  • Preimmune sera from animals to be immunised are screened against the immunogen to ensure that there is no endogenous cross reactivity.
  • Animals typically sheep, rabbits or mice
  • the resulting sera is affinity purified using the immunogen cross-linked to a chromatography matrix.
  • purification of the antibody fraction from the seram e.g. using protein G or protein A cross-linked to a matrix, may be sufficient.
  • Monoclonal antibody production proceeds by methods familiar to those skilled in the art.
  • the specificities of the resulting polyclonal and/or monoclonal antibodies are checked by ELISA and/or western blotting using the immunogen, related constracts or whole cell lysates and extracts as targets.
  • Negative controls such as paralogous proteins, different constracts or different species are also employed to test specificity and/or to determine the range of species and/or genus cross-reactivity.
  • Example 10 Production of fungi in which the gene of interest is functionally disabled.
  • a BAG (bacterial artificial chromosome) clone library containing the A. fumigatus genome, partially digested with BamffJ and inserted into the vector pBACe3.6 was purchased from the Sanger Centre, Cambridge, UK.
  • the BAC clone containing the gene to be inactivated is identified by bioinformatics (BLAST searching of Sanger BAC and related databases) and the glycerol stock of the clone grown up in 50 ml LB, 20 ⁇ g/ml chloramphenicol at 37°C overnight. The overnight culture is centrifuged at 4,500 rpm for 15 min.
  • the bacterial pellet is resuspended in 4 ml of Buffer PI (Qiagen plasmid miniprep kit) and then 4 ml of buffer P2 (Qiagen plasmid miniprep kit, lysis buffer) is added and mixed gently by inverting 3-6 times. Proteins and genomic DNA are precipitated by adding 4 ml of buffer P3 (Qiagen plasmid miniprep kit, neutralizing buffer) and incubating on ice for 10 minutes.
  • Buffer PI Qiagen plasmid miniprep kit
  • buffer P2 Qiagen plasmid miniprep kit, lysis buffer
  • the supernatant is transfened into a 50 ml falcon tube, an equal volume of phenol/chlorophorm (1:1) mixture is added, and the mixture centrifuged for 15 min at 4500 rpm. The supernatant is then transfened into an Oakridge tube and 0.7 volumes isopropanol are added. After mixing, the tube is centrifuged at 10,000 rpm (Beckman centrifuge, rotor JA-17) for 30 min at 4°C. The resulting pellet is washed with 2 ml 70% ethanol at the same speed. The resulting BAC DNA is resuspended in 100 ⁇ l buffer EB.
  • the transposition reaction is carried out as follows. 7 ⁇ l purified BAC, 1 ⁇ l transposon pZVK2 (an engineered plasmid the sequence of which is given as SEQ LD No. 71), containing the mosaic ends of pMOD2 (Epicenter), a kanamycin resistance gene and a Zeocin resistance gene under the control of fungal promoter) and 1 ⁇ l EZ:TN transposase (Epicenter) are incubated at 37°C for two hrs after which 1 ⁇ l stop solution (1%) SDS) is added and the mixture heated to 70°C for 10 minutes. Electrocompetent GeneHogs E.
  • coli cells (Invitrogen) are then transformed with the transposed BAC, the cells plated onto LB agar, 25 ⁇ g/ml kanamycin, 20 ⁇ g/ml chloramphenicol, and plates incubated overnight at 37°C.
  • BAC DNA is then purified using the Millipore montage 96 BAC KIT using a MWG ROBOSEQ 4200 robot.
  • BACs containing the transposon inserted into the gene of interest are identified by PCRs both spanning the gene of interest and extending from the transposon into the BAC. Insertion into the gene of interest is manifested as an increase in product size. Southern blots are also carried out to ensure that the transposon has only inserted once into the BAC.
  • A. fumigatus (haploid) protoplasts are prepared using 5% Glucanex (Novo Nordisk A/S) solution (in 0.6 M KCI) and shaking for 2 h at 80 rpm in 30°C .
  • the protoplasts were washed with 0.6 M KCI and then with STC (Sorbitol, Tris, CaCl 2 ).
  • STC Session Control Tube
  • zeocin resistant colonies are picked and checked for presence of the knocked-out gene by PCR using primers which specifically amplify the whole gene of interest. Usually 10-20 transformants are checked.
  • the ectopic integration of the BAC gives two bands by PCR, one for the endogenous gene and one for the BAC/transposon construct, which has a higher molecular weight. Replacement of the endogenous gene with the transposon-modified gene results in a single band of higher molecular weigh by PCR. If none of the transformants show the disrupted endogenous gene, the gene of interest may be essential, with the knock-out cells having died and only cells where replacement is unsuccessful surviving. In this case, the transformation is canied out on diploids using the same method of transformation. Essentiality of the gene is then tested by rehaploidisation, and examining the segregation pattern in haploids.

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Abstract

Method of identifying an anti-fungal agent which targets an essential protein or gene of a fungus comprising contacting a candidate substance with (i) a protein which comprises the sequence shown by SEQ ID NOS: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90 (ii) a protein which has 50% identity with (i), (iii) a protein comprising a fragment of (i) or (ii) which fragment has a length of at least 50 amino acids, (iv) a polynucleotide that comprises sequence which encodes (i), (ii) or (iii), (v) a polynucleotide comprising sequence which has at least 70% identity with the coding sequence of (iv), and determining whether the candidate substance binds or modulates (i), (ii), (iii), (iv), or (v), wherein binding or modulation of (i), (ii), (iii), (iv), or (v) indicates that the candidate substance is an anti-fungal agent.

Description

Anti-Fungal Target Genes
Field of the invention
The present invention relates to a method of screening for an anti-fungal agent, to fungal genes, and to diagnosis and therapy of fungal infections.
Background of the invention
Invasive fungal infections are well recognised as diseases of the immunocompromised host. Over the last twenty years there have been significant rises in the number of recorded instances of fungal infection (Groll et al., 1996. Trends in the postmortem epidemiology of invasive fungal infections at a university hospital. J Infect 33, 23-32).
In part this is due to increased awareness and improved diagnosis of fungal infection.
However, the primary cause of this increased incidence is the vast rise in the number of susceptible individuals. This is due to a number of factors including new and aggressive immunosuppressive therapies, increased survival in intensive care, increased numbers of transplant procedures and the greater use of antibiotics worldwide. In certain patient groups, fungal infection occurs at high frequency; lung transplant recipients have a frequency of up to 20% colonisation and infection with a fungal organism and fungal infection in allogenic hoemopoetic stem transplant recipients is as high as 15% (Ribaud et al., 1999, Survival and prognostic factors of invasive aspergillosis after allogeneic bone marrow transplantation. Clin Infect Dis.
28:322-30). Currently only four classes of antifungal drug are available to treat systemic fungal infections. These are the polyenes (e.g., amphotericin B), the azoles (e.g., ketoconazole or itraconazole) the echinocandins (e.g., caspofungin) and flucytosine. The polyenes are the oldest class of antifungal agent being first introduced in the
1950's. The exact mode of action remains unclear but polyenes are only effective against organisms that contain sterols in their outer membranes. It has been proposed that amphotericin B interacts with membrane sterols to produce pores allowing leakage of cytoplasmic components and subsequent cell death. Azoles work by inhibition of the 14α-demethylase via a cytochrome P450 dependent mechanism. This leads to a depletion of the membrane sterol ergosterol and the accumulation of sterol precursors resulting in a plasma membrane with altered fluidity and structure. Echinocandins work by the inhibition of the cell wall synthetic enzyme β-glucan synthase. This leads to abnormal cell wall formation, osmotic sensitivity and cell lysis. Flucytosine is a pyrimidine analogue interfering with cellular pyrimidine metabolism as well DNA, RNA and protein synthesis. However widespread resistance to flucyotosine limits its therapeutic use. It can be seen that to date the currently available antifungal agents act primarily against only two cellular targets; membrane sterols (ployenes and azoles) and β-glucan synthase (echinocandins). Resistance to both azoles and polyenes has been widely reported leaving only the recently introduced echinocandins to combat invasive fungal infections. As the use of echinocandins increases resistance by fungi will inevitably occur. The identification of new classes of antifungal agent with novel modes of action is required to give the promise of positive therapeutic outcomes to patients. Novel fungal-specific genes are likely to present the best opportunity for the development of effective novel antifungal agents. To be effective drug targets, such genes should confer an essential function within the fungal cell.
Summarv of the invention
The inventors have found a set of twenty two genes which are essential for the viability of fungal cells. This finding allows the identification of anti-fungal agents based on their ability to target these genes.
The invention provides a set of twenty two proteins which can be used to screen for anti-fungal agents. In particular a set of twenty two proteins from Aspergillus fumigatus (see Table I) is provided.
Accordingly the invention provides the following: - a method of identifying an anti-fungal agent which targets an essential protein or gene of a fungus comprising contacting a candidate substance with (i) a protein which comprises the sequence shown by SEQ LD NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90 (ii) a protein which has 50% identity with (i), (iii) a protein comprising a fragment of (i) or (ii) which fragment has a length of at least 50 amino acids, (iv) a polynucleotide that comprises sequence which encodes (i), (ii) or (iii), (v) a polynucleotide comprising sequence which has at least 70% identity with the coding sequence of (iv), and determining whether the candidate substance binds or modulates (i), (ii), (iii), (iv) or (v), wherein binding or modulation of (i), (ii), (iii), (iv) or (v) indicates that the candidate substance is an anti-fungal agent, - use of (i), (ii), (iii), (iv) or (v) as defined above to identify or obtain an anti-fungal agent, - use of an anti-fungal agent identified by the method of the invention in the manufacture of a medicament for prevention or treatment of fungal infection, - a method of detecting the presence of a fungus in a sample comprising detecting the presence in the said sample of a protein or polynucleotide of the invention,
- an isolated protein or polynucleotide of the invention, - an organism which is transgenic for a polynucleotide of the invention, - an organism which has been genetically engineered to render a polynucleotide or protein of the invention non-functional or inhibited. - an antibody which is specific for a protein of the invention, - a method for preventing or treating a fungal infection comprising administering an anti-fungal agent identified by the screening method of the invention, and a fungus which has been killed, or whose growth has been impaired, by inhibition of the expression or activity of a protein or polynucleotide of the invention.
Detailed description of the invention
As mentioned above the invention relates to use of particular proteins and polynucleotide sequences (termed "proteins of the invention" and "polynucleotides of the invention" herein), including homologues and/or fragments of the fungal proteins and polynucleotides, to identify anti-fungal agents.
An essential fungal gene may be defined as one which, when disrupted genetically (for example when not expressed) in a fungus, prevents survival or significantly retards growth of the cell on minimal or defined medium, or in guinnea pigs, mice, rabbits or rats infected with the fungus. In one embodiment, the protein of the invention is able to complement such an effect of the genetic disruption. Thus the protein may cause survival (viability) of a fungal cell which does not express its native protein.
A protein or polynucleotide of the invention may be defined by similarity in sequence to a another member of the family. As mentioned above this similarity may be based on percentage identity (for example to the sequences shown in the sequence listing).
The protein or polynucleotide of the invention may align with other proteins or polynucleotides of the invention (as shown in SEQ LD Nos. 1-3, 5-7, 9-11, 13-15, 17- 19, 21-23, 25-27, 29-31, 33-35, 37-19, 41-43, 45-17, 49-51, 53-55, 57-59, 61-63, 65- 67, 72-74, 76-78, 80-82, 84-86, 88-90).
The protein or polynucleotide of the invention may be in isolated form (such as non- cellular form), for example when used in the method of the invention. The polynucleotide may comprise native, synthetic or recombinant polynucleotide, and the protein may comprise native, synthetic or recombinant protein. The polynucleotide or protein may comprise combinations of native, synthetic or recombinant polynucleotide or protein, respectively. The polynucleotides and proteins of the invention may have a sequence which is the same as, or different from, naturally occurring polynucleotides and proteins.
It is to be understood that the term "isolated from" may be read as "of herein. Therefore references to polynucleotides and proteins being "isolated from" a particular organism include polynucleotides and proteins which were prepared by means other than obtaining them from the organism, such as synthetically or recombinantly.
Preferably, the polynucleotide or protein, is isolated from a fungus, more preferably a filamentous fungus, even more preferably an Ascomycete.
Preferably, the polynucleotide or protein, is isolated from an organism selected from Aspergillus; Blumeria; Candida; Colletotrichium; Cryptococcus; Encephalitozoon; Fusarium; Histoplasma; Leptosphaeria; Magnaporthe; Mycosphaerella; Neurospora, Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Saccharomyces; Schizosaccharomyces, Trichophyton; and Ustilago.
Preferably, the polynucleotide or protein, is isolated from Aspergillus.
Preferably, the polynucleotide or protein, is isolated from an organism selected from the species Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans;
Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria graminis;
Candida albicans; Candida cruzei; Candida glabrata; Candida parapsilosis; Candida tropicalis; Colletotrichium trifolii; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium graminarium; Fusarium solani; Fusarium sporotrichoides; Histoplasma capsulata; Leptosphaeria nodorum; Magnaporthe grisea;
Mycosphaerella graminicola; Neurospora crassa; Phytophthora capsici;
Phytophthora infestans; Plasmopara viticola; Pneumocystis jiroveci; Puccinia coronata; Puccinia graminis; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Saccharomyces cerevisiae; Schizosaccharomyces pombe; Trichophyton inter digitale; Trichophyton rubrum; and Ustilago maydis.
Preferably, the polynucleotide or protein, is isolated from an organism selected from Aspergillus fumigatus;.
The polynucleotide, and preferably the protein, may be isolated from A. fumigatus AF293. Variants of the above mentioned polynucleotides and proteins are also provided, and are discussed below.
Table I. Sequences claimed and their relationship to sequences given in the sequence listing
Figure imgf000008_0001
Figure imgf000009_0001
(1)Numbers after SEQ ID Nos. correspond to bases of genomic DNA encoding the protein in cases where introns are present. ( :>RNA sequences are given in the sequence listing with Thymidine (T), although it is understood that in vivo Undine (U) would be present. (3)Base within plasmid rescue sequence at which A. fumigatus sequence starts. (4)Site of insertion in the gDNA sequence. A minus sign indicates that the point of insertion was up-stream of the 5 'ATG.
In one embodiment, the protein of the invention may comprise an amino acid sequence substantially as set out and independently selected from any of SEQ ID Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90, or variants thereof.
The polynucleotide of the invention may comprise DNA, such as genomic DNA. The polynucleotide may comprise a sequence substantially as set out and independently selected from any of SEQ ID Nos. 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 72, 76, 80, 84 or 88, or complements, or variants thereof.
The polynucleotide may comprise RNA, preferably mRNA, preferably spliced mRNA. Preferably, the polynucleotide comprises substantially the sequence shown as SEQ ID Nos: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 73, 77, 81, 85 or 89, or a complement, or a variant thereof.
Preferably, the protein is encoded by the regions of sequences SEQ LD Nos. 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 72, 76, 80, 84 or 88, as described in the column "gDNA" in Table I, or a complement, or a variant thereof.
Preferably, the polynucleotide encodes a protein which comprises substantially the amino acid sequences SEQ TD Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90, or a variant thereof.
By the term "native amino acid/polynucleotide/protein", is meant an amino acid, polynucleotide or protein produced naturally from biological sources either in vivo or in vitro.
By the term "synthetic amino acid/polynucleotide/protein", is meant an amino acid, polynucleotide or protein which has been produced artificially or de novo using a DNA or protein synthesis machine known in the art.
By the term "recombinant amino acid/polynucleotide /protein", is meant an amino acid, polynucleotide or protein which has been produced using recombinant DNA or protein technology or methodologies which are known to the skilled technician.
The term "variant", and the terms "substantially the amino acid/polynucleotide/protein sequence" are used herein to refer to related sequences. As discussed below such related sequences are typically homologous to (share percentage identity with) a given sequence, for example over the entire length of the sequence or over a portion of a given length. The related sequence may also be a fragment of the sequence or of a homologous sequence. A variant protein may be encoded by a variant polynucleotide.
By the term "variant", and the terms "substantially the amino acid/polynucleotide/ protein sequence", we mean that the sequence has at least 30%, preferably 40%, more preferably 50%, and even more preferably, 60% sequence identity with the amino acid/polynucleotide/protein sequences of any one of the sequences referred to. A sequence which is "substantially the amino acid/polynucleotide/peptide sequence" may be the same as the relevant sequence.
Calculation of percentage identities between different amino acid/polynucleotide/ protein sequences may be carried out as follows. A multiple alignment is first generated by the ClustalX program (pairwise parameters: gap opeining 10.0, gap extension 0.1, protein matrix Gonnet 250, DNA matrix IUB; multiple parameters: gap opening 10.0, gap extension 0.2, delay divergent sequences 30%>, DNA transition weight 0.5, negative matrix off, protein matrix gonnet series, DNA weight IUB; Protein gap parameters, residue-specific penalties on, hydrophilic penalties on, hydrophilic residues GPSNDQERK, gap separation distance 4, end gap separation off). The percentage identity is then calculated from the multiple alignment as (N/T)*100, where N is the number of positions at which the two sequences share an identical residue, and T is the total number of positions compared. Alternatively, percentage identity can be calculated as (N/S)*100 where S is the length of the shorter sequence being compared. The amino acid/polynucleotide/protein seqences may be synthesised de novo, or may be native amino acid/polynucleotide/protein sequence, or a derivative thereof. An amino acid/polynucleotide/protein sequence with a greater identity than 65% to any of the sequences referred to is also envisaged. An amino acid/polynucleotide/ protein sequence with a greater identity than 70% to any of the sequences referred to is also envisaged. An amino acid/polynucleotide/protein sequence with a greater identity than 75% to any of the sequences referred to is also envisaged. An amino acid/polynucleotide/protein sequence with a greater identity than 80% to any of the sequences referred to is also envisaged. Preferably, the amino acid polynucleotide/protein sequence has 85% identity with any of the sequences referred to, more preferably 90% identity, even more preferably 92% identity, even more preferably 95% identity, even more preferably 97% identity, even more preferably 98% identity and, most preferably, 99% identity with any of the referred to sequences.
The above mentioned percentage identities may be measured over the entire length of the original sequence or over a region of 15, 20, 50 or 100 amino acids/bases of the original sequence. In a preferred embodiment percentage identity is measured with reference to SEQ LD Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90. Preferably the variant protein has at least 40% identity, such as at least 60% or at least 80% identity with SEQ ID Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90, or a portion of SEQ LD Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90.
Alternatively, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID Nos. 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 72, 73, 76, 77, 80, 81, 84, 85, 88 or 89, or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 6x sodium chloride/sodium citrate (SSC) at approxmiately 45°C followed by at least one wash in 0.2x SSC/0.1% SDS at approximately 5-65°C. Alternatively, a substantially similar protein may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90. Such differences may each be additions, deletions or substitutions.
Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
Other suitable variants are those having homologous nucleotide sequences "but comprising all, or portions of, sequence which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and metliionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Certain organisms, including Candida are known to use non-standard codons compared to those used in the majoxϊty of eukaryotes. Furthermore, mitocliondrial sequences may use the TGA codon for tryptophan rather than as a stop codon. Any comparisons of polynucleotides and proteins from such organisms and/or organelles with the sequences given here should take these differences into account.
In accurate alignment of protein or DNA sequences the trade-off between optimal matching of sequences and the introduction of gaps to obtain such a match is important. In the case of proteins, the means by which matches are scored is also of significance. The family of PAM matrices (e.g., Dayhoff, M. et al, 1978, Atlas of protein sequence and structure, Natl. Biomed. Res. Found.) and BLOSUM matrices quantitate the nature and likelihood of conservative substitutions and are used in multiple alignment algorithms, although other, equally applicable matrices will be known to those skilled in the art. The popular multiple alignment program ClustalW, and its windows version ClustalX (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al, 1997, Nucleic Acids Research, 24, 4876-4882) are efficient ways to generate multiple alignments of proteins and DNA.
Use of the Align program is also preferred (http://www.gwdg.de/~dhepper/download/; Hepperle, D., 2001: Multicolor Sequence Alignment Editor. Institute of Freshwater Ecology and Inland Fisheries, 16775 Stechlin, Germany), although others, such as JalView or Cinema are also suitable.
Calculation of percentage identities between proteins occurs during the generation of multiple alignments by Clustal. However, these values need to be recalculated if the alignment has been manually improved, or for the deliberate comparison of two sequences. Programs that calculate this value for pairs of protein sequences within an alignment include PROTDIST within the PHYLIP phylogeny package (Felsenstein; http://evolution.gs.washington.edu/ phylip.html) using the "Similarity Table" option as the model for amino acid substitution (P). For DNA RNA, an identical option exists within the DNADIST program of PHYLLP.
Other modifications in protein sequences are also envisaged and within the scope of the claimed invention, i.e. those which occur during or after translation, e.g. by acetylation, amidation, carboxylation, phosphorylation, proteolytic cleavage or linkage to a ligand.
The term "variant", and the terms "substantially the amino acid/polynucleotide/protein sequence" also include a fragment of the relevant polynucleotide or protein sequences, including a fragment of the homologous sequences (which have percentage identity to a specified sequence) referred to above. A polynucleotide fragment will typically comprise at least 10 bases, such as at least 20, 30, 50, 100, 200, 500 or 1000 bases. A protein fragment will typically comprise at least 10 amino acids, such as at least 20, 30, 50, 80, 100, 150, 200, 300, 400 or 500 amino acids. The fragments may lack at least 3 amino acids, such as at least 10, 20 or 30 amino acids of the amino acids from either end of the protein. The invention provides a method of screening which may be used to identify modulators of the proteins or polynucleotides of the invention, such as inhibitors of expression or activity of the proteins or polynxicleotides of the invention. In one embodiment of the method a candidate substance is contacted with a protein or polynucleotide of the invention and whether or not the candidate substance binds or modulates the protein or polynucleotide is detennined.
The modulator may promote (agonise) or inhibit (antagonise) the activity of the protein. A therapeutic modulator (against fungal infection) will inhibit the expression or activity of protein or polynucleotide of the invemtion.
The method may be carried out in vitro (inside or outside a cell) or in vivo. In one embodiment the method is carried out on a cell, c ell culture cell extract. The cell may or may not be a cell in which the polynucleotide or protein is naturally present. The cell may or may not be a fungal cell, or may or may not be a cell of any of the fungi mentioned herein. The protein or polynucleotide may be present in a non-cellular form in the method, thus the protein may be in the form of a recombinant protein purified from a cell.
Any suitable binding or activity assay may be used. Methods which determine whether a candidate substance is able to bind the protein or polynucleotide may comprise providing the protein or polynucleotide to a candidate substance and determining whether binding occurs, for example by measαiring the amount of the candidate substance which binds the protein or polynucleotide. The binding may be determined by measuring a characteristic of the protein or polynucleotide that changes upon binding, such as spectroscopic changes.
The assay format may be a 'band shift' system. This involves determining whether a test candidate advances or retards the protein or polynucleotide on gel electrophoresis relative to the absence of the compound. The method may be a competitive binding method. This determines whether the candidate is able to inhibit the binding of the protein or polynucleotide to an agent which is known to bind to the protein or polynucleotide, such as an antibody specific for the protein.
Whether or not a candidate substance modulates the activity of the protein may be determined by providing the candidate substance to the protein under conditions that permit activity of the protein, and determining whether the candidate substance is able to modulate the activity of the product.
The activity which is measured may be any of the activities of the proteins of the invention mentioned herein, including dehydrogenase, galactosidase, glycophosphatidyl inositol derivitisation, kinase, helicase, mediu-m acidification, nucleotide polymerase, oxidoreductase, transferase or vesicle motility activities. In one embodiment the screening method comprises carrying out a reaction in the presence and absence of the candidate substance to determine whether the candidate substance inhibits the activity of the protein of the invention.
In a further embodiment of the method, a candidate substance is contacted with a cell heterozygous for an underexpressed, mutated, disrupted or deleted copy of the gene, and the extent to which the candidate substance inhibits growth of the cell is determined by any suitable means and compared to the effects of the candidate substance on cells homozygous for unaltered copies of the gene. The heterozygous cell will show greater sensitivity to substances that inhibit the gene or its ene product.
Suitable candidate substances which can tested in the above methods include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies). Furthermore, combinatorial libraries, defined chemical identities, peptide and peptide numerics, oligonucleotides and natural product libraries , and display libraries (e.g. phage display libraries) may also be tested. The candidate substances may be chemical compounds. Batches of the candidate substances may be used in an initial screen of, for example, ten substances per reaction, and the substances from batches which show inhibition tested individually.
According to a further aspect of the present invention, there is provided a polynucleotide or protein of the invention for use as a medicament or in diagnosis.
The polynucleotide or protein may be modified prior to use, preferably to produce a derivative or variant thereof. The polynucleotide or protein may be derivatised. The protein may be modified by epitope tagging, addition of fusion partners or purification tags such as glutathione S-transferase, multiple histidines or maltose binding protein, addition of green fluorescent protein, covalent attachment of molecules including biotin or fluorescent tags, incorporation of selenomethionine, inclusion or attachment of radioisotopes or fluorescent/non-fuorescent lanthanide chelates. The polynucleotide may be modified by methylation or attachment of digoxygenin (DIG) or by addition of sequence encoding the above tags, proteins or epitopes.
Preferably, the medicament is adapted to retard or prevent a fungal infection. The fungal infection may be in human, animal or plant. The polynucleotide or protein may be used for the development of a drug. The polynucleotide or protein may be used in, or for the generation of, a molecular model of said polynucleotide or said protein.
According to a further aspect of the present invention, there is provided use of a polynucleotide or protein of the invention for the preparation of a medicament for the treatment of a fungal infection.
The polynucleotide or protein may be modified prior to use, preferably to produce a derivative or variant thereof. The polynucleotide or protein may be derivatised. The polynucleotide or protein may not be modified or derivatised.
Preferably, the medicament is adapted to retard or prevent a fungal infection. The treatment may comprise retarding or preventing fungal infection. Preferably, the drug and/or medicament comprises an inhibitor. Preferably, the drug or medicament is adapted to inhibit expression and/or activity of the polynucleotide of the invention-, or a fragment thereof, and/or the function of the protein of the invention or a fragpnent thereof.
Preferably, the fungal infection comprises an infection by a fungus, more preferably an Ascomycete, and even more preferably, an organism selected from the genera Aspergillus; Blumeria; Candida; Colletotrichium; Cryptococcus; Encephalitozoon; Fusarium; Histoplasma; Leptosphaeria; Magnaporthe; Mycosphaerella; Neurospora, Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Trichophyton; and Ustilago.
Preferably, the fungal infection comprises an infection by an organism of the genus Aspergillus.
Preferably, the fungal infection comprises an infection by an organism selected from the species Aspergillus flavus; Aspergillus fumigatus; Aspergillus niάvdans;
Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria graminis;
Candida albicans; Candida cruzei; Candida glabrata; Candida parapsilosis; Candida tropicalis; Colletotrichium trifolii; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium graminarium; Fusarium solani; Fusarium sporotrichoides;
Histoplasma capsulata; Leptosphaeria nodorum; Magnaporthe gr~isea;
Mycosphaerella graminicola; Phytophthora capsici; Phytophthora infestans;
Plasmopara viticola; Pneumocystis jiroveci; Puccinia coronata; Puccinia graminis;
Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Trichophyton inter dig~itale; Trichophyton rubrum; and Ustilago maydis.
Preferably, the fungal infection comprises an infection by Aspergillus fumigatus.
According to another aspect of the present invention, there is provided a method of detecting the presence of a fungal infection in an individual, said method comprisϊng:- (i) obtaining a sample from an organism; and (ii) detecting in the said sample the presence of a polynucleotide or protein of the invention.
The individual may be a person (human) or animal (such as a mammal or bird) or a plant. The fungal infection may arise from infection with an organism s. elected from the genera Aspergillus; Blumeria; Candida; Colletotrichium; Cryptococcus; Encephalitozoon; Fusarium; Histoplasma; Leptosphaeria; dagnaporthe; Mycosphaerella; Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Trichophyton; and Ustilago
The fungal infection may arise from infection with an organism selected from the species Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans - Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria gramirzis; Candida albicans; Candida cruzei; Candida glabrata; Candida parapsiloszs; Candida tropicalis; Colletotrichium trifolii; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium graminarium; Fusarium solani; Fusarium sporotrichoides; Histoplasma capsulata; Leptosphaeria nodorum; Magnaporthe grisea; Mycosphaerella graminicola; Phytophthora capsici; Phytophthorcz infestans; Plasmopara viticola; Pneumocystis jiroveci; Puccinia coronata; Puccirt ia graminis; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Trichophyton znterdigitale;
Trichophyton rubrum; and Ustilago maydis.
Preferably, the sample comprises a biological sample which, preferably, comprises nucleic acid and/or protein. In one embodiment of the method the nucleic acid or protein is purified (at least partially) from the sample before the detection is performed.
Where the organism is Aspergillus fumigatus, Aspergillus nidulans OJT Aspergillus niger, the sample may comprise sputum, bronchoalveloar lavage, urine, respiratory specimens, endotracheal aspirates, sterile specimens obtained by an invasrve procedure such as vitreous tap, tympanocentesis, brain biopsy or aspiration, na_sal or sinus specimens, blood, tissue or autopsy. Where the organism is Magnaporthe grisea the sample may comprise rice leaf or rice stem.
Preferably, said detecting of the presence in the said sample of a polynucleoticLe as defined by the invention comprises use of at least one oligonucleotide pair adapted to be used for amplification of DNA, preferably genomic, more preferably, fu-ngal genomic DNA. The amplification may be PCR amplification.
Preferably, said detecting comprises subjecting the amplified DNA to size analysis, preferably, electrophoresis and, preferably, comparing the results to a positive control and, preferably, a negative control. Said detecting may also comprise sequencing of the amplified DNA to demonstrate the correct sequence.
Preferably, said detecting of the presence in the said sample of a protein comprises use of a monoclonal or polyclonal antibody directed to part or all of the protein of the invention.
According to a further aspect of the present invention, there is provided a recombiznant DNA molecule or vector comprising a polynucleotide of the invention.
The recombinant DNA molecule or vector may comprise an expression cassette. Preferably, the recombinant DNA molecule or vector comprises an expression vector. Preferably, the polynucleotide sequence is operatively linked to an expression control sequence. A suitable control sequence may comprise a promoter, an enhancer etc.
According to another aspect of the present invention, there is provided a cell containing a polynucleotide, recombinant DNA molecule or vector of the invention.
The cell may be transformed or transfected with the polynucleotide, recombinant DNA molecule or vector by suitable means. Preferably, the cell produces a recombinant protein of the invention. The invention also provides an organism which is transgenic for the polynucleotide of the invention (whose cells may be the same as the cells of the invention mentioned herein). Such an organism is typically a fungus, such as any genera or species of fungus mentioned herein. The organism may be microorganism, such as a bacterium, virus or yeast. The organism may be a plant, animal (including birds and mammals), such as any of the animals mentioned herein.
The organism may be produced by introduction of the polynucleotide of the invention into a cell of the organism, and in the case of a multicellular organism allowing the cell to grow into a whole organism.
According to a further aspect of the present invention, there is provided a cell in which a native polynucleotide or protein of the invention protein is non-functional and/or inhibited. The cell may be of, or present in, a multicellular organism.
The cell may be a mutant cell. The cell is typically a fungal cell, such as of any genera or species of fungus mentioned herein. A preferred means of generating the cell is to modify the polynucleotide of the invention, such that the polynucleotide is non-functional. This modification may be to cause a mutation, which disrupts the expression or function of a gene product. Such mutations may be to the nucleic acid sequences that act as 5' or 3' regulatory sequences for the polynucleotide, or may be a mutation introduced into the coding sequence of the polynucleotide. Functional deletion of the polynucleotide may be, for example, by mutation of the polynucleotide in the form of nucleotide substitution, addition or, preferably, nucleotide deletion.
The polynucleotide may be made non- functional and/or inhibited by: (i) shifting the reading frame of the coding sequence of the polynucleotide; (ii) adding, substituting or deleting amino acids in the protein encoded by the polynucleotide; or (iii) partially or entirely deleting the DNA coding for the polynucleotide and/or the upstream and or downstream regulatory sequences associated with the polynucleotide. (iv) inserting DNA into the coding or non-coding regions.
A preferred means of introducing a mutation into a polynucleotide is to utilize molecular biology techniques specifically to target the polynucleotide which is to be mutated. Mutations may be induced using a DNA molecule. A most prefened means of introducing a mutation is to use a DNA molecule that has been especially prepared such that homologous recombination occurs between the target polynucleotide and the DNA molecule. When this is the case, the DNA molecule, which may be double stranded, may contain base sequences similar or identical to the target polynucleotide to allow the DNA molecule to hybridize to (and subsequently recombine with) the target.
It is also possible to provide a cell in which the polynucleotide is non-functional and/or inhibited without introducing a mutation into the gene or its regulatory regions. This may be done by using specific inhibitors. Examples of such inhibitors include agents that prevent transcription of the polynucleotide, or prevent translation, expression or disrupt post-translational modification. Alternatively, the inhibitor may be an agent that increases degradation of the gene product (e.g. a specific proteolytic enzyme). Equally, the inhibitor may be an agent which prevents the polynucleotide product from functioning, such as neutralizing antibodies. The inhibitor may also be an antisense oligonucleotide, or any synthetic chemical capable of inhibiting expression of the gene or the stability and/or function of the protein. The inhibitor may also be a protein which interacts with a protein of the invention to prevent its function. The inhibitor may also be an RNA molecule which causes inhibition by RNA interference. In one embodiment the antisense polynucleotide or RNA molecule which causes RNA interference is an example of a polynucleotide of the invention.
According to a further aspect, there is provided an antibody exhibiting immunospecificity for a protein of the invention. The antibody may be used as a diagnostic reagent. The antibody may be monoclonal or polyclonal, and may be raised in mouse, rat, rabbit, chicken, turkey, horse, goat or donkey. The antibody may be raised against one or all of the proteins together, or may be raised against proteolytic or recombinant fragments.
For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments which bind a protein of the invention. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR- grafted antibodies or humanised antibodies.
Administration
The formulation of any of the therapeutic substances (e.g. proteins, polynucleotides or modulators) mentioned herein will depend upon factors such as the nature of the substance and the condition to be treated. Any such substance may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The substance may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.
Typically the substance is formulated for use with a pharmaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, com starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol. Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymefhylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
A therapeutically effective non-toxic amount of substance is administered. The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about O.lmg/kg to lOmg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are
Figure imgf000024_0001
Agricultural use
Modulators identified by the method of the invention may be administered to plants in order to prevent or treat fungal infections. The modulators are normally applied in the form of compositions together with one or more agriculturally acceptable carriers or diluents and can be applied to the crop area or plant to be treated, simultaneously or in succession 'with further compounds.
The modulators of the invention can be applied together with carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and diluents coreespond to substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
A prefened method of applying the modulators of the present invention or an agrochemical composition which contains them is leaf application. The number of applications and the rate of application depend on the intensity of infection by the fungus. However, the active ingredients can also penetrate the plant through the roots via the soil (systemic action) by impregnating the locus of the plant with a liquid composition, or by applying the compounds in solid form to the soil, e.g. in granular fomi (soil application). The active ingredients may also be applied to seeds (coating) by impregnating the seeds either with a liquid formulation containing active ingredients, or coating them with a solid formulation. In special cases, further types of application are also possible, for example, selective treatment of the plant stems or buds.
The active ingredients are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances. Like the nature of the compositions, the methods of application, such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances. Advantageous rates of application are normally from 50g to 5kg of active ingredient (a.i.) per hectare ("ha", approximately 2.471 acres), preferably from lOOg to 2kg a.i./ha, most preferably from 200g to 500g a.i./ha. The formulations, compositions or preparations containing the active ingredients and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, for example by homogeneously mixing and/or grinding active ingredients with extenders, for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).
Suitable solvents include aromatic hydrocarbons, preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol, monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl formamide, as well as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil; or water.
The solid carriers used e.g. for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand, hi addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.
Depending on the nature of the active ingredient to be used in the fonnulation, suitable surface-active compounds are nonionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants.
Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds. Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon atoms), for example the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained for example from coconut oil or tallow oil. The fatty acid methyltaurin salts may also be used.
More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates. The fatty sulfonates or sulfates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammoniums salts and have a 8 to 22 carbon alkyl radical which also includes the alkyl moiety of alkyl radicals, for example, the sodium or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p- nonylphenol with 4 to 14 moles of ethylene oxide.
Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 3O glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.
Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit. Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.
Cationic surfactants are preferably quaternary ammonium salts which have, as N- substituent, at least one C8-C22 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium bromide.
The surfactants customarily employed in the art of formulation are described, for example, in "McCutcheon's Detergents and Emulsifiers Amiual", MC Publishing Corp. Ringwood, New Jersey, 1979, and Sisely and Wood, "Encyclopaedia of Surface Active Agents," Chemical Publishing Co., Inc. New York, 1980.
The agrochemical compositions usually contain from about 0.1 to about 99% preferably about 0.1 to about 95%, and most preferably from about 3 to about 90% of the active ingredient, from about 1 to about 99.9%, preferably from about 1 to 99%, and most preferably from about 5 to about 95%> of a solid or liquid adjuvant, and from about 0 to about 25%, preferably about 0.1 to about 25%>, and most preferably from about 0.1 to about 20% of a surfactant. Whereas commercial products are preferably formulated as concentrates, the end user will normally employ dilute formulations.
All of the features described herein may be combined with any of the above aspects, in any combination.
Embodiments of the invention will now be described by way of example. EXAMPLES
Example 1. Identification of essential genes in. Aspergillus fumigatus Essential regions of the A. fumigatus genome were identified using the mycobank technology as described in patent WO00177295 Al with the following modifications:
Re-haploidisation (section 1.6):
P24 lines 11-18: Conidia {A. fumigatus) were collected from a stable diploid transformant colony and approximately 3xlO4 spores were used to inoculate 1 ml of SAB broth containing lmg/ l FPA. This culture was incubated with shaking (200 rpm) at 37°C for 20 hours. lOOμl of the culture was spread onto complete media containing 0.2 mg/ml FPA and incubated at 37 °C for 3 days or until rapidly growing sectors emerged. Conidia were collected from each sector and plated onto nitrate, nitrite and hypoxanthine media and the nitrogen utilisation profiles of the resulting conidia assessed. Colonies with the nitrogen utilisation profiles of the parental strains indicated breakdown of the diploid to a haploid. Multiple haploid sectors were isolated. None of the haploids were hygromycin resistant indicating the insertion of the hph gene into a portion of the genome required for function.
Transformation (section 1.7):
P25 line 9: Plasmid pAN7-l linearised with Hindlll was used as the transforming vector. PAN7-1 carries the hph gene which confers hygromycin resistance. P25 lines 17-20: 1 ml of cold YED was added to the cuvette and incubated at 37 °C for 1 h. Aliquots were spread on selective agar (complete media with 250 μg/ml hygromycin). Colonies growing on selective media were deemed putative transformants.
The points of insertion were identified using the plasmid rescue method outlined on page 31 lines 5-17. The insertion sites were confirmed by employing PCR: Using the sequence obtained from plasmid rescue data, complementary primers were designed within the predicted sequence near the point of insertion. A primer corresponding to the sequence of pAN7-l was also used. Genomic DNA samples isolated from diploids of experiments 1745, 1775, 1784, 1859, 1871, 2008, 2009, 2022, 2083, 2139, 2610, 2905, 2975, 2994, 2998, 3026, 3034, 3336, 3345, 3508, 3524 and 3640 were used as templates. The resulting sequences are given as SEQ J-D Nos. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 75, 79, 83, 87 and 91 respectively. The relationship between the individual experiments and the SEQ LD numbers is given in Table I.
Example 2. Characterisation of the essential genes 2.1 Genome analysis The A. fumigatus database (www.TIGR.org or www.sanger.ac.uk) was searched (blastn) with the sequences SEQ LD Nos. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 75, 79, 83, 87 and 91 identified in Example 1 above, and matching contigs identified. In each case, the appropriate region of the contig sequence was down-loaded and gene predictions carried out using Genscan (genes.mit.edu/GENSCAN.html; Settings; organism = vertebrate; Suboptimal exon cutoff = 1.00).
The ab initio prediction of genes from genomes is known to be an inaccurate process (Burset, M. and Guigό, 1996, Genomics, 34, 353-367) and this is particularly so when the programs used have not been specifically trained for the genome under examination (as is the case here). It is therefore necessary to carefully examine the predictions, to compare any predicted genes with any homologous proteins, and to exploit the operative's knowledge of fungal gene structure, and thus to arrive at an informed prediction. The predicted genes were therefore compared with similar sequences using blastp (http:// blast.genome.ad.jp/), the multiple alignment program ClustalX (Thompson et al, 1997, Nucleic Acids Research, 24:4876-4882), and the alignment editor/ viewer Align (http:// www.gwdg.de/~dhepper/download/; Hepperle, D., 2001: Multicolor Sequence Alignment Editor. Institute of Freshwater Ecology and Inland Fisheries, 16775 Stechlin, Germany). Gene structures were visualised and modified using Artemis (http://www.sanger.ac.uk/Software/Artemis/; Rutherford et al., 2000, Bioinformatics 16, 944-945). The genes adjacent to the insertion sites are listed in Table I along with the location of the insertion relative to the gene.
2.2 Genomic Sequencing of Genes The genomic sequences of the genes identified in Example 2.1 above can be determined experimentally as follows:
2.2.1 Bacterial and Fungal Strains
For bacterial cloning, E. coli Select96 cells (Promega) are used in accordance with manufacturers' instructions.
A. fumigatus clinical isolate AF293 (ref. No. NCPF7367; available to the public from the NCPF repository; Bristol, U.K.); the CBS repository (Belgium) or from Dr. David Denning' s clinical isolate culture collection, Hope Hospital, Salford. U.K.) is the prefened strain according to the present invention. AF293 was isolated in 1993 from the lung biopsy of a patient with invasive aspergillosis and aplastic anaemia. It was donated by Shrewsbury PHLS.
2.2.2 Purification of A. fumigatus genomic DNA To obtain mycelial material for genomic DNA isolation, approximately IO7 A. fumigatus conidia are inoculated in 50 ml of Vogel's minimal medium and incubated with shaking at 200 rpm until late exponential phase (18-24 h) at 37°C. Mycelium is dried down onto Whatmann 54 paper using a Buchner funnel and a side-arm flask attached to a vacuum pump and washed with PBS/Tween. At this point, the mycelium can be freeze-dried for extraction at a later date.
The mycelium (fresh or freeze dried) is ground to a powder using liquid nitrogen in a - 20°C cooled mortar. The ground biomass is transfened to 50 ml tubes on ice up to the 10 ml mark. An equal volume of extraction buffer (0.7 M NaCl; 0.1 M Na2SO3; 0.1 M Tris-HCl pH 7.5; 0.05 M EDTA; l%(w/v) SDS; pre-warmed to 65°C) is then added to each tube, mixed thoroughly with a pipette tip and incubated at 65°C for 20 minutes in a water bath. A volume of chloroform/isoamyl alcohol (24:1) equivalent to the volume of the original biomass is then added to each tube, tubes are mixed thoroughly and incubated on ice for 30 min. Tubes are then centrifuged at 3,500 x g for 30 min and the aqueous phase carefully transferred to fresh 50 ml tubes without disturbing the interface.
An equal volume of chloroform/isoamyl alcohol (24:1) is added, the tubes vortexed and incubated on ice for 15 minutes. Tubes are then spun at 3,500 x g for 15 minutes. After this spin, if large amounts of precipitate are still present, the supernatant is removed and the chloroform:isoamyl alcohol step repeated. The supernatant is removed and placed in clean sterile Oak Ridge tubes. An equal volume of isopropanol is added and mixed gently. Tubes are incubated at room temperature for at least 15 minutes. Tubes are then centrifuged at 3,030 x g for 10 minutes at 4°C to pellet the DNA. The supernatant is removed and the pellet allowed to air dry for 10-25 minutes. The pellet is suspended in 2 ml sterile water. 1 ml of 7.5 M ammonium acetate is added, mixed and incubated on ice for 1 hour. Tubes are centrifuged at 12,000 x g for 30 min, the supernatants transfened to a fresh tube and 0.54 volumes of isopropanol are added, mixed and incubated at room temperature for at least 15 minutes. Tubes are then centrifuged at 5,930 x g for IO min, the supernatant is removed and the pellet washed in 1 ml of 70%> ethanol. Tub>es are centrifuged at 5,930 x g for 10 min and all the ethanol is removed. The pellet is air dried for 20-30 minutes at room temperature and suspended in 0.5-1.0 ml of TE (L 0 mM Tris-HCl pH 7.5; lmM EDTA) Finally, the DNA is treated with RNase A (5 μl of 1 mg/ml stock).
2.2.3 PCR Reactions Primers pairs are designed to the upstream and downstream regions of the A. fumigatus AF293 genes: The 200 base regions flanking the predicted gene of interest are used as input sequence for Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3 www. cgi) to provide a primer pair that spans the gene. If the gene is particularly long, it may be necessary to design primer pairs with internal sequences and thus sequence the gene in parts.
The following reagents and conditions are used: PCR Master Mix
1 Ox high fidelity PCR buffer 5 μl dNTP (clontech: lOmM) 1 μl nH2O 39 μl
Pfu Ultra Polmerase (2.5U/μl) 1 μl
Primer pairs (10 pmol/μl stock) 1 μl each gDNA (1:30 dilution of stock) 2 μl
PCR Cycle
1) 95° C 2 min
2) 95° C 30 sec
3) 54° C 30 sec
4) 72° C 2 min 5) 72° C 10 min
6) 8° C Hold
40 cycles of steps 2-4 are carried out and the PCR products are run on a gel. The product band is excised from the gel and purified using Qiagen's QIAquick Gel Extraction Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, West Sussex, RH10 9AX, UK) according to the manufacturers instructions and eluted into 30 μl of sterile water (BDH molecular biology grade/filter sterile).
2.2.4 Genomic DNA Cloning and Sequencing Since the gDNA is amplified using Pfu ultra polymerase which produces blunt ends it is necessary to add 'A' overhangs before ligating in to pGEM Teasy. 12.5 μl of purified PCR product is incubated with 12.5 μl 2x PCR Reddy Mix (ABGene) 12.5 μl at 70° C for 30 minutes. The sample is then purified using Qigen Qiaquick gel extraction kit and eluted in 30 μl of molecular biology grade water.
The PCR product is then ligated into pGEM-Teasy (Promega) using the following ligation mixture: 2x Buffer 5 μl pGEM Teasy 1 μl
PCR product 3 μl T4 DNA Ligase 1 μl
The reaction is incubated over-night at 4° C.
2 μl of the ligation mix are then added to Select 96 cells (Promega) and incubated for 20 min on ice. Cells are then heat shocked at 42° C for 45 sees and placed back on ice. 250 ml of room temp. SOC medium is then added and the cells incubated for 1 hour at 37° C, with shaking at 220 rpm. 50 and 200 μl amounts are then plated on to LB agar plates containing ampicillin (100 μg/ml), 50 μl X-gal (4%) and 10 μl LPTG (100 mM) and incubated over night at 37° C.
Individual white colonies are picked from each transformation inoculated into LB with ampicillin (100 μg/ml) and incubated over-night at 37° C, with shaking at 220 rpm. Plasmid DNA is extracted using Qiagen miniprep kit according to the manufacturers instructions. 1 μl of plasmid DNA is digested with restriction enzymes for 1 hour at 37° C. Results are compared with the predicted sizes for constructs and clones showing the correct restriction digest pattern are sequenced at MWG Biotech UK Ltd, Waterside House, Peartree Bridge, Milton Keyn s, MK6 3BY.
Example 3. cDNA sequencing and RACE The internal sequences of the genes of interest are experimentally determined by cloning and sequencing cDNA, and the 5' and 3 ' ends of the genes are determined by RACE (Rapid Amplification of cDNA Ends).
3.1 cDNA cloning and sequencing 3.1.1 Preparation of A. fumigatus RNA and cDNΛ. Fungal cultures are prepared as described in Example 2.2.2. Cultures are harvested by filtration, then washed twice with DEPC-treated water and transfened to a 50 ml Falcon tube. Samples are frozen in liquid nitrogen and stored at -80°C until required. To prepare RNA, fungal samples are ground to a fine powder under liquid nitrogen. RNA is then extracted using the Qiagen RNeasy Plant Mini Kit following the protocol for isolation of total RNA from filamentous fungi in the RNeasy Mini Handbook (06/2001, Pages 75-78, http://www.qiagen.com/literature/ handbooks/ma/mamini/1016272HBRNY_062001WW.pdf). The following modifications are used: At step 3, RLC is used as the Lysis buffer of choice; At step 7, the Rneasy column is incubated for 5 min at room temperature after addition of RW1; The optional step 9a is canied out; At step 10, 30μl _RNase-free water is added, the samples incubated for 10 min at room temperature, and then centrifuged; At step 11, the elution step is repeated to give a total volume of 60 μl RNA.
DNA contamination is removed from the RNA by the addition of Dnase, using 2 μl DNase per μg RNA, in the presence of 10X DNase buffer and incubating at 37° C for 2h. DNase-treated RNA is cleaned up using the RNeasy Plant Mini Kit following the RNeasy Mini Protocol for RNA Cleanup (RNeasy Mind Handbook 06/2001, pages 79- 81).
To synthesise cDNA from the above RNA the following reaction mixture is prepared: lOOng-lμg of DNA-free RNA, 3μl oligo (dT) (100 ng/μl), and DEPC-treated water to a total volume of 42 μl. Samples are incubated in a heat block at 65°C for 5 min after which they are allowed to cool slowly to room temperature. Then 2 μl Ultrapure dNTPs, lμl reverse transcriptase (Stratascript) and 5 μl 10X reverse transcriptase reaction buffer (Stratascript) are added. Samples -ure incubated at 42°C for lh, denatured at 90°C for 5 min and then cooled on ice.
3.1.2 Production of cDNA constructs PCR is carried out using the cDNA above to generate cDNA fragments. Primers are designed based on the 5' and 3' ends of the predicted genes. PCR reactions are carried out using the following reagents and conditions: PCR Master Mix
1 Ox high fidelity PCR buffer 5 μl dNTP (clontech: lOmM) 1 μl
MgSO4 (50 mM) 2 μl nH2O 37.8μl
Platinum TAQ Polmerase (5U/μl) 0.2μl
Primer pairs (10 pmol/μl stock) 1 μl each cDNA 2 μl
PCR Cycle
1) 94° C 5 min
2) 94° C 30 sec 3) 53° C 30 sec
4) 68° C 90 sec
5) 68° C 10 min
6) 8° C Pause
Cycles 2-4 are run 40 times in total, bp. The PCR products are purified using Qiagen' s QIAquick PCR Purification Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, West Sussex, RH10 9AX, UK) according to the manufacturers instructions and run on agarose gels.
PCR products are ligated into pGEM-Teasy, used to transform Select 96 cells, and sequenced as described in 2.2.4 above.
3.2 RACE
To determine the 5' and 3' ends of the genes, RACE (Rapid Amplification of cDNA Ends) is carried out, using the GeneRacer™ Kit (-Ihvitrogen; cat. No. L1502-01), essentially as per manufacturers instructions. 3.2.1 Preparation of RNA
A. fumigatus biomass is prepared as described in 2.2.2. RNA is prepared using the FastRNA kit (QBIOgene) following the manufacturer's instructions (Revision 6030- 999-1J05) with the following amendments: At step 1 40 mg of biomass is used per extraction; At step 2, samples are processed for 20 seconds at speed 5, incubated on ice for 3 minutes, and processed again for 20 seconds at speed 5; At step 3 samples are centrifuged for 5 minutes; At step 5, 500 μl DLPS are added, mixed, and incubated- at room temperature for 2 minutes. Samples are mixed again and incubated for a furthex 2 minutes; At step 6 two washes in 250 μl SEWS are carried out; At step 7, the pellet is disolved in 50 μl SAFE buffer.
3.2.2 RACE
1 μg total RNA prepared as described above is de-phosphorylated in a 10 μl reaction using 10 units of calf intestinal phosphate (CIP), 1 μl 10X CIP buffer and 40U RNaseOut™ (made up to 10 μl in DEPC water) at 50°C for 1 hour. Samples are tnen made up to 100 μl with DEPC water and the RNA extracted with 100 μl (25:24 :1) phenohchloroform: isoamyl alcohol. RNA is then precipitated by the addition of 2 μl mussel glycogen (lOmg/ml), 10 μl 3M sodium acetate, pH 5.2 and 220 μl 95% ethanol and the sample frozen on dry ice for 10 minutes. RNA is pelleted by centrifugatiori- at 14,500 rpm for 20 minutes at 4°C, washed with 70% ethanol, air dried and ore- suspended in 8 μl DEPC water.
De-phosphorylated RNA (7 μl) is de-capped in a 10 μl reaction with 0.5 U tobacco acid pyrophosphatase (TAP), 1 μl lOx TAP buffer and 40 U RnaseOut™ for 1 hour- at 37°C. RNA is extracted with phenohchloroform and precipitated as above, and then ire- suspended in 7 μl DEPC-treated water.
De-phosphorylated, de-capped RNA (7 μl) is added to the pre-aliquoted GeneRacerr™
RNA Oligo (0.25 μg) and incubated at 65°C for 5 minutes. A 10 μl ligation reaction is then set up by the addition of 1 μl lOx ligase buffer, 1 μl 10 mM ATP, 40 U
RnaseOut™ and 5 U T4 RNA ligase and incubated at 37°C for 1 hour. RNA is extracted and precipitated as described previously and re-suspended in 11 μl DEPC- treated water.
First-strand cDNA is prepared by the addition of 1 μl GeneRacer™ Oligo dT primer and 1 μl dNTP mix (lOmM each) to 10 μl ligated RNA and incubated at 65°C for 5 minutes. The following reagents are added to the 12 μl ligated RNA and primer mix; 4 μl 5x first strand buffer, 2 μl 0.1 M DTT, 1 μl RNaseOut™ and 1 μl Superscript™ II RT (200 U/μl) and incubated first at 42°C for 50 minutes and then, to stop the reaction, at 70°C for 15 minutes. 2 U RNase H is added to the reaction mix and incubated at 37°C for 20 minutes.
To amplify the 5'cDNA ends a 50 μl PCR reaction is set up using 1 μl of the RACE- ready cDNA prepared above, 1 μl GeneRacer™ 5' primer, 1 μl reverse gene-specific primer (designed against the complementary strand of the coding sequence: 5 pmol/μl stock), 1 μl dNTP solution (10 mM each), 2 μl 50 mM MgSO4, 5 μl High Fidelity PCR buffer, 0.5 μl Platinum® Taq DNA Polymerase High Fidelity (5 U/μl) and 38.5 μl sterile water. Cycling parameters are given in Table II below.
A second, nested PCR stage may also be carried out. This is set up using 1 μl of the RACE cDNA from the first stage above, 1 μl Nested 5' primer (supplied with kit), 1 μl second reverse gene-specific primer (designed against the complementary strand of the coding sequence and nested with respect to the above primer: 5 pmol/μl stock), 1 μl dNTP solution (10 mM each), 2 μl 50 mM MgSO4, 5 μl High Fidelity PCR buffer, 0.5 μl Platinum® Taq DNA Polymerase High Fidelity (5 U/μl) and 38.5 μl sterile water. Cycling parameters are given in Table II below.
To amplify 3' ends a 50 μl PCR reaction is set up using 1 μl of the RACE-ready cDNA prepared above, 1 μl GeneRacer™ 3' primer (10 μM), 1 μl forward gene-specific primer (designed against the coding strand of the coding sequence : 5 pmol/μl stock), 1 μl dNTP solution (10 mM each), 2 μl 50 mM MgSO4, 5 μl High Fidelity PCR buffer, 0.5 μl Platinum® Taq DNA Polymerase High Fidelity (5 U/μl) and 38.5 μl sterile water. Cycling parameters are given in Table II below: A second, nested PCR stage may also be carried out. This is set up using 1 μl of the 3' RACE cDNA from the first stage above, 1 μl Nested 3' primer (supplied with kit), 1 μl reverse gene-specific primer (designed against the coding strand of the coding sequence and nested with respect to the above primer: 5 pmol/μl stock), 1 μl dNTP solution (10 mM each), 2 μl 50 mM MgSO4, 5 μl High Fidelity PCR buffer, 0.5 μl Platinum® Taq DNA Polymerase High Fidelity (5 U/μl) and 38.5 μl sterile water. Cycling parameters are given in Table II below.
Table II. Cycling parameters for 5' and 3 'RACE
Figure imgf000039_0001
5' and 3' RACE identify the 5' ATG and 3' stop codons as well as giving the 5' and 3' untranslated regions of the genes.
Example 4. Identification of fungal homologs of genes of interest
Homologs of the proteins or polynucleotides of the invention are identified in other fungi by means of bioinformatics analysis. Sequences identified by bioinformatics can be used to design primers which in turn can be used in PCR to generate DNA coding for the homologs.
Alternatively, degenerate PCR can be used to obtain sequence, which can then be used to generate probes for screening cDNA or genomic libraries of the organism of interest to identify clones containing the homologs. As a further alternative, Southern blots using fragments of genes from one species as probes can be used to identify the presence of a homolog in the genome of a second species. The same probe can then be used to screen cDNA or genomic DNA libraries. Once clones conesponding to the novel genes have been identified they can be expressed for functional characterisation of the protein.
4.1 Identification of homologs by bioinformatics Homologs of the proteins and polynucleotides of the invention can be identified by searching locally held databases, as detailed in Table III, using BLAST with SEQ ID Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90 as the query sequence. Where necessary, matching contigs are down-loaded and genes predicted from genomic DNA by Genscan analysis, blast searches, alignment and visualisation with Artemis as described in Example 2.
Table III. Sources of data for local BLAST searches
Figure imgf000040_0001
Figure imgf000041_0001
This dataset contains ESTs from the following plant pathogen fungi: Blumeria graminis, Botryotinia, Cladosporium fulvum, Colletotrichum trifolii, Cryphonectria parasitica, Fusarium sporotrichioides, Gibberella zeae, Leptosphaeria maculans, Magnaporthe grisea, Mycosphaerella graminicola, Phytophthora infestans, Phytophthora sojae, Sclerotinia sclerotiorum, Ustilago maydis and Verticillium dahliae
Protein and nucleic acid multiple alignments are generated by means of programs such as ClustalX (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882;) and/or using manual alignment editors such as Align (http://www.gwdg.de/~dhepper/ download/; Hepperle, D., 2001: Multicolor Sequence Alignment Editor. Institute of Freshwater Ecology and Inland Fisheries, 16775 Stechlin, Germany).
The relationships between SEQ ID Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90, and hits identified from blast searches above can be clarified by phylogenetic analysis, for example using the PHYLLP suite of programs (Felsenstein, Felsenstein, J., 2002. PHYLIP (Phylogeny Inference Package) version 3.6a3. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle). A distance matrix is generated using PROTDIST with the Jones- Taylor-Thornton model and a tree infened using FITCH with global reanangements and 10 jumbles of input order. 100 bootstrap replicates are generated using SEQBOOT, distance matrices generated using PROTDIST as above, trees infened using NEIGHBOUR, and then bootstrap values and the consensus trees are calculated using CONSENSE. Trees are viewed using TREEVIEW (Page, 1996 Page, R. D. M., 1996. TREEVLEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357-358.)
Alternatively , the relationship between SEQ ID Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90 and homologs can be clarified using reciprocal blast hits as described by e.g., Wall et al. (Bioinformatics 19, 1710-1711).
4.2 Identification of homologs by degenerate PCR
4.2.1. Preparation of genomic DNA from organism of interest
Fungal cultures are prepared using methods suitable for particular species. For example, Aspergillus and Candida species, Cryptococcus neoformans, Fusarium solani and Trichophyton species are maintained on Sabouraud dextrose agar at 30- 35°C; Leptosphaeria nodorum on Malt agar medium (30 g/L malt extract; 15 g/L Bacto-agar, pH 5.5), 24.0°C; Magnaporthe grisea on Oatmeal agar (6.7 g/L agar, 53.3 g/L instant oatmeal) 25.0°C, or Cornmeal agar (Difco 0386), 26.0 C; Phytophthora capsici cultures were maintained on on V-8 agar at 24°C; Pyricularia oryzae cultures were maintained on rice polish agar at 24°C under white fluorescent lights (12 hr artificial day), and were subculrured every 7 - 14 days by the transfer of mycelial plugs to fresh plates; Pythium ultimum cultures were maintained on PDA at 24°C, and subcultured every 7 days by the transfer of aerial mycelium to fresh plates with an inoculating needle; Rhizoctonia solani cultures were maintained on PDA at 24°C under fluorescent lights (12 h artificial day), and subcultured every 7 days by the transfer of mycelial plugs to fresh plates; Ustilago maydis cultures were maintained on PDY agar at 30°C in the dark, and subcultured by re-streaking.
Genomic DNA is prepared from cultures using standard methodologies, e.g. using the Qiagen DNeasy Plant Kit, or using methods described in Example 2.2. 4.2.2 PCR
Primers are designed to conespond to regions conserved between the gene of interest and its homologs (identified as described above). Those skilled in the art will appreciate that it may be necessary to try a range of primer pairs. PCR reactions using the primer pairs are set up as follows:
12.5 μl 2x ReddyMix PCR mastermix (ABgene) 1 μl each primer (5 pmol) template gDNA (1.5-4 μg/ml) nuclease-free water to give a final volume of 25 μl
The reactions are run using the following conditions on a Biometra personal PCR cycler (Thistle Scientific Ltd, DFDS House, Goldie Road, Uddington, Glasgow, G71 6NZ):-
Stepl 95°C 5min
Steρ2 95°C lmin
Step3 53°C lmin 30sec
Step4 68°C 2min 30sec
Step5 72°C lOmin
Step6 4°C Hold
30 cycles of steps 2-4 are carried out. The PCR products are purified (to remove residual enzymes and nucleotides) using Qiagen' s QIAquick PCR Purification Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, West Sussex, RHIO 9AX, UK) according to the manufacturers instructions and eluted into 40 μl of sterile water (BDH molecular biology grade/filter sterile). The purified PCR products are examined on 1% agarose gels.
Those skilled in the art will appreciate that degenerate PCR may require variations in a number of parameters in the attempt to generate a product. These include primer concentration, template concentration, concentration of Mg2+ ions, elongation and annealing times, and annealing temperature. Variations in temperature can be accomodated by the use of a gradient PCR machine.
The purified PCR products are cloned into pPEM-Teasy (Promega) and then transformed into XLIO-Gold® Kan ultracompetent E. coli cells according to the manufacturers instructions. The transformation reactions are then plated onto LB agar plates containing ampicillin (100 μg/ml), 50 μl X-gal (4%) and 10 μl LPTG (100 mM). Following overnight incubation at 37°C, individual white colonies from each transformation are sub-cultured into LB broth containing ampicillin (100 μg/ml). After overnight incubation at 37°C with shaking, plasmids are extracted using Qiagen spin mini plasmid extraction kits according to the manufacturers instructions and sent away for full-length sequencing.
4.3 Identification of homologs by Southern Blotting
4.3.1 Digestion of genomic DNA and transfer to nylon membranes Genomic DNA from the fungi of interest are digested with the appropriate restriction enzyme and run on 0.8 % agarose gel. The gel is then submerged in 250 mM HCl for no more than 10 mins, with shaking, at room temperature, after which the gel is rinsed with sterilised RO water.
Transfer of the DNA onto nylon membrane is carried out using 0.4 M NaOH. Transfer protocols and apparatus are well known and are described in e.g. Sambrook et al., (1989), Molecular Cloning, 2nd Edition., Cold Spring Harbor Laboratory Press. After transfer, the DNA is fixed to the membrane by baking at 120°C for 30 min. The membrane can then be used immediately, or stored dry for future use.
4.3.2. Preparation of probe
Probes are generated either by restriction digests of DNA or by PCR of an appropriate region. A suitable probe can be generated by PCR using a primer pair designed using Primer3(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_ www.cgi), A. fumigatus genomic DNA, and the methods give in 4.2.2. 1 μg DNA template is diluted in molecular biology water to a total volume of 16 μl, denatured in a boiling water bath for 10 mins, and quickly chilled on ice. 4 μl DIG- High Prime (1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM alkali-labile-digoxygenin-11-dUTP, 1 U/μl labelling grade Klenow enzyme, 5 x reaction buffer, in 50%> (v/v) glycerol) is then added and the reaction incubated at 37°C for 20 hours, after which 2 μl of 200 mM EDTA pH 8.0 is added to terminate the labelling reaction. The labelling efficiency is estimated by comparison with DIG- labelled control DNA.
4.3.3. Prehybridisation and Hybridisation
The membrane is placed in a hybridisation tube containing 20 ml of prehybridisation solution (DIG Easy Hyb, Roche) per 100cm2 of membrane surface area and prehybridised at 42°C for 2 hours in a hybridisation oven. The DIG- labelled probe is denatured by heating in a boiling water bath for 10 min and then chilled directly on ice. The probe is then diluted to -200 ng/mL in hybridisation solution (Easy Hyb, Roche; at least 5 mL of hybridisation solution is required per hybridisation). The prehybridisation solution is discarded from the hybridization tube and the hybridisation solution containing the DIG-labelled probe added quickly. The hybridisation then proceeds overnight at a 42°C in the hybridisation oven. The optimum temperature is dependant on probe size and homology with target sequence and is determined empirically.
After hybridisation, the membrane is washed twice at 42°C, 5 mins per wash, with 50 mL of stringency wash solution (3 x SSC, 0.1% SDS; where 20 x SSC buffer is 3 M NaCL, 300 mM sodium citrate, pH 7.0), followed by two washes at RT, 15 min per wash, in 50 mL stringency wash solution. The stringency of these washes can be decreased by increasing the SSC concentration to 6 x SSC, 0.1% SDS and/or decreasing the wash temperatures.
4.3.4. Detection The membrane is washed in 20 mL washing buffer (100 mM maleic acid, 150 mM NaCl; pH 7.5; 0.3% v/v Tween 20), and then incubated successively with the following; 20 mL blocking solution (1% w/v blocking reagent for nucleic acid hybridisation, Roche, dissolved in 100 mM maleic acid, 150 mM NaCl, pH 7), for 30 min at room temperature; Anti-DIG-alkaline phosphatase (Roche) diluted 1:5,000 in blocking buffer, 30 min at room temperature; Washing buffer, two washes each of 15 min at room temperature; Detection buffer (lOOmM Tris-Hcl, 100 mM NaCl; pH 9.5), 2 min at room temperature. The membrane is then removed, placed on top of an acetate sheet, and ~ 0.5 ml (per 100cm2) of CSPD or CDP-star added to the top of the membrane. A second sheet of acetate is then placed over the surface of the membrane, the assembly incubated for 5 min at room temperature and then sealed in a plastic bag. The assembly is then exposed to X-ray film for between 15 min and 1 hour. Optimal exposure time is determined empirically by increasing exposure time up to 24 hours.
The presence of a band on the gel is evidence of a gene in the genomic DNA of interest. The molecular weight of the band depends on the size of the restriction fragment that contains the gene.
Example 5. Expression during infection of wax moth larvae (Galleria melonella) and mice infected with A. fumigatus
5.1 Preparation of cDNA from infected wax-moth larvae
Wax moth larvae have been shown to be good model systems in which to study Candida infection (Cotter et al., 2000, FEMS Immunol Med Microbiol 27, 163-9; Brennan et al., 2002, FEMS Immunol Med Microbiol 34, 153-7). We have found that this insect system is also a good system in which to study Aspergillus infection (D. Law and J. Rooke, manuscript in preparation).
5.1.1 Growth and infection of wax-moth larvae Spores of A. fumigatus (AF293), grown on Sabaraud Dextrose agar, were harvested and re-suspended in PBS/Tween 80. Spores were washed and the concentration adjusted such that a 10 μl inoculum will cause death in 90% of the test group 3-4 days after infection (for AF293 this is 5.0-7.0x108 cfu/ml). Inoculum concentration was estimated using an improved Neubauer haemocytometer cotinting chamber and confirmed by TVC enumeration.
Wax moth larvae were purchased from Livefood UK, Somerset, UK (www.livefood.co.uk), and were maintained in the dark at room temperature in wood shavings prior to infection. Healthy larvae (250 mg +/- 50 mg) were selected and incubated at 4°C for 10 minutes immediately prior to infection to immobilise them. Larvae were then injected through the cuticle of the left last pro-leg with 10 μl spore suspension (lOOx stock), using a sterile Hamilton syringe. Larvae were then transfereed to a sterile Petri dish. The following controls were also established: Larvae injected with 10 μl PBS/Tween only; larvae injected with 10 μl heat killed spores (killed by incubation for 20 min 100°C); larvae pierced but not injected; and untouched larvae. Larvae were incubated at 30°C and monitored at least twice daily. All treatments and controls were carried out on batches of 10 larvae. Larval deaths and general health condition was recorded every 24 hrs and dead or moribund larvae were removed from the test group.
5.1.2 Preparation of DNA-free RNA from Aspergillus fumigatus-infected wax moth larvae. cDNA was prepared from the following sources: Uninfected larvae; larvae after 48h infection with A. fumigatus (early infection); larvae after 72h infection with A. fumigatus (late infection); larvae infected with heat-killed A. fumigatus spores; and A. fumigatus grown in Sabaraud Dextrose agar broth for 16hr.
Frozen larvae were ground to a fine powder under liquid nitrogen in a mortar and pestle previously baked at 22°C overnight, treated with RNaseZAP, rinsed with DEPC- treated water (0.1% (v/v) DEPC, stured for lh and autoclaved for lh) and cooled with liquid nitrogen. Ground sample was transfened to Eppendorf tubes (no more than 50 mg per tube) and total RNA extracted using the Qiagen RNeasy Plant Mini Kit following the protocol for isolation of total RNA from filamentous fungi in the RNeasy Mini Handbook (06/2O01, Pages 75-78, http ://www.qiagen.com/literature/handbooks/ma/rnamini/l 016272HBRNY _062001 W W.pdf).
The following modifications were used: At step 3, 600 μl RLT was added to each 50 mg tissue and vortexed; At step 4, samples were centrifuged for 3 min at maximum speed; At step 6, all samples from the same tissues were applied to the same RNeasy column; At step 7, RNeasy column was incubated for 5 min at room temperature after addition of RWl; Optional step 9a was canied out twice; At step 10, 30 μl RNase-free water was added, samples incubated for 10 min at room temperature, and then centrifuged for 1 min at 14,000 RPM; At step 11, the elution step was repeated to give a total volume of 60 μl RNA. A sample of the RNA was run on a 1.5% agarose gel and the amount of RNA quantified using the molecular marker. RNA was then stored at - 80°C.
A portion of the RNA was Dnase treated using 2 μl RNase-free DNase (Promega) per μg RNA, in the presence of 1 OX DNase buffer (Promega) at 37°C for 4h. The RNA was then cleaned up using the Qiagen RNeasy Plant Mini Kit following the RNeasy Mini Protocol for RNA Cleanup (RNeasy Mini Handbook 06/2001, pages 79-81), but including a further DNase treatment step during clean-up as in the Rneasy handbook.
The following modifications were made: Optional step 5a was carried out; At step 6, 30μl RNase-free water was added, samples incubated for 10 min at room temperature and then centrifuged for 1 min at 14,000 RPM; At step 7, the eluate from step 6 was transfened onto the RNeasy column, incubated for 10 min at room temperature, and then centrifuged for 1 min at 14,000 RPM. A sample of the DNase-treated RNA was run on an agarose gel, quantified and stored at -80°C.
5.1.3 Checking RNA samples for DNA contamination
To verify the absence of genomic DNA from the RNA samples, PCR was carried out using primers that amplify the μ-tubulin gene (SEQ LD Nos. 69 and 70). In the absence of a reverse-transcription step, only gDNA will be detected and thus any gDNA contamination will be revealed. The following reaction mixture was set up:
12.5 μl 2x ReddyMix PCR mastermix (ABgene) 1 μl each primer (5 pmol) template gDNA (1.5-4 μg /ml) nuclease-free water to give a final volume of 25 μl
The reactions were ran using the following conditions on a Biometra personal PCR cycler (Thistle Scientific Ltd, DFDS House, Goldie Road, Uddington, Glasgow, G71 6NZ):-
Stepl 95°C 5min
Step2 90°C lmin
Step3 51°C lmin
Step4 68°C lmin
Step5 68°C lOmin
Step6 4°C Hold
40 cycles steps 2-4
If a PCR product was observed, genomic DNA was present and the sample was DNase-treated again. If the PCR was negative, no DNA was present in the sample.
5.1.4 Preparation ofcDNA 300 μg DNA-free RNA and 3 μl oligo (dT) (100 ng/μl) were added to an RNase-free 0.5 ml microcentrifuge tube, and made up a total volume of 42 μl with DEPC-treated water. Samples were mixed and incubated in a heat block at 65°C for 5 min and then slowly cooled to room temperature. 2 μl Ultrapure dNTPs (10 mM each, Clontech), 1 μl stratascript reverse transcriptase (Stratagene) and 5 μl 10X reverse transcriptase reaction buffer were then added. The samples were incubated at 42°C for lh, denatured at 90°C for 5 min and then cooled on ice. Samples were dispensed in 5-10 μl aliquots and stored at -20°C. 5.2. Preparation of cDNA from infected mice
5.1.1 Infection of mice with A. fumigatus and extraction of tissues.
Mice were infected with Aspergillus fumigatus and organs harvested as follows. Thirteen male CDl mice were injected with the immunosuppressant cyclophosphamide (0.025 g/ml; 200 mg/kg) LV via the tail vein. After 72 hours, twelve mice were injected with 0.15 ml Aspergillus fumigatus AF293 conidia (7.5 x 105/ml). 11 hours after infection, four mice were sacrificed with an overdose of inhaled halothane. The brain, lungs, liver and kidney were removed, frozen by immersion in liquid nitrogen, and stored at -70°C. A further four mice were also sacrificed at 24 and 48 hours after infection.
RNA was prepared from mouse tissues as described for wax moth larvae above (5.1.2 and 5.1.3).
5.2.2 Preparation ofcDNAfrom DNA-free RNA. cDNA was prepared from DNA-free RNA using the Promega Reverse Transcription kit, following the protocol as supplied with the product (Technical Bulletin No. 099, http://www.promega.com/tbs/tb099/tb099.pdf). In a modification to the protocol, the cDNA synthesis reaction was incubated for 60 min at 42°C rather than for the suggested 15 min. Samples were stored in 5-1 Oμl aliquots at -20°C.
5.3 Design and optimisation of primers
Primers are designed against cDNA sequences conesponding to the polynucleotides of the invention using Beacon Designer 2.1 (Premier Biosoft, http://www.premierbiosoft.com) with the following parameters; Target Tm = 58 ± 8°C; Length of primers = 16-24; Amplicon length = 75-150 bp. All other settings are default. Care is taken to choose primers that will not form dimers or other secondary structures. The secondary structures of amplicons is calculated using mfold (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/forml .cgi) and primer sets giving an amplicon with little or no secondary structure are chosen. To determine optimum annealing temp for the primer set, a gradient PCR is run on an Icycler PCR machine (Biorad), using A. fumigatus AF293 genomic DNA as a template and the following reaction mixture:
112.5ul Abgene PCR Reddymix 9ul each primer (5 pm/μl) 85.5ul H2O 9ul AF293 gDNA (lOng/ul)
For the negative control, the gDNA is omitted and the amount of water increased conespondingly.
For each mix, 25 μl 1 is pipetted into 8 wells on a multiwell plate, and each well run at a different temperature (between 50 and 65°C) with the following conditions:
Stepl. 95°C - 5 min
Step2. 95°C - l min
Step3. Gradient 50-65°C - 1.5 min
Step4. 72°C - 1 min Step5. 72°C - 10 min
Step6. 8°C - hold
Steps 2-4 are run for 30 cycles
The PCR products are run on a 2% agarose gel.
5.4 Testing species-specificity of primers
The real-time primers designed above are further tested to ensure that mouse nucleic acid is not amplified using these primers. Four reactions are set up, each containing the following: 12.5 μl Abgene Reddymix 1 μl each primer 9.5 μl H2O and either; 1 μl infected mouse kidney cDNA (50 ng/ul; experimental); 1 μl uninfected mouse kidney cDNA (50 ng/ul; uninfected control); 1 μl AF293 gDNA (10 ng/μl; positive control); 1 μl water (negative control).
The following PCR settings are used:
Stepl 95°C - 5 min Step2 95°C - 1 min
Step3 63°C - 1.5 min
Step4 72°C - 1 min
Step5 72° C - 10 min
Step6 8°C - hold Steps 2-4 were run 40 times
The PCR products are run on a 2% agarose gel. If primers are species-specific, bands should be seen with A. fumigatus genomic DNA as template, but not with uninfected mouse cDNA as template.
5.5 Real-time PCR to detect expression in cDNA from infected larvae or infected mouse kidney
PCR reactions are set up using the Biorad iQ SYBR green supermix as follows:
14 μl each primer 175 μl SYBR mix 133 μl H2O
Four reactions are set up containing 72 μl of the above mix and either; 3 μl H2O; 3 μl uninfected larvae cDNA (50 ng/μl ); 3 μl AF293 gDNA (5 ng/μl); or 3 μl infected larvae cDNA (50 ng/μl. For mouse kidney samples, the four reactions contain 72 μl of the above SYBR mix and either; 3 μl uninfected mouse kidney (50 ng/μl); 3 μl infected mouse kidney - 48h post-infection (50ng/ul); or 3 μl AT293 cDNA (5ng/μl).
3 x 25 μl aliquots of each reaction are aliquoted into an Abgene multiwell plate, the plate sealed with optical sealing tape (Biorad), then placed in a Biorad Icycler realtime PCR machine. Reactions are run with the following conditions:
Stepl. 95.0°C 3 min
SStteepp22.. 9955..00°°CC 30 sec
Step3. 63.0°C 30 sec
Data collection and real-time analysis enabled.
Step4. 72.0°C 15 sec
60 cycles of steps 2-4. Step5. 95.0°C 30 sec
Step6. 50.0°C 30 sec
Step7. 50.0°C 10 sec
90 cycles of step 7 are carried out with the temperature increased by 0.5°C after each cycle starting with cycle 2. Melt curve data collection and analysis enabled.
Lower Ct values for infected tissue compared to uninfected tissue and other controls indicate expression. Comparison of expression levels between samples from A. fumigatus cultures and samples from infected larvae or mice (using the levels of house-keeping genes to normalise data) can be used to indicate whether the genes of interest are up-regulated during infection. Such up-regulated genes are suitable targets for an anti-fungal drugs. Example 6. Expression of recombinant proteins and/or fragments
Recombinant proteins or fragments are expressed to enable detailed study of function and for the development of an in vitro high-throughput screen for inhibitory compounds.
PCR is carried out using cDNA, prepared as described above, to generate polynucleotides encoding protein sequence essentially conesponding to SEQ LD Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90.
Primers are designed to encode the 5' and 3' ends of the coding sequences (SEQ LD Nos. 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 73, 77, 81, 85 or 89), with the addition of a 5' C in the forward primer and a 5' AA in the reverse primer. If the protein has an N-terminal leader peptide, this should be excluded. If the protein is made up of multiple domains, it may be desirable or necessary to express only a limited number of domains, or even a single domain. In these cases, primers are designed to conespond to domain boundaries.
PCR reactions are carried out using the following reaction mixture and conditions. All Reagents are present in the KOD kit (Novagen).
5 μl lOx PCR Buffer 5 μl dNTPs (2mM) 2 μl MgSO4 (25mM) 3 μl each primer (5 pmol) 1 μl template Af293 cDNA 30 μl nuclease-free water 1 μl KOD Hot Start DNA Polymerase
PCR reactions are run using the following conditions :-
Step 1 94°C 2 min Step 2 94°C 15 sec
Step 3 53°C 30 sec
Step 4 68°C 1 min
Step 5 68°C 10 min Step 6 10°C Hold
30 cycles of steps 2-4 are carried out and the PCR products purified using Qiagen' s QIAquick PCR Purification Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, West Sussex, RH10 9 AX, UK) according to the manufacturers instructions. The purified PCR products are examined on agarose gels.
To prepare the insert (PCR product), it must be subjected to end conversion to give blunt phosphorylated ends. The following reaction is set up:
1 μl PCR product
4 μl water
5 μl End conversion mix (Novagen)
The reaction mixture is incubated at 22°C for 15 mins, denatured at 75°C for 5 minutes, and then placed on ice for 2 mins. 1 μl T4 DNA ligase and 1 μl Blunt pETBlue-2 vector are then added and the ligation reaction carried out at 14°C overnight.
Ligation mixtures are then transformed into Nova Blue chemically competent E. coli cells, and plated on to a prewarmed LBAmp selection plate. After an overnight incubation at 37° C, ampicillin-resistant colonies are selected and grown up in ampicillin-containing LB medium. Plasmid DNA is isolated using the P*lasmid Mini Kit (Qiagen). Confirmation of the presence and conect orientation of the inserts is determined by restriction analysis and sequencing of the construct.
Purified plasmid DNA, which has been confirmed to be of the conect sequence and orientation, is transformed into chemically competent Tuner(DE3)pLacI E. coli cells (Novagen) and grown overnight at 37°C. 500 μl of an over-night culture is used to inoculate 10 ml of LB, 1% glucose, 500 μg/mL ampicillin-, 34 μg/ml chloramphenicol, and the cultures incubated at 37° C, 220 rpm until the cell density reaches an optical density of 0.6 (approximately 3 hours). Expression of the recombinant protein is then induced with IPTG (ImM) for 21 hours.
Samples are removed from the induction at 0, 1, 2, 3, 4, 5, and 21 hours and bacteria harvested by centrifugation at 1300 rpm for 5 minutes. Lysis buffer (1 ml Bugbuster (Novagen), 1 μl Benzonase (Novagen), 10 μl protease inhibitor cocktail (Novagen)) is added to each pellet; 50 μL for 1 OD6oo of culture, and samples incubated for 20 minutes at room temperature. Samples are then reduced b;y the addition of 1.5 μL lOx reducing mix (Invitrogen) and 3.5 μL sample loading buffer (Invitrogen) to a 10 μL sample, incubated at 70°C for 10 minutes, and then placed on ice. Reduced samples are run on an SDS-PAGE gel and stained with Coomassie. In this way the optimal duration of induction is determined. Larger cultures, e.g. 0.5 L final volume can then be prepared in the same way.
Recombinant proteins are purified from pellets as follows : Supernatant from the lysis stage above is added to prewashed Ni-Nta resin at a concentration of 5-10 mg protein per ml of resin and allowed to bind for 1 hour at 4° C. Protein-resin mix is then poured into a column, washed twice with 4 ml of wash buffer (2.5 ml 1M phosphate buffer pH8 , 6.25 ml 4M NaCl, 1 ml 1M imidazole pH8, 0.5 ml 10% Tween 20; made up to 50 mis in n.H2O) and then eluted in 4x 0.5 ml fractions with elution buffer (250 μl 1M phosphate buffer pH8, 625 μl 4M NaCl, 1.25 ml 1M imidazole pH8, 50 μl 10% Tween 20, made up to 5 ml in n.H2O). Fractions containing purified protein are detected by SDS-PAGE and Western blotting using an HSV-tag HRP conjugate (Novagen). Fractions containing purified recombinant protein are concentrated using YM10 columns (Millipore)
Alternative expression systems can be used for expression in bacteria, such as the glutathione S-transferase or mannose-binding fusion-protein system. Example 7. Assays for the identification of inhibitors 7.1 Biochemical assays for the identification of inhibitors
Recombinant proteins can be assayed using an assay type specific for the particular protein. The assay can then be used to screen for inhibitors. For example:
Galactosidases can be screened by incubating the galactosidase with fluorogenic substrates such as fluorescein digalactoside, methylumbelliferyl galactoside or fluorescent glycosphingolipid and monitoring the increase in fluorescence. Candidate substances can be included and examined for an effect on- the increase in fluorescence.
Helicases can be screened by incubating the helicase with an oligonucleotide containing a fluorescent analog such as 2-aminopurine and monitoring the unwinding mediated by the helicase using fluorescence spectroscopy. The degree of inhibition by candidate compounds can be assessed.
Kinases can be screened by incubating the kinase with [32PJ-ATP, substrate, and candidate substance, and measuring the decrease of 32P-label incorporation into the substrate due to the presence of the substance. Suitable substrates may include myelin basic protein, glycogen synthase and enolase.
Oxidoreductases can be screened by carrying out a redox reaction and measuring the inhibition of the reaction by detecting the amount of NADH or NADPH oxidation, for example by measuring the generation of the oxidised forms of NADH and NADPH spectroscopically at 340nm. Alternatively, a suitable colourimetric oxidoreductase substrate may be used to measure inhibition, such as methylene blue, phenazine methosulphate or 2, 6-dichlorophenolindophenol. 7.2 Genetic screen for the identification of inhibitors
In the case of proteins for which a function is not known or obvious, the m- cobank mutants can be screened for inhibitors using a generic genetic screen. These mutants are heterozygotes, that is, they cany one normal and one disrupted version of a gene. In most cases this should result in less gene product being made by the hete-rozygote than the wild type diploid. If the gene is essential for growth then the hetexozygote should be more sensitive to a compound that targets the product of that gene. This phenomenon is called haploinsufficiency and has been demonstrated in yeast (Genomic profiling of drag sensitivities via induced haploinsufficiency. Giaever G, Shoemaker DD, Jones TW, Liang H, Winzeler EA, Astromoff A, Davis RW. Nat Genet. 1999 21:278-83.)
The primary screen for genes of unknown function involves monitoring the growth of the mycobank heterozygous mutant versus the growth of the wild type diploid strain of Aspergillus fumigatus, in the presence and absence of a panel of compounds. Spore suspensions of these strains are set up in RPMI 1640 medium in 96-well plates. lxlO4 cfu/ml is the inoculum used. Potential inhibitors are added to give a final concentration of 32 μg/ml. The plates are then incubated at 37°C for 48h. The OD485 of the cultures is then measured using a plate reading spectrophotometer.
If both heretozygote and wild-type are unaffected no further work is carried out on the compound. If there is (a) growth of the wild type but no growth of the heteroz^gote, or (b) no growth of both strains, the Minimal Inhibitory Concentration (MIC) for the compound in each strain is determined as follows:
The heterozygote mycobank mutant and the wild type diploid are incubated in the presence of a range of concentrations of the chemical. The lowest concentration of chemical that prevents growth of the organism (the Minimal Inhibitory Concentration, MIC) is calculated for both strains. Doubling dilutions of the compound of interest are prepared in RPMI 1640 medium in 96-well plates starting at 50 μg/ml doλv to 0.1 μg/ml in duplicate. Each well is inoculated with either wild type or mutant ^Aspergillus fumigatus and the plate incubated at 37°C for 24/48h prior to measuring the D485.
An inhibitor of the product of the gene of unknown function will have a lower MIC in the mutant strain than in the wild type strain, i.e., a 2-fold or more difference in MIC between the 2 strains. This anti-fungal compound can then be used as the basis for chemistry approaches to improve the specificity, potency and other properties of the compound.
Example 8. Method for detecting fungal infection
The sequences described in the invention can be exploited to diagnose fungal infections. Samples from patients potentially canying an infection with A. fumigatus, A. nidulans, or C. albicans or rice leaves or stem potentially infected with MX- grisea, or of alfalfa infected with C. trifolii, or wheat infected with F. graminearum, F. sporotrichioides, or M. graminicola, or other organisms, are processed to exitract DNA using the DNAeasy Tissue kit or QIAamp DNA Blood Mini kit(Quiagen, Crawley, UK), although other DNA preparation methods are available and suitable.
Once DNA has been prepared, PCR reactions are set up as follows:
Reaction mix:
12.5 μl 2x ReddyMix PCR mastermix (ABgene) 1 μl each primer (5 pmol) 5 μl template DNA
5.5 μl nuclease-free water
Suiable primer pairs are designed using Primer3 (http://frodo.wi.init.edu/cgi- bin/primer3/primer3_www.cgi). Appropriate controls include; (i) template DNA but no primers; primers but no template (negative controls); (ii) cDNA encoding the gene of interest or DNA from fungal cultures instead of patient DNA (positive control). PCR reactions are run as follows:
Stepl 95°C 5 min
Step2 95°C 1 min Step3 53°C 1 min 30sec
Step4 72°C 1 min 30sec
Step5 72°C 10 min
Step6 4°C Hold
30 cycles of steps 2-4 are carried out and the PCR products examined on agarose gels. The production of a band of the conect molecular weight is diagnostic of the presence of the particular fungus. It may be additionally necessary to carry out diagnostic restriction digests of the PCR products. If necessary, PCR products are subcloned into a vector, such as pGEM-Teasy (Promega), and sequenced to verify that the PCR products are from the appropriate fungus.
Alternatively, the presence of an infection with A. fumigatus, A. nidulans, C. albicans or M. grisea, C. trifolii, F. graminearum, F. sporotrichioides or M. graminicola, or other organisms is detected by means of antibodies raised against the fungal protein. One suitable means is the use of a capture ELISA. Here, microtitre plates are coated with a monoclonal antibody raised against the fungal protein. Then the plates are incubated with diluted patient samples, or appropriate protein extracts of samples (particularly if the samples are biopsies or plant tissues). Plates are then incubated with a polyclonal antibody (again against the fungal protein). Finally, binding of the second antibody was detected by means of an enzyme-coupled or fluorescently-labelled antibody directed against the polyclonal. In practise, two monoclonal or polyclonal antibodies or various combinations may be used.
Example 9. Production of an antibody
Antibodies against the proteins of the invention will be of considerable use as diagnostic reagents (see example 8 above). As an immunogen, recombinant proteins are used (as described in Example 6). Alternatively, synthetic proteins or polypeptides encoding regions either unique to the individual proteins, or likely to provide cross- reactivity within a set of homologs are used. Peptides may need to be conjugated to carrier proteins before immunization.
Preimmune sera from animals to be immunised are screened against the immunogen to ensure that there is no endogenous cross reactivity. Animals (typically sheep, rabbits or mice) are then immunised. For polyclonal antibody production, the resulting sera is affinity purified using the immunogen cross-linked to a chromatography matrix. Alternatively, purification of the antibody fraction from the seram, e.g. using protein G or protein A cross-linked to a matrix, may be sufficient. Monoclonal antibody production proceeds by methods familiar to those skilled in the art.
The specificities of the resulting polyclonal and/or monoclonal antibodies are checked by ELISA and/or western blotting using the immunogen, related constracts or whole cell lysates and extracts as targets. Negative controls, such as paralogous proteins, different constracts or different species are also employed to test specificity and/or to determine the range of species and/or genus cross-reactivity.
Example 10. Production of fungi in which the gene of interest is functionally disabled. A BAG (bacterial artificial chromosome) clone library containing the A. fumigatus genome, partially digested with BamffJ and inserted into the vector pBACe3.6 was purchased from the Sanger Centre, Cambridge, UK. The BAC clone containing the gene to be inactivated is identified by bioinformatics (BLAST searching of Sanger BAC and related databases) and the glycerol stock of the clone grown up in 50 ml LB, 20 μg/ml chloramphenicol at 37°C overnight. The overnight culture is centrifuged at 4,500 rpm for 15 min. The bacterial pellet is resuspended in 4 ml of Buffer PI (Qiagen plasmid miniprep kit) and then 4 ml of buffer P2 (Qiagen plasmid miniprep kit, lysis buffer) is added and mixed gently by inverting 3-6 times. Proteins and genomic DNA are precipitated by adding 4 ml of buffer P3 (Qiagen plasmid miniprep kit, neutralizing buffer) and incubating on ice for 10 minutes. Following the centrifugation of the mixture at 4500 rpm for 30 min, the supernatant is transfened into a 50 ml falcon tube, an equal volume of phenol/chlorophorm (1:1) mixture is added, and the mixture centrifuged for 15 min at 4500 rpm. The supernatant is then transfened into an Oakridge tube and 0.7 volumes isopropanol are added. After mixing, the tube is centrifuged at 10,000 rpm (Beckman centrifuge, rotor JA-17) for 30 min at 4°C. The resulting pellet is washed with 2 ml 70% ethanol at the same speed. The resulting BAC DNA is resuspended in 100 μl buffer EB.
The transposition reaction is carried out as follows. 7 μl purified BAC, 1 μl transposon pZVK2 (an engineered plasmid the sequence of which is given as SEQ LD No. 71), containing the mosaic ends of pMOD2 (Epicenter), a kanamycin resistance gene and a Zeocin resistance gene under the control of fungal promoter) and 1 μl EZ:TN transposase (Epicenter) are incubated at 37°C for two hrs after which 1 μl stop solution (1%) SDS) is added and the mixture heated to 70°C for 10 minutes. Electrocompetent GeneHogs E. coli cells (Invitrogen) are then transformed with the transposed BAC, the cells plated onto LB agar, 25 μg/ml kanamycin, 20 μg/ml chloramphenicol, and plates incubated overnight at 37°C.
At least 96 colonies are picked and grown up in 96-well plates in 2xLB (double concentrated LB), 20 μg/ml chloramphenicol, at 37°C overnight. BAC DNA is then purified using the Millipore montage 96 BAC KIT using a MWG ROBOSEQ 4200 robot. BACs containing the transposon inserted into the gene of interest are identified by PCRs both spanning the gene of interest and extending from the transposon into the BAC. Insertion into the gene of interest is manifested as an increase in product size. Southern blots are also carried out to ensure that the transposon has only inserted once into the BAC.
The BAC is then linearised using a restriction enzyme determined to cut in the vector backbone but not the BAC DNA, and used to transform A. fumigatus strain Af293. A. fumigatus (haploid) protoplasts are prepared using 5% Glucanex (Novo Nordisk A/S) solution (in 0.6 M KCI) and shaking for 2 h at 80 rpm in 30°C . The protoplasts were washed with 0.6 M KCI and then with STC (Sorbitol, Tris, CaCl2). The washed protoplasts are diluted in STC to 105/ml and 100 μl transfened into 14 ml falcon tubes. 7 μl of linearised BAC are added to the tube and the whole mixture incubated on ice for 20 min. Transformation is carried out by adding 200 μl of PEG 8000 solution (60%w/v, pH 7.5) drop-wise over 2 min and then adding 800 μl PEG. The mixture is left at room temperature for 20 min. Transformed protoplasts are washed with STC, resuspended in 1 ml STC, spread onto CM-sorbitol- Zeocin (250 μg/ml) plates and incubated at 37 ° C.
After 4-10 days of incubation, zeocin resistant colonies are picked and checked for presence of the knocked-out gene by PCR using primers which specifically amplify the whole gene of interest. Usually 10-20 transformants are checked. The ectopic integration of the BAC gives two bands by PCR, one for the endogenous gene and one for the BAC/transposon construct, which has a higher molecular weight. Replacement of the endogenous gene with the transposon-modified gene results in a single band of higher molecular weigh by PCR. If none of the transformants show the disrupted endogenous gene, the gene of interest may be essential, with the knock-out cells having died and only cells where replacement is unsuccessful surviving. In this case, the transformation is canied out on diploids using the same method of transformation. Essentiality of the gene is then tested by rehaploidisation, and examining the segregation pattern in haploids.
The reader's attention is directed to all papers and documents which are filed concunently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1. Method of identifying an anti-fungal agent which targets an essential protein or gene of a fungus comprising contacting a candidate substance with (i) a protein which comprises the sequence shown by SEQ LD NOS: 3, 7, 11, 15,
19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 74, 78, 82, 86 or 90 (ii) a protein which has 50% identity with (i), (iii) a protein comprising a fragment of (i) or (ii) which fragment has a length of at least 50 amino acids, (iv) a polynucleotide that comprises sequence which encodes (i), (ii) or (iii), (v) a polynucleotide comprising sequence which has at least 70% identity with the coding sequence of (iv), and detennining whether the candidate substance binds or modulates (i), (ii), (iii), (iv), or (v), wherein binding or modulation of (i), (ii), (iii), (iv), or (v) indicates that the candidate substance is an anti-fungal agent.
2. Method according to the preceding claims comprising carrying out a reaction in the presence and absence of the candidate substance to determine whether the candidate substance inhibits the activity of the protein as defined in the preceding claim.
3. Method according to any one of the preceding claims wherein the (i), (ii), (iii), (iv) or (v) is from Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans; Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria graminis; Candida albicans; Candida cruzei; Candida glabrata; Candida parapsilosis; Candida tropicalis; Colletotrichium trifolii; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium graminarium; Fusarium solani; Fusarium sporotrichoides; Histoplasma capsulata; Leptosphaeria nodorum; Magnaporthe grisea; Mycosphaerella graminicola; Neurospora crassa; Phytophthora capsici; Phytophthora infestans; Plasmopara viticola; Pneumocystis jiroveci; Puccinia coronata; Puccinia graminis; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Saccharomyces cerevisiae; Schizosaccharomyces pombe; Trichophyton inter digitale; Trichophyton rubrurn; or Ustilago maydis.
4. Method according to any one of the preceding claims which further comprises formulating the identified anti-fungal agent into a agricultural or pharmaceutical composition.
5. Method according to any one of claims 1 to 3 which further comprises killing or impairing the growth of a fungus by contacting the fungus with the identified antifungal agent.
6. Use of (i), (ii), (iii), (iv) or (v) as defined in any one of claims 1 to 3 to identify or obtain an anti-fungal agent.
7. Use of an anti-fungal agent identified by the method of any one of claims 1 to 3 in the manufacture of a medicament for prevention or treatment of fungal infection.
8. Method of detecting the presence of a fungus in a sample comprising detecting the presence in the said sample of a protein or polynucleotide as defined in any one of claims 1 or 3.
9. Method according to claim 8 wherein the sample is from an human, animal or plant individual who is suspected of having a fungal infection.
10. An isolated protein or polynucleotide as defined in any one of claims 1 or 3
11. A vector comprising a polynucleotide as defined in any one of claims 1 or 3.
12. A recombinant cell comprising a polynucleotide as defined in any one of claims 1 or 3 or a vector according to claim 11.
13. A method of obtaining a protein as defined in any one of claims 1 or 3 comprising expressing the protein from a polynucleotide as defined in any one of claims 1 or 3 or a vector according to claim 11.
14. A method of obtaining a polynucleotide as defined in claim 1 or 3 comprising replication of a vector as defined in claim 11 or synthesis of the polynucleotide by condensation of nucleotides.
15. An organism which is transgenic for a polynucleotide as defined in any one of claims 1 or 3.
16. An organism which has been genetically engineered to render a polynucleotide or protein as defined in any one of claims 1 or 3 non-functional or inhibited.
17. An antibody which is specific for a protein as defined in any one of claims 1 or 3.
18. A method for preventing or treating a fungal infection comprising administering an anti-fungal agent identified by the method of any one of claims 1 to 3.
19. A method for preventing or treating a fungal infection comprising administering a protein or polynucleotide as defined in any one of claims 1 or 3.
20. A method of killing, or impairing the growth of, a fungus comprising inhibiting the expression or activity of a polynucleotide or protein as defined in any one of claims l or 3.
21. A method according to claim 20 wherein the fungus has infected a human, animal or plant individual.
22. A fungus which has been killed, or whose growth has been impaired, by inhibition of the expression or activity of a protein or polynucleotide as defined in any one of claims 1 or 3.
PCT/GB2005/001289 2004-04-02 2005-04-04 Anti-fungal target genes WO2005095975A2 (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001002550A2 (en) * 1999-07-01 2001-01-11 Janssen Pharmaceutica N.V. Cell death related drug targets in yeast and fungi
WO2001077295A1 (en) * 2000-04-11 2001-10-18 F2G Limited Mutant bank
US20020025524A1 (en) * 1998-09-30 2002-02-28 Millennium Pharmaceuticals, Inc., A Delaware Corporation Aspergillus N-myristoyl transferase genes and polypeptides and uses thereof
WO2002053728A2 (en) * 2000-12-29 2002-07-11 Elitra Pharmaceuticals, Inc. Gene disruption methodologies for drug target discovery
US20020119509A1 (en) * 1997-11-07 2002-08-29 Millennium Pharmaceuticals, Inc., A Massachusetts Corporation Essential fungal genes and their use
US20030119013A1 (en) * 2001-04-23 2003-06-26 Bo Jiang Identification of essential genes of Aspergillus fumigatus and methods of use

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020119509A1 (en) * 1997-11-07 2002-08-29 Millennium Pharmaceuticals, Inc., A Massachusetts Corporation Essential fungal genes and their use
US20020025524A1 (en) * 1998-09-30 2002-02-28 Millennium Pharmaceuticals, Inc., A Delaware Corporation Aspergillus N-myristoyl transferase genes and polypeptides and uses thereof
WO2001002550A2 (en) * 1999-07-01 2001-01-11 Janssen Pharmaceutica N.V. Cell death related drug targets in yeast and fungi
WO2001077295A1 (en) * 2000-04-11 2001-10-18 F2G Limited Mutant bank
WO2002053728A2 (en) * 2000-12-29 2002-07-11 Elitra Pharmaceuticals, Inc. Gene disruption methodologies for drug target discovery
US20030119013A1 (en) * 2001-04-23 2003-06-26 Bo Jiang Identification of essential genes of Aspergillus fumigatus and methods of use

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