WO2004104193A2 - Phospholipase c - Google Patents

Phospholipase c Download PDF

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Publication number
WO2004104193A2
WO2004104193A2 PCT/GB2004/002223 GB2004002223W WO2004104193A2 WO 2004104193 A2 WO2004104193 A2 WO 2004104193A2 GB 2004002223 W GB2004002223 W GB 2004002223W WO 2004104193 A2 WO2004104193 A2 WO 2004104193A2
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Prior art keywords
plc
polypeptide
polynucleotide
variant
seq
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WO2004104193A3 (fr
Inventor
Michael Birch
Sandra Elizabeth Lavens
Catherine Elaine Rycroft
Daniel Scott Tuckwell
Geoffrey David Robson
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F2G Ltd
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F2G Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04003—Phospholipase C (3.1.4.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to phospholipase C (PLC) enzymes.
  • PLC phospholipase C
  • the invention relates to fungal extracellular PLC genes, and encoded proteins thereof, including variants, derivatives and analogues thereof, and their uses in diagnosis and therapy.
  • Phospholipases are a broad group of enzymes that break down phospholipids, a ubiquitous class of molecule found in both eukaryotes and prokaryotes. Phospholipids are important structural molecules, forming the lipid bilayers of membranes. Phospholipids and their break down products also play a pivotal role in many signalling pathways.
  • Phospholipids are amphipathic molecules and are generally assembled into bilayers or micelles with very little free phospholipid present within cells. They are defined by their polar head group, the most common of which is phosphatidylcholine
  • PC phosphatidylethanolamine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phsphatidylglycerol
  • Phosphodiesterases such as phospholipases C and D, hydrolyse the phosphate bond
  • acyl hydrolases such as phospholipases Al, A2 and B and lysophospholipase, cleave the fatty acid substituents from the glycerophosphatidyl backbone.
  • Phospholipase C enzymes PLC; EC. 3.1.4.3 hydrolyse the phosphodiester bond between the phosphate oxygen and the glycerol carbon, liberating a diacylglycerol (DAG) molecule and headgroup-phosphate complex.
  • DAG diacylglycerol
  • PLCs may have wide substrate specificity acting on a variety of phospholipid classes including sphingomyelin (which is based on the amino alcohol sphingosine, rather than glycerol), although activity is often optimal against a specific class of phospholipid.
  • sphingomyelin which is based on the amino alcohol sphingosine, rather than glycerol
  • PLCs are subdivided into intracellular and extracullar PLCs.
  • Intracellular PLCs act exclusively within a cell, controlling phospholipid turnover and have important signalling roles, activating protein kinase C and the arachidonic acid cascade through the formation of DAG.
  • These PLCs are found in both prokaryotes and eukaryotes and are exemplified by the mammalian phosphoinositol specific PLC (PI-PLC).
  • Extracellular PLCs are secreted phospholipases and have been found in bacteria (Titball, 1993, Microbiol. Rev. 57, 347-366).
  • Fungal PLCs could play an important role in the progression and pathogenesis of fungal infections.
  • the invasion of host cells by fungi involves the penetration and damage of cell membranes by physical or enzymatic means. Since phospholipids are major components of the cell membrane, the production of extracellular phospholipases by an invading organism may be a significant factor in pathogenesis, as has been demonstrated for bacterial infections.
  • the presence of extracellular PLCs may be of particular significance since the lung and the alveoli are coated with lung surfactant consisting of up to 80% phospholipid. Breakdown of this lung surfactant, by extracellular PLCs could lead to a breakdown in localised oxygen tensions.
  • Phospholipases also have many industrial applications, including the modification of phospholipid emulsifiers.
  • An example of a phospholipid emulsifier is lecithin, which is a mixture of both polar and neutral lipids in which the content of polar lipids is at least 60%.
  • Phospholipid emulsifiers have many food and non-food applications, for example egg-lecithin is used as an emulsifier in for example dairy products, specifically mayonnaise, dressings, pastry, etc., soya lecithin for example, is for example used as an emulsifier in (low calorie) sauces, bread, margarine, cosmetics etc, other lecithins are used in for example chocolates, calf feed.
  • Modification of phospholipid emulsifiers by phospholipases may cause an increased emulsification of the oil/water mixture. Modification of phospholipid emulsifiers by phospholipases may increase the stability of the emulsions resulting from the addition of the modified phospholipid emulsifiers for a wider or different pH and/or temperature range. Modification of phospholipid emulsifiers by phospholipases may increase the stability of the emulsions, resulting from the addition of modified phospholipid emulsifiers, in the presence of Ca2+ or Mg2+ ions.
  • phospholipases can be used for the degumming of vegetable oils in the processing of these oils.
  • lecithins are removed from vegetable oils, for example soy oils, rapeseed (canola) oils, linseed oils, sunflower oils, to increase among others the stability of the vegetable oil, by washing the oil phase with water, wherein mixing of the water and oil under high shear conditions forces the bulk of the lecithins into the aqueous phase, which is subsequently removed in a separator.
  • phospholipases are used to remove the precipitate that occurs during the saccharification (with the aid of a-amylase and glucoamylase) of wheat gluten or wheat starch to produce glucose syrups. The removal of the precipitate considerably speeds up the subsequent filtration of the resulting glucose syrups.
  • the above-mentioned industrial applications of the phospholipase enzyme are only a few examples and this listing is not meant to be restrictive.
  • phospholipases are particularly useful in baking applications to improve dough or baked product quality.
  • Wheat flour contains approximately 2.2-2. 9% lipids.
  • the flour lipids can be divided into starch lipids (0.8-0. 9 %) and non-starch lipids (1.4-2. 0 %>).
  • starch lipids consist mainly of polar lysophospholipids
  • non- starch lipids consist of about 40 % neutral triglycerides and 40 % polar phospho-and glycolipids.
  • PLC extracellular PLC
  • extracellular PLC polypeptide extracellular PLC protein
  • extracellular PLC protein are defined as nucleic acids or proteins which: (a) match the PFAM matrix PF04185 with an E-value of O.00001 (http://www.sanger.ac.u Sofrvare/Pfam/search.shtml); and/or (b) match the Pseudomonas aeruginosa PLC protein (PHLC_PSEAE) or DNA (M13047) on BLAST with E-values of O.00001; and/or (c) have the enzyme activity defined by EC number 3.1.4.3, noting that the substrate may not be defined as, or restricted to, phosphatidylcholine, but may include other phospholipids or sphingomyelin http://www.chem.qmul.ac.Uk/iubmb/enzyme/EC3/l/ 4/3.html); and/or (d) show enzymic
  • PLCs can thus be identified either by sequence similarity to a known member of the family or by enzymic activity, since only a few of the available sequences relate to functionally characterised molecules (as is the case with most proteins). Extracellular PLCs identified on the basis of sequence similarity are validated by alignment of the sequence with known extracellular PLCs.
  • an isolated polynucleotide comprising a fungal PLC gene.
  • the PLC gene may comprise a PLC polynucleotide that comprises:
  • the PLC polypeptide may comprise:
  • amino acid sequence independently selected from at least one of SEQ ID NOS: 3, 6, 9, 12, 14 or 16; or
  • the inventors have isolated three extracellular PLCs genes (arbitrarily named PLC-A, PLC-B and PLC-C) in A. fumigatus, and also the corresponding polypeptides, i.e. the PLC proteins, which each of these three genes encode. Furthermore, the inventors have isolated PLC genes from Aspergillus nidulans, Aspergillus niger and Magnaporthe grisea.
  • the isolated polynucleotide comprises a PLC gene.
  • the isolated polypeptide comprises a PLC.
  • the PLC is an extracellular PLC, preferably when functional.
  • the polynucleotide may comprise native, synthetic or recombinant polynucleotide, and the polypeptide may comprise native, synthetic or recombinant polypeptide.
  • the polynucleotide or polypeptide may comprise combinations of native, synthetic or recombinant polynucleotide or polypeptide, respectively.
  • the polynucleotides and polypeptides of the invention may have a sequence which is the same as, or different from, naturally occurring PLC polynucleotides and polypeptides.
  • references to polynucleotides and polypeptides being "isolated from” a particular organism include polynucleotides and polypeptides which were prepared by means other than obtaining them from the organism, such as synthetically or recombinantly.
  • the isolated polynucleotide, and preferably the isolated polypeptide is isolated from a filamentous fungus, more preferably an Ascomycete.
  • the isolated polynucleotide, and preferably the isolated polypeptide is isolated from an organism independently selected from a group of genera consisting of Aspergillus; Blumeria; Candida; Cryptococcus; Encephalitozoon;
  • the isolated polynucleotide, and preferably the isolated polypeptide is isolated from an organism independently selected from a group of genera consisting of Aspergillus and Magnaporthe.
  • the isolated polynucleotide, and preferably the isolated polypeptide is isolated from an organism independently selected from a group of species consisting of Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans; Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria graminis; Candida albicans; Candida cruzei; Candida glabrata; Candida parapsilosis; Candida tropicalis; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium solani; Leptosphaeria nodorum; Magnaporthe grisea; Phytophthora capsici; Phytophthora infestans; Plasmopara viticola; Pneumocystis jiroveci; Puccinia coronata; Puccinia graminis; Pyricularia oryzae; Pyth
  • the isolated polynucleotide, and preferably the isolated polypeptide is isolated from an organism independently selected from a group of species consisting of Aspergillus fumigatus; Aspergillus nidulans; Aspergillus niger, and Magnaporthe grisea.
  • the polynucleotide, and preferably the polypeptide may be isolated from
  • Aspergillus preferably from Aspergillus fumigatus, and more preferably, from A. fumigatus AF293.
  • the polynucleotide, and preferably the polypeptide may be isolated from Aspergillus nidulans, or Aspergillus niger.
  • the polynucleotide, and preferably the polypeptide may be isolated from Magnaporthe, and more preferably, from Magnaporthe grisea .
  • Bioinformatics analysis was carried out to identify functionally important regions within the fungal PLCs.
  • the PFAM phosphoesterase profile for PLCs (PF04185; http://www.sanger.ac.uk/Sofhvare/Pfam ) also detects acid phosphatases, indicating a functional similarity between these protein families. Since acid phosphatases typically show only 15-20% identity to PLCs at the amino acid level (i.e, they are not closely related), conserved or similar regions of sequence are likely to be- important for basic functional properties. Therefore, a multiple alignment of fungal PLCs, plant PLCs, bacterial PLCs and related acid phosphatases was generated (see Figure 1 and Example 4 hereinafter). From this it was possible to identify five regions or motifs (Boxes II, III, V, VI, and X) that are conserved between all PLCs and acid phosphatases and are therefore predicted to play an important role in PLC function.
  • the isolated polypeptide may comprise a region or motif independently selected from at least one of the following:- (i) a region comprising an amino acid sequence
  • At least one region or motif may be functional.
  • the isolated polypeptide may comprise a region or motif independently selected from at least one of the following:- (i) a region comprising an amino acid sequence substantially as set out as row PLC-B in box LI as shown in Figure 1, or a variant thereof;
  • the isolated polypeptide may comprise a region or motif independently selected from at least one of the following:- (i) a region comprising an amino acid sequence substantially as set out as row PLC-C in box 13 as shown in Figure 1, or a variant thereof;
  • the isolated polypeptide may comprise a region or motif comprising an amino acid sequence substantially as set out as row Anidl in box X as shown in Figure 1, or a variant thereof.
  • the isolated polypeptide may comprise a region or motif independently selected from at least one of the following: -
  • the isolated polypeptide may comprise a region or motif comprising an amino acid sequence substantially as set out as row Anigl in box VI as shown in Figure 1, or a variant thereof; and (i) a region comprising an amino acid sequence substantially as set out as row Anigl in box X as shown in Figure 1, or a variant thereof.
  • the isolated polypeptide may comprise a region or motif comprising an amino acid sequence substantially as set out as row
  • fungus-specific residues have been identified, which are conserved between most or all fungal PLCs, and which are absent from other PLCs. These residues, at positions 236, 265, 279, 365, 368, 369, 535 and 537, represent sites at which the putative active-site regions identified by homology between PLCs and acid phosphatases may acquire fungus-specific function (as shown in Figure 1).
  • the isolated polypeptide may comprise an amino acid sequence independently selected from at least one of the following: -
  • Analysis of the multiple alignment given in Figure 1 identified regions that were conserved between bacterial, plant and fungal PLCs, but not acid phosphatases (Boxes I, VII, and IX, as shown in Figure 1). This conservation suggests that these regions are important for the specific function of PLCs, such as binding of the lipid moiety.
  • the isolated polypeptide may comprise an amino acid sequence independently selected from at least one of the following: - (i) a PLC-specificity region shown in rows PLC-A/PLC-B/PLC-C/Mporl of box I as shown in Figure 1, or a variant thereof; (ii) a PLC-specificity region shown in rows PLC-A/PLC-B/PLC-C/Anigl of box VLT as shown in Figure 1, or a variant thereof; and (iii) a PLC-specificity region shown in rows PLC-A/PLC-B/PLC-C/Anigl of box IX as shown in Figure 1 , or a variant thereof.
  • the isolated polypeptide may comprise an amino acid sequence independently selected from at least one of the following:-
  • the protein or polynucleotide also comprises sequence flanking the region, motif or residue as shown in Figures 1 or Table LT such as sequences of length at least 10, 20 or 30 amino acids/nucleotides flanking the N terminal side and/or C terminal side, or 5' and/or 3' side, of the region, motif or residue; or sequence which has percentage identity with the flanking sequence.
  • the isolated polynucleotide may comprise DNA, preferably genomic DNA.
  • the isolated polynucleotide comprises substantially the sequence as shown by bases 1658-2959 of SEQ ID No.1 , or a complement, or a variant thereof.
  • the isolated polynucleotide comprises substantially the sequence as shown by bases 1313-2733 of
  • the isolated polynucleotide comprises a first exon as shown by bases 1313-1873 of SEQ ID No.4, and preferably, a second exon as shown by bases 1924-2733 of SEQ LD No.4.
  • the isolated polynucleotide comprises substantially the sequence as shown by bases 948-2376 of
  • the isolated polynucleotide comprises a first exon as shown by bases 948-1526 of SEQ LD No.7, and preferably, a second exon as shown by bases 1576-2376 of SEQ LD No.7.
  • the isolated polynucleotide comprises substantially the sequence as shown by bases 141-315 of SEQ ID No.10, or a complement, or a variant thereof.
  • the isolated polynucleotide comprises substantially the sequence as shown by bases 1-404 of SEQ LD No.13, or a complement, or a variant thereof.
  • the isolated polynucleotide comprises substantially the sequence as shown by bases 21-517 of SEQ LD No.15, or a complement, or a variant thereof.
  • the isolated polynucleotide encodes a PLC polypeptide which, in a first embodiment, A. fumigatus PLC-A, preferably, comprises substantially the amino acid sequence as shown as SEQ ID No.3, or a variant thereof.
  • A. fumigatus PLC-B the polynucleotide encodes a PLC polypeptide which encodes a PLC polypeptide which, preferably comprises substantially the amino acid sequence as shown as SEQ ID No.6, or a variant thereof.
  • A. fumigatus PLC-C the polynucleotide encodes a PLC polypeptide which, preferably comprises substantially the amino acid sequence as shown as SEQ LD No.9, or a variant thereof.
  • the polynucleotide encodes a PLC polypeptide which preferably comprises substantially the amino acid sequence as shown as SEQ LD No.12, or a variant thereof.
  • the polynucleotide encodes a PLC polypeptide, which preferably comprises substantially the amino acid sequence as shown as SEQ ID No.14, or a variant thereof.
  • the polynucleotide encodes a PLC polypeptide which, preferably comprises substantially the amino acid sequence as shown as SEQ LD No.16, or a variant thereof.
  • the isolated polynucleotide may comprise RNA, preferably mRNA which is, preferably spliced mRNA.
  • RNA preferably mRNA which is, preferably spliced mRNA.
  • A. fumigatus PLC-A the isolated polynucleotide comprises substantially the sequence shown as SEQ LD No.2, or a complement, or a variant thereof.
  • A. fumigatus PLC-B the isolated polynucleotide comprises substantially the sequence shown as SEQ LD No.5, or a complement, or a variant thereof.
  • A. fumigatus PLC-C the isolated polynucleotide comprises substantially the sequence shown as SEQ LD No.8, or a complement, or a variant thereof.
  • the isolated polynucleotide comprises substantially the sequence shown as SEQ LD No.11, or a complement, or a variant thereof.
  • the isolated polynucleotide comprises substantially the sequence shown as SEQ LD No.13, or a complement, or a variant thereof.
  • the isolated polynucleotide comprises substantially the sequence shown as SEQ LD No.15, or a complement, or a variant thereof.
  • A. nidulans PLC the isolated polynucleotide comprises substantially the sequence shown as SEQ LD No.11, or a complement, or a variant thereof.
  • A. niger PLC the isolated polynucleotide comprises substantially the sequence shown as SEQ LD No.13, or a complement, or a variant thereof.
  • M. grisea PLC the isolated polynucleotide comprises substantially the sequence shown as SEQ LD No.15, or a complement, or a variant thereof.
  • A. nidulans PLC the isolated polyn
  • the polypeptide comprises substantially the sequence as shown as SEQ LD No.3, or a variant thereof.
  • the polypeptide comprises substantially the sequence as shown as SEQ LD No.6, or a variant thereof.
  • the polypeptide comprises substantially the sequence as shown as SEQ LD No.9, or a variant thereof.
  • the polypeptide comprises substantially the sequence as shown as SEQ LD No.12, or a variant thereof.
  • A. nidulans PLC the polypeptide comprises substantially the sequence as shown as SEQ LD No.12, or a variant thereof.
  • A. nidulans PLC comprises substantially the sequence as shown as SEQ LD No.12, or a variant thereof.
  • the polypeptide comprises substantially the sequence as shown as SEQ LD No.14, or a variant thereof.
  • the polypeptide comprises substantially the sequence as shown as SEQ LD No.16, or a variant thereof.
  • the isolated polypeptide is encoded by the sequence shown as bases 1658-2959 of SEQ LD No.l or a complement, or a variant thereof.
  • the isolated polypeptide is encoded by exon sequences of the polynucleotide shown in SEQ LD No.4, preferably bases 1313-1873, and preferably bases 1924-2733, or a complement, or a variant therof.
  • A. fumigatus PLC-A the isolated polypeptide is encoded by the sequence shown as bases 1658-2959 of SEQ LD No.l or a complement, or a variant thereof.
  • the isolated polypeptide is encoded by exon sequences of the polynucleotide shown in SEQ LD No.4, preferably bases 1313-1873, and preferably bases 1924-2733, or a complement, or a variant therof.
  • A. fumigatus PLC-A the isolated polypeptide is encoded by the sequence shown as bases 1658-2959 of SEQ LD No.
  • the polypeptide is encoded by the exon sequences of the polynucleotide shown in SEQ LD No.7, preferably bases 948-1526, and preferably bases 1573-2376, or a complement, or a variant thereof.
  • the isolated polypeptide is encoded by the sequence shown as bases 141-315 of SEQ LD No.10 or a complement, or a variant thereof.
  • the isolated polypeptide is encoded by the sequence shown as bases 1-404 of SEQ LD No.13 or a complement, or a variant thereof.
  • the isolated polypeptide is encoded by the sequence shown as bases 21-517 of SEQ LD No.15 or a complement, or a variant thereof.
  • an isolated polynucleotide comprising substantially a nucleotide sequence independently selected from at least one sequence referred to in Table II, or a complement, or a variant thereof.
  • the isolated polynucleotide comprises a PLC gene.
  • the isolated polynucleotide comprises substantially a nucleotide sequence independently selected from at least one of the following:- (i) bases 1658-2959 of SEQ LD No.l or a complement, or a variant thereof; (ii) bases 1313-2733 of SEQ LD No.4 or a complement, or a variant thereof; (iii) bases 948-2376 of SEQ LD No.7 or a complement, or a variant thereof;
  • bases 141-315 of SEQ LD No.10 or a complement, or a variant thereof (v) bases 1-404 of SEQ D No.13. or a " complement, or a variant thereof; and (vi) bases 21-517 of SEQ LD No.15, or a complement, or a variant thereof.
  • the isolated polynucleotide encodes a gene product, which gene product comprises at least one amino acid sequence independently selected from SEQ LD No.3, SEQ LD No.6, SEQ LD No.9, SEQ LD No.12, SEQ LD No.14 and SEQ LD No.16, or variants thereof.
  • an isolated polypeptide comprising substantially an amino acid sequence independently selected from at least one of SEQ LD No.3, SEQ LD No.6, SEQ LD No.9, SEQ LD No.12, SEQ LD No.14, and SEQ LD No.16, or variants thereof.
  • the isolated polypeptide comprises a PLC.
  • the polypeptide is encoded by a polynucleotide which polynucleotide comprises substantially a sequence independently selected from at least one of the following:-
  • nucleic acid/polynucleotide/polypeptide we mean an amino acid, polynucleotide or polypeptide produced naturally from biological sources either in vivo or in vitro.
  • amino acid/polynucleotide/polypeptide we mean an amino acid, polynucleotide or polypeptide which has been produced artificially or de novo using a DNA or protein synthesis machine known in the art.
  • recombinant amino acid polynucleotide /polypeptide we mean an amino acid, polynucleotide or polypeptide which has been produced using recombinant DNA or protein technology, or methodologies which are known to the skilled technician.
  • variants and the terms “substantially the amino acid/polynucleotide/polypeptide 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 polypeptide may be encoded by a variant .polynucleotide.
  • a polynucleotide fragment will typically comprise at least 10 bases, such as at least 20, 30, 50, 100, 200, 500 or 1000 bases.
  • a polypeptide 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.
  • variant and the terms “substantially the amino acid/polynucleotide/polypeptide sequence” we mean that the sequence has at least
  • Calculation of percentage identities between different amino acid/polynucleotide/polypeptide 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 LUB; 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/polypeptide seqences may be synthesised de novo, or may be native amino acid/polynucleotide/polypeptide sequence, or a derivative thereof.
  • An amino acid/polynucleotide/polypeptide sequence with a greater identity than 65% to any of the sequences referred to is also envisaged.
  • An amino acid/polynucleotide/polypeptide sequence with a greater identity than 70%> to any of the sequences referred to is also envisaged.
  • An amino acid/polynucleotide/polypeptide sequence with a greater identity than 75% to any of the sequences referred to is also envisaged.
  • An amino acid/polynucleotide/polypeptide sequence with a greater identity than 80% to any of the sequences referred to is also envisaged.
  • the amino acid/polynucleotide/polypeptide 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.
  • a sequence which is "substantially the amino acid/polynucleotide/peptide sequence" may be the same as the relevant sequence.
  • 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.
  • identity is measured with reference to SEQ LD Nos: 3, 6, or 9.
  • the variant polypeptide has at least 40% identity, such as at least 60% or at least 80% identity with SEQ ID NOS: 3, 6 or 9 or a portion of SEQ LD NOS: 3, 6 or 9.
  • Polynucleotide sequences which encode such variant polypeptides are also preferred.
  • a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ LD Nos. 1, 2, 4, 5, 7, 8, 10, 11, 13 or 15 or their complements under stringent conditions.
  • 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.
  • a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ LD Nos. 3, 6, 9, 12, 14 or 16.
  • 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 methionine.
  • 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 majority of eukaryotes. Any comparisons of polynucleotides and polypeptides from such organisms with the sequences given here should take these differences into account.
  • the accurate alignment of protein or DNA sequences is a complex process which has been investigated in detail by a number of researchers. Of particular importance is the trade-off between optimal matching of sequences and the introduction of gaps to obtain such a match. In the case of proteins, the means by which matches are scored is also of significance.
  • Align 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.
  • variant polypeptide is able to complement the activity of the original PLC polypeptide in a fungal cell, for example the presence of the variant polypeptide in a fungal cell whose native equivalent PLC gene has been rendered non-functional, may rescue a nutrient deficiency, reconstitute a nutrient scavenging pathway, restore physiological properties, or allow the cell to remain alive.
  • the polynucleotide or polypeptide may be modified prior to use, preferably to produce a derivative or variant thereof.
  • the polynucleotide or polypeptide may be derivatised.
  • the polypeptide may be modified by epitope tagging, addition of fusion partners or purification tags such as glutathione S-transferase 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-fluorescent 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 polynucleotide defined in the first- or third aspect, or the polypeptide defined in the second or fourth aspect, may not be modified or derivatised.
  • the medicament is adapted to retard or prevent a fungal infection.
  • the fungal infection may be in human, animal or plant.
  • the polynucleotide or polypeptide may be used for the development of a drug.
  • the polynucleotide or polypeptide may be used in, or for the generation of, a molecular model of said polynucleotide or said polypeptide.
  • the invention provides a method of screening which may be used to identify modulators of PLC genes or PLC polypeptides.
  • a candidate substance is contacted with a polynucleotide or polypeptide of the invention and whether or not the candidate substance binds or modulates the polynucleotide, or polypeptide is determined.
  • the modulator may increase or decrease expression of the polynucleotide, or may promote (agonise) or inhibit (antagonise) the activity of the polypeptide.
  • a therapeutic modulator (against fungal infection) will generally causes cause decreased expression of a PLC polypeptide or will inhibit the activity of a PLC polypeptide.
  • the method may be carried out in vitro (inside or outside a cell) or in vivo.
  • the method is carried out on a cell, cell culture or cell extract.
  • the cell may or may not be a cell in which the polynucleotide or polypeptide 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.
  • Whether or not a candidate substance modulates the activity of the polypeptide may be determined by providing the candidate substance to the polypeptide under conditions that permit activity of the polypeptide, and determining whether the candidate substance is able to modulate the activity of the polypeptide.
  • the activity which is measured may be any of the activities of the polypeptide of the invention mentioned herein, such as phospholipase C activity.
  • the screening method comprises carrying out a phospholipase C assay in the presence and absence of the candidate substance to determine whether the candidate substance inhibits the phospholipase activity of the protein of the invention, wherein the phospholipase assay is carried out by contacting said protein with a phospholipase C substrate such as pNitro phenylphosphorylcholine (pNPPC), under conditions in which in the absence of the candidate substance the protein catalyses cleavage of the substrate.
  • a phospholipase C substrate such as pNitro phenylphosphorylcholine (pNPPC)
  • the inhibition of the phospholipase C reaction is measured by detecting the amount of pNPPC cleaved. This can be done spectroscopically by measurement at 405 nm (see Example 7).
  • Suitable candidate substances which can tested in the above methods include naturally occuring or synthesised phospholipids or phospholipid derivatives, combinatorial libraries, defined chemical identities, peptide and peptide mimetics, oligonucleotides, natural product libraries and display libraries (e.g. phage display libraries).
  • 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.
  • antibody products for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies may also be tested.
  • the polynucleotide or polypeptide may be modified prior to use, preferably to produce a derivative or variant thereof.
  • the polynucleotide or polypeptide may be derivatised.
  • the polynucleotide or polypeptide 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, preferably a PLC inhibitor.
  • the drug or medicament is adapted to inhibit expression and/or activity of the polynucleotide or a fragment thereof, and/or the function of the polypeptide or a fragment thereof.
  • the fungal infection comprises an infection by a filamentous fungus, more preferably an Ascomycete, and even more preferably, an organism independently selected from a group of genera consisting of Aspergillus; Blumeria; Candida; Cryptococcus; Encephalitozoon; Fusarium; Leptosphaeria; Magnaporthe; Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Trichophyton; and Ustilago.
  • a filamentous fungus more preferably an Ascomycete
  • an organism independently selected from a group of genera consisting of Aspergillus; Blumeria; Candida; Cryptococcus; Encephalitozoon; Fusarium; Leptosphaeria; Magnaporthe; Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia
  • the fungal infection comprises an infection by an organism independently selected from a group of genera consisting of Aspergillus and Magnaporthe.
  • the fungal infection comprises an infection by an organism independently selected from a group of species consisting of Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans; Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria graminis; Candida albicans; Candida cruzei; Candida glabrata; Candida parapsilosis; Candida tropicalis; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium solani; Leptosphaeria nodorum; Magnaporthe grisea; Phytophthora capsici; Phytophthora infestans; Plasmopara viticola; Pneumocystis ⁇ roveci; Puccinia coronata; Puccinia graminis; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; T
  • the fungal infection comprises an infection by an organism independently selected from a group of species consisting of A. fumigatus; A. nidulans; A. niger; and M. grisea.
  • a method of detecting the presence of a fungal infection in an individual comprising: - (i) obtaining a sample from an organism; and
  • the individual may be a person (human) or animal (such as a mammal or bird) or a plant.
  • the individual may be of any of the species of animal mentioned below in regard to the therapeutic aspects of the invention.
  • the fungal infection may arise from infection with an organism independently selected from a group of genera consisting of Aspergillus; Blumeria; Candida; Cryptococcus; Encephalitozoon;
  • the fungal infection may arise from infection with an organism independently selected from a group of species consisting of Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans; Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria graminis; Candida albicans; Candida cruzei; Candida glabrata; Candida parapsilosis; Candida tropicalis; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium solani; Leptosphaeria nodorum; Magnaporthe grisea; Phytophthora capsici; Phytophthora infestans; Plasmopara viticola; Pneumocystis jiroveci; Puccinia coronata; Puccinia graminis; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Trich
  • the sample comprises a biological sample which, preferably, comprises nucleic acid and/or polypeptide.
  • the nucleic acid or polypeptide 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 invasive procedure such as vitreous tap, tympanocentesis, brain biopsy or aspiration, nasal 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 polynucleotide as defined by the first or third aspect comprises use of at least one oligonucleotide pair adapted to be used for amplification of DNA, preferably genomic, more preferably, fungal genomic DNA.
  • the amplification may be PCR amplification.
  • the PCR amplification employs at least one primer pair comprising a polynucleotide selected from the group consisting of:
  • Aspergillus fumigatus SEQ LD Nos 64 + 65 (216bp product); SEQ LD Nos 66 + 67 (264 bp product), SEQ LD Nos 68 + 69 (211 bp product); Aspergillus nidulans: SEQ LD Nos. 70 + 71 (147 bp product); Aspergillus niger. SEQ LD Nos. 72 + 73 (139 bp product); and Magnaporthe grisea: SEQ LD Nos. 744- 75 (120 bp product)
  • 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 polypeptide as defined by the second or fourth aspect comprises use of a monoclonal or polyclonal antibody directed to part or all of the polypeptide as defined by the second or fourth aspect.
  • a recombinant DNA molecule or vector comprising a polynucleotide as defined in the first or third aspect, or fragment or variant thereof.
  • the fragment may be a functional fragment of the polynucleotide.
  • the fragment of the polynucleotide may comprise a polynucleotide sequence encoding any region of DNA shown in Tables I or II, or any combination thereof.
  • the polynucleotide may encode a motif, region or domain or residue selected from Table
  • the recombinant DNA molecule may comprise an expression cassette.
  • the recombinant DNA molecule 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 recombinant DNA molecule defined by the eighth aspect may be transformed or transfected with the recombinant DNA molecule by suitable means.
  • the cell produces a recombinant protein of the invention.
  • the cell of the ninth aspect produces a recombinant polypeptide.
  • the recombinant polypeptide may comprise the isolated polypeptide defined by the second or fourth aspect, or fragment thereof.
  • the fragment is a functional fragment.
  • 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 a 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 polynucleotide defined by the first or third aspect, or variant thereof, or a polypeptide defined by the second or fourth aspect, or variant thereof, is nonfunctional and/or inhibited.
  • 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 according to the first or third aspect, 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;
  • 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 preferred 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.
  • 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.
  • the polynucleotide is non-functional and/or inhibited without introducing a mutation into the PLC gene or its regulatory regions.
  • This may be done by using specific inhibitors.
  • 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 fromfunctioning, such as neutralizing antibodies (for instance an anti-PLC antibody).
  • the inhibitor may also be an antisense oligonucleotide, an oligonucleotide that mediates RNAi, or any synthetic chemical capable of inhibiting expression of the gene or the stability and/or function of the polypeptide.
  • the inhibitor may also be a polypeptide which interacts with the PLC to prevent its function.
  • the inhibitor may an RNA molecule which causes inhibition by RNA interference.
  • the antisense polynucleotide or RNA molecule which causes RNA interference are examples of polynucleotides of the invention.
  • an antibody exhibiting immunospecificity for a polypeptide of the second or fourth aspect, or fragment or variant thereof.
  • 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 PLC proteins together, or may be raised against proteolytic or recombinant fragments.
  • the term "antibody”, unless specified to the contrary, includes fragments which bind a polypeptide 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.
  • a method for treating a fungal infection comprising administering to an individual a polynucleotide defined in the first or third aspect, or variant thereof, or a polypeptide defined in the second or fourth aspect, or variant thereof, each being optionally modified.
  • the polynucleotide may be modified prior to use, and may include derivatisation.
  • the polynucleotide or polypeptide may not be modified or derivatised prior to administration to the individual.
  • the method of treating may comprise antifungal therapy.
  • the individual may be a person (human), animal (such as a mammal or bird), or a plant.
  • the animal is typically an agricultural animal such as a pig, cow, horse, sheep, goat, camel, chicken, duck, turkey or goose.
  • the animal may be a cat or a dog.
  • the polypeptides, polynucleotides, vectors, cells or antibodies of the invention may be present in a substantially isolated form. They may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95% > , 98%o or 99%>, of the proteins, polynucleotides, cells or dry mass of the preparation.
  • a fungal PLC inhibitor According to a thirteenth aspect, there is provided a fungal PLC inhibitor.
  • the inhibitor is adapted to inhibit, or render non-functional, a fungal
  • the fungal PLC gene may comprise a polynucleotide defined in the first or third aspect, or variant thereof.
  • the PLC gene product may comprise a polypeptide defined in the second or fourth aspect, or variant thereof.
  • 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, corn 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, carboxymethylcellulose, 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 hydrochlori.de.
  • 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 from 5 mg to 2 g.
  • 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 correspond to substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
  • a preferred 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 form (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.
  • 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).
  • 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 mono ethyl 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
  • 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.
  • 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 30 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 2 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%o, 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%o 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 surfactant preferably formulated as concentrates, the end user will normally employ dilute formulations.
  • the phospholipases of the present invention may be used for degumming an aqueous carbohydrate solution or slurry to improve its filterability, particularly, a starch hydrolysate, especially a wheat starch hydrolysate which is difficult to filter and yields cloudy filtrates.
  • the treatment may be performed using methods well known in the art.
  • Phospholipases of the present invention may be used in a process to reduce the phospholipid content in edible oil by treating the oil with the polypeptide to hydrolyse a major portion of the phospholipid and separating an aqueous phase containing the hydrolysed phospholipid from the oil. Such a process is applicable to the purification of any edible oil that contains phospholipid, e. g.
  • the oil Prior to phospholipase treatment, the oil is preferably pre-treated to remove slime (mucilage), e. g. , by wet refining. Typically, the oil will contain 50-250 ppm of phosphorus as phospholipid at the beginning of the treatment with the phospholipase, and the treatment may reduce the phosphorus value to below 5-10 ppm.
  • the phospholipase treatment is conducted by dispersing an aqueous solution of the phospholipase, preferably as droplets with an average diameter below 10 um. The amount of water is preferably 0.5-5%o by weight in relation to the oil.
  • An emulsifier may optionally be added.
  • the phospholipase treatment can be conducted at a pH in the range of about 3.5 to about 5 to maximize the enzyme's performance, or a pH in the range of about 1.5 to about 3 (e. g. , 2-3) may be used in order to suppress the alkaline hydrolysis of triglycerides (saponification).
  • the pH may be adjusted by adding citric acid, a citrate buffer, or hydrochloric acid.
  • a suitable temperature is generally 30-70 C (particularly 30-45 C, e. g., 35-40 C).
  • the reaction time will typically be 1-12 hours (e. g. , 2-6 hours).
  • a suitable enzyme dosage will usually be 0.1-10 mg per liter (e. g.
  • the phospholipase treatment may be conducted batchwise, e. g. , in a tank with stirring, or it may be continuous, e. g. , a series of stirred tank reactors.
  • the phospholipase treatment is followed by separation of an aqueous phase and an oil phase.
  • the separation may be performed by conventional means, e. g: , centrifugation.
  • the aqueous phase will contain phospholipase, and the enzyme may be re-used to improve the process economy.
  • the treatment may be performed using any of the methods known in the art. See, for example, U. S. Patent No. 5,264, 367, EP-A-654,527, JP-A-2-153997.
  • the proteins of the invention may be incorporated into a dough or baked product to improves one or more properties of the dough or the baked product obtained from the dough relative to a dough or a baked product in which the polypeptide is not incorporated.
  • the phospholipase according to the invention may be added in any step of the dough preparation and may be added in one, two or more steps.
  • the phospholipase according to the invention may be added to the ingredients of a dough that is kneaded and baked to make the baked product using methods well known in the art. See, for example, U. S. Patent No. 4,567, 046, EP-A-426, 211, JP-A-60-78529, JP-A-62- 111629, and JP-A-63-258528.
  • the improved property may include, but is not limited to, increased strength of the dough, increased elasticity of the dough, increased stability of the dough, reduced stickiness of the dough, improved extensibility of the dough, improved flavour of the baked product, improved anti-staling of the baked product.
  • the improved property may be determined by comparison of a dough and/or a baked product prepared with and without addition of a polypeptide of the present invention.
  • Phospholipases of the present invention may be in any form suitable for the use in question, e. g. , in the form of a dry powder, agglomerated powder, or granulate, in particular a non-dusting granulate, liquid, in particular a stabilized liquid, or protected enzyme.
  • Granulates and agglomerated powders may be prepared by conventional methods, e. g. , by spraying the phospholipase according to the invention onto a carrier in a fluid-bed granulator.
  • the carrier may consist of particulate cores having a suitable particle size.
  • the carrier may be soluble or insoluble, e. g.
  • a salt such as NaCI or sodium sulphate
  • sugar such as sucrose or lactose
  • sugar alcohol such as sorbitol
  • starch rice, corn grits, or soy.
  • additional enzymes may be contained in slow-release formulations. Methods for preparing slow-release formulations are well known in the art. Adding nutritionally acceptable stabilizers such as sugar, sugar alcohol, or another polyol, and/or lactic acid or another organic acid according to established methods may for instance, stabilize liquid enzyme preparations.
  • the present invention also relates to methods for preparing a baked product comprising baking a dough obtained by a method of the present invention to produce a baked product.
  • the baking of the dough to produce a baked product may be performed using methods well known in the art.
  • the present invention also relates to doughs and baked products, respectively, produced by the methods of the present invention. 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, with reference to the accompanying drawings in which:-
  • Figure 1 illustrates a multiple sequence alignment of amino acid sequences corresponding to fungal, plant and bacterial PLCs, and acid phosphatases
  • Figure 2 illustrates an electrophoretic gel on which has been run the products of
  • Figure 4 illustrates plasmid pZVK2
  • FIG. 5 illustrates an SDS-PAGE gel on which have been run fractions from the purification of soluble recombinant PLC-A from E. coli.
  • E1-E3 shows elution of PLC-A (marked) from a Ni column with imidazole buffer. Molecular weight markers are also shown.
  • the predicted genes were therefore compared with known sequences of bacterial and plant PLCs, 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 Lnland Fisheries, 16775 Stechlin, Germany). This resulted in an optimal prediction for each gene.
  • FIG. 1 there is shown a multiple alignment of the PLC-A, PLC- B and PLC-C proteins from A. fumigatus along with PLCs from A. nidulans, A. niger and M. grisea. PLCs from plants and bacteria and fungal acid phosphatases are also shown (see also Example 4).
  • Boxed regions refer to regions conserved between PLCs and acid phosphatases (boxes LJ, III, V, VI, X), regions conserved between PLCs only (boxes I, VII and IX) and regions conserved between fungal PLCs only (boxes JV and VIII). Highlighted amino acids outside the boxes indicate fungus-specific residues.
  • Fungal PLCs include: PLC-A, A. fumigatus PLC-A, SEQ LD No. 3; PLC-B, A. fumigatus PLC-B SEQ LD No. 6; PLC-C, A. fumigatus PLC-C, SEQ LD No. 9; Anidl, A. nidulans EST AA966809 SEQ LD No. 12; Anigl, A. niger EST BE758821, SEQ LD No. 13; Mporl, M. Grisea EST BM862140, SEQ LD No. 15; Plant PLCs include: A. thaliana, Athall, T02648; Athal2, AB084295_1;
  • Bacterial PLCs include: C_cresc, E87624, Caulobacter crescens;
  • PHLA_MYCTU M. tuberculosis
  • PHL-B_MYCTU M. tuberculosis
  • PHLC_MYCTU M. tuberculosis
  • PHL-D_MYCTU M. tuberculosis
  • PHLNJBURPS B. pseudomallei
  • PHL-N PSEAE P. aeruginosa
  • PLCH JPSEAE P.
  • Acid phosphatases include: AP_Km, JC7179 K. marxianus; AP_Af, AF462065_1, A. fumigatus; AP_Pc, P. chrysogenum; AP_An, A. niger, PHOA_ASPNG.
  • PLC-A, PLC-B and PLC-C align well with each other and were of very similar length. In addition, all three PLCs have good signal peptides. The three proteins show -50 % ⁇ identity to each other, and ⁇ 20%o identity to P. aeruginosa PLC, over the region which they align. PLC-A, PLC-B and PLC-C were shorter at the C-terminus than the Pseudomonas PLC. Further searching of the A. fumigatus database (www.tigr.org) with PLC-A, PLC-B or PLC-C did not show any other PLCs (tblastn; no match with E value less than 0.39). The Pseudomonas PLC requires the presence of the PLC accessory protein,
  • PLCs have been identified in bacteria other than P. aeruginosa, such as Clostridium toxin (PI 5310), and B. cereus phospholipase (PS0197). Although these proteins show PLC activity, they do not show appreciable sequence homology to the P. aeruginosa and A. fumigatus PLC described here. Searches of the A. fumigatus genome with these sequences did not reveal any matches with E values ⁇ 0.86.
  • E. coli strains Top 10 (Inv ⁇ trogen) and select96 (Promega) were 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 preferred 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.
  • the mycelium (fresh or freeze dried) was ground to a powder using liquid nitrogen in a -20°C cooled mortar.
  • the ground biomass was transferred 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 ⁇ DTA; l%(w/v) SDS; pre- warmed to 65°C) was 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 was then added to each tube, tubes were mixed thoroughly and incubated on ice for 30 min. Tubes were 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 was suspended in 2 ml sterile water. 1 ml of 7.5 M ammonium acetate was added, mixed and incubated on ice for 1 hour. Tubes were centrifuged at 12,000 x g for 30 min, the supernatants transferred to a fresh tube and 0.54 volumes of isopropanol were added, mixed and incubated at room temperature for at least 15 minutes. Tubes were then centrifuged at 5,930 x g for 10 min, the supernatant was removed and the pellet washed in 1 ml of 70% ethanol. Tubes were centrifuged at 5,930 x g for 10 min and all the ethanol was removed.
  • the pellet was air dried for 20-30 minutes at room temperature and suspended in 0.5-1.0 ml of TE (10 mM Tris-HCl pH 7.5; lmM EDTA) Finally, the DNA was treated with RNase A (5 ⁇ l of lmg/ml stock).
  • PLC-A cloning primer pair SEQ LD Nos. 17 (Mbplca-genFl: 1) + 18 (Mbplca- genFl: 3490) - product 3490 bases. Numbers in brackets after primer code correspond to 5 ' base of oligonucleotide SEQ LD No. 1.
  • PLC-B Cloning primer pair SEQ LD Nos. 25 (mbplcb-genFl : 1) + 26 (mbplcb- genRl: 864 bases after end of gene) - product 3596 bases bases. Numbers in brackets after primer code correspond to 5' base of oligonucleotide SEQ LD No. 4.
  • PLC-C Cloning primer pair SEQ LD Nos. 33 (mbplcc-genFl: 1483 bases up- stream of start of gene) + 34(mbplcc-genRl: 3062) - product -3600 bases. Numbers in brackets after primer code correspond to 5' base of oligonucleotide SEQ LD No. 7.
  • PCR reactions using the PLC-A, PLC-B or PLC-C primer pair were set up using the Hifidelity 2x PCR master mix (ABGene, Blenheim Road, Epsom, Surrey, KT19 9AP, UK) according to the manufacturer's instructions. The following reagent concentrations and volumes were used: Master mix 1
  • Reverse primer (10 pmol/ ⁇ l) 1 ⁇ l
  • AF293 genomic DNA 1.5-4 ⁇ g/ml
  • the product band (3.6 kb) was excised from the gel and purified using Qiagen's
  • lane 1 there is shown a band at -3600 bp signifying the amplification of PLC-A (1302 bp) and a total of 2188 bp of upstream and downstream sequence.
  • lane 2 there is shown a band at -3600 bp signifying the amplification of PLC-B (1420 bp) and a total of 2176 bp of upstream and downstream sequence.
  • lane 3 there is shown a band at -3600 bp signifying the amplification of PLC-C (1428 bp) and a total of 2168 bp of upstream and downstream sequence.
  • the purified PCR products shown in Figure 2 were cloned into pGEM- Teasy (Promega) and then transformed into select 96 E. coli cells (Promega) according to the manufacturer's instructions.
  • the transformation reactions were 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 were sub-cultured into LB broth containing ampicillin (100 ⁇ g/ml). After overnight incubation at 37°C with shaking, plasmids were extracted using Qiagen spin mini plasmid extraction kits accordmg to the manufacturers instructions.
  • Genomic DNA clones were sequenced by primer walking (MWG Biotech UK Ltd, Waterside House, Peartree Bridge, Milton Keynes, MK6 3BY).
  • the experimentally determined sequences of PLC-A and PLC-C were identical in the coding regions to those identified by bioinformatic analyses as shown as SEQ LD No.l and SEQ LD No.7, respectively.
  • SEQ LD No.l SEQ LD No.7
  • position 2208 was experimentally determined to be a T, although it is a C on the TIGR database. This mutation has no effect on the amino acid specified by the codon.
  • Five single base changes in non-coding sequence were observed in PLC-B (G7->A, G52- >A, A253->G, A980->G, C3030->T; in each case the experimentally determined sequence is given second).
  • the internal sequence of the PLC-A and PLC-B messages were determined by cloning and sequencing cDNA.
  • the 5' and 3' ends of the PLC-A gene and the 5' end of the PLC-B gene were determined by RACE (Rapid Amplification of cDNA Ends). 3.1 cDNA cloning and sequencing
  • A. fumigatus RNA and cDNA 20 ml of Vogels medium, 0.5 % (w/v) soybean phospholipid, and phosphate-free Vogels medium, 0.5 % (w/v) soybean phospholipid were inoculated with A. fumigatus spores and incubated with shaking at 37° C. Cultures were harvested after 24 hrs (phosphate-free Vogels medium) or 48 hrs (Vogels medium) by filtration, then washed twice with DEPC- treated water and transferred to a 50ml Falcon tube. Samples were frozen in liquid nitrogen and stored at -80°C until required.
  • RNA was 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 was used as the lysis buffer of choice;
  • step 7 the Rneasy column was incubated for 5 min at room temperature after addition of RW1;
  • step 9a was carried out;
  • step 10 30 ⁇ l RNase-free water was added, the samples incubated for 10 min at room temperature, and then centrifuged;
  • step 11 the elution step was repeated to give a total volume of 60 ⁇ l RNA.
  • DNA contamination was 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.
  • PCR was carried out using the cDNA above to generate cDNA fragments corresponding to PLC-A and PLC-B. Primer pairs were designed to amplify the cDNA sequence for PLC-A and PLC-B (see below). PCR reactions were carried out using the following reaction mixture and conditions:
  • Reaction mix 12.5 ⁇ l 2x Hifidelity mastermix (ABgene) 1 ⁇ l primer A (10 pmol) 1 ⁇ l primer B (10 pmol) 5 ⁇ l template cDNA 5.5 ⁇ l nuclease-free water
  • PCR products were purified using Qiagen' s QIAquick PCR Purification Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, Westshire, RHIO 9 AX, UK) according to the manufacturers instructions.
  • the purified PCR products were examined on agarose gels. cDNA fragments were then cloned in to the pGEM-Teasy vector (Promega).
  • the plasmids were then transformed in to select 96 chemically competent E.coli cells and plated on to a pre-warmed LB, ampicillin, X-Gal selection plate.
  • Plasmid DNA was isolated using the Plasmid Mini Kit (qiagen). Confirmation of the presence and correct orientation of the inserts was determined by restriction analysis. Constructs were sequenced from the ends of the vector using standard sequencing primers SP6 and T7 at MWG Biotech UK Ltd, Waterside House, Peartree Bridge, Milton Keynes, MK6 3BY. Three clones were sequenced and the consensus sequence taken.
  • PLC-A SEQ LD 19 (Mbplca-rtFl: 1655) + 20 (Mbplca-rtRl : 2953). Numbers in brackets after primer code refer to position in SEQ LD No.l at which oligonucleotides bind.
  • PLC-B SEQ LD 27 (Mbplcb-rtFl: 1307) + 28 (Mbplcb-rtRl: 2718) Numbers in brackets after primer code refer to position in SEQ LD No. 4 at which oligonucleotides bind.
  • cDNA sequence obtained for PLC-A and PLC-B agreed with the predicted cDNA sequence.
  • cDNA sequence of PLC-A confirmed bases 18-1278 of SEQ LD
  • cDNA sequence of PLC-B confirmed bases 15-1336 of SEQ LD No. 5.
  • the C- >T substitution seen in the genomic DNA was not seen in the cDNA and may therefore have been a sequencing error.
  • the cDNA sequence for PLC-C can be obtained in the same way as described above, using the primer pair SEQ LD 35 (Mbplcc-rtFl: 948) + 36 (Mbplcc-rtRl : 2372).
  • the numbers in brackets after primer code refer to position in SEQ LD No. 7 at which oligonucleotides bind.
  • RACE Rapid Amplification of cDNA Ends
  • RNA was prepared as described in 3.1.1. RNA was 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 was used per extraction; At step 2, samples were 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 were centrifuged for 5 minutes; At step 5, 500 ⁇ l DLPS were added, mixed, and incubated at room temperature for 2 minutes. Samples were mixed again and incubated for a further 2 minutes; At step 6 two washes in 250 ⁇ l SEWS were carried out; At step 1, the pellet was disolved in 50 ⁇ l SAFE buffer.
  • RNA was then precipitated by the addition of 2 ⁇ l mussel glycogen (lOmg/ml), 10 ⁇ l 3M sodium acetate, pH5.2 and 220 ⁇ l 95%o ethanol and the sample frozen on dry ice for 10 minutes.
  • CIP calf intestinal phosphate
  • 10X CIP buffer 1 ⁇ l 10X CIP buffer
  • 40U RNaseOutTM made up to 10 ⁇ l in DEPC water
  • Samples were then made up to 100 ⁇ l with DEPC water and the RNA extracted with 100 ⁇ l (25:24:1) phenolxhloroform: isoamyl alcohol.
  • RNA was then precipitated by the addition of 2 ⁇ l mussel glycogen (lOmg/ml), 10 ⁇ l 3M sodium acetate, pH5.2 and 220 ⁇ l 95%o ethanol and the sample frozen on dry ice for 10 minutes.
  • RNA was pelleted by centrifugation at 14,500 rpm for 20 minutes at 4°C, washed with 70%> ethanol, air dried and re-suspended in 8 ⁇ l DEPC water.
  • De-phosphorylated RNA (7 ⁇ l) was de-capped in a 10 ⁇ l reaction with 0.5 U tobacco acid pyrophosphatase (TAP), 1 ⁇ l lOx TAP buffer and 40U RnaseOutTM for 1 hour at 37°C.
  • TAP tobacco acid pyrophosphatase
  • RNA was extracted with phenol: chloroform and precipitated as above, and then re-suspended in 7 ⁇ l DEPC-treated water.
  • First-strand cDNA was 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 were added to the 12 ⁇ l ligated RNA and primer mix; 4 ⁇ l 5x first strand buffer, 2 ⁇ l 0.1M DTT, 1 ⁇ l RNaseOutTM and 1 ⁇ l SuperscriptTM II RT (200U/ ⁇ l) and incubated first at 42°C for 50 minutes and then, to stop the reaction, at 70°C for 15 minutes. 2U RNase H was added to the reaction mix and incubated at 37°C for 20 minutes.
  • a 50 ⁇ l PCR reaction was set up using 1 ⁇ l of the RACE-ready cDNA prepared above, 3 ⁇ l GeneRacerTM 5' primer (10 ⁇ M), 1 ⁇ l reverse gene-specific primer (10 ⁇ M; see Table IV below), 1 ⁇ l dNTP solution (lOmM each), 2 ⁇ l 50 mM MgSO 4 , 5 ⁇ l High Fidelity PCR buffer, 2.5 U Platinum® Taq DNA Polymerase High Fidelity and 36.5 ⁇ l sterile water. Cycling parameters are given in Table TV below.
  • PCR reaction was set up using 1 ⁇ l of the RACE- ready cDNA prepared above, 3 ⁇ l GeneRacerTM 3' primer (10 ⁇ M), 1 ⁇ l forward gene-specific primer (10 ⁇ M; see Table IV below), 1 ⁇ l dNTP solution (lOmM each), 2 ⁇ l 50 mM MgSO 4 , 5 ⁇ l High Fidelity PCR buffer, 2.5 U Platinum® Taq DNA Polymerase High Fidelity and 36.5 ⁇ l sterile water. Cycling parameters are given in Table TV below:
  • Numbers in brackets after primer SEQ LDs refer to position of 5' end of primer on SEQ LD No. 1 (PLC-A) or SEQ LD No. 4 (PLC-B).
  • the cDNA sequence for the 5' and 3' ends of the PLC-A message was identical to that given in SEQ LD No. 2.
  • the 5' and 3' UTR were also determined; the longest message corresponding to bases 1427 to 3058 of SEQ LD No. 1. Taking together the cDNA sequencing and RACE data, the predicted cDNA sequence for PLC-A given in SEQ LD No. 2 was fully confirmed.
  • 5' RACE confirmed the sequence for the 5' end of the PLC-B gene given as SEQ LD No. 5. 130 bases of 5' UTR were also identified, matching identically the corresponding sequence for SEQ LD No. 4.
  • PLC-B 3'RACE SEQ LD Nos. 49 (2144), 50 (2538), and 51 (1738). Numbers in brackets refer to position of 5' base in SEQ LD No. 4.
  • PLC-C 5' RACE SEQ LD Nos. 52 (1437), 53 (1884), 54 (1729) and 55 (1874). Numbers in brackets refer to position of 5' base in SEQ LD No. 7.
  • PLC-C 3 'RACE SEQ LD Nos. 56 (951), 57 (1411), 58 (1862) and 59 (2150). Numbers in brackets refer to position of 5' base in SEQ LD No. 7.
  • RACE confirmed the sequence for the 3' end of the PLC-B message and identified a 113 base 3' UTR.
  • PCR conditions for PLC-B 3 'RACE were as follows: 94°C 2 min; 94°C 30 sec, 72°C 1 min, 5 cycles; 94°C 30 sec, 70°C 1 min, 5 cycles; 94°C 30 sec, 62°C 30 sec, 68°C 1 min, 25 cycles; 68°C 10 min.
  • Example 4 Ldentification of other fungal PLCs
  • Homologs of the A. fumigatus PLCs may be identified in other organisms, particulaly fungi, by means of bioinformatics analysis, degenerate PCR or Southern blotting. Sequences identified by bioinformatics can be used to design primers which can in turn be used in PCR to generate DNA coding for the PLC homolog. Degenerate PCR enables sequence to be obtained for novel genes. This sequence information can be used to generate probes by PCR which can then used to screen cDNA or genomic libraries of the organism of interest to identify clones containing the PLC homolog. Southern blots using fragments of PLC genes from one species as probes can identify the presence of a PLC homolog in the genome of a second species. The same probe can then be used to screen cDNA or genomic DNA libraries. Once clones corresponding to the novel genes have been identified they can be expressed for functional characterisation of the protein, and their expression during infection studied.
  • A. fumigatus PLC-A sequence was used to search the non-redundant protein sequence database (nr) at http://blast.genome.ad.jp (blastp) to identify any related genes. Typically these identified matches in plant and bacterial genomes (Table V). This indicates that the fungal PLCs have homologs in plants (Arabidopsis and Oryza), and bacteria (e.g. Burkholderia pseudomallei, Caulobacter crescentus, Mycobacterium tuberculusosum, Pseudomonas aeruginosa, Ralstonia solanacearum, Streptomyces coelicolor). There were no matches to human/mammalian proteins with E ⁇ 5.4.
  • Candida albicans (alces.med.umn.edu/gbsearch/ybc.html); Cryptococcus neoformans: www.genome.ou.edu/cneo_h99_blast.html; www.genome.ou.edu/cneo_B3501_blast.html;
  • Schizzosaccharomyces pombe (www.sanger.ac.uk/Projects/S_pombe/blast_server.shtml).
  • Fungal cultures can be 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 (6.1 g/L agar, 53.3 g/L instant oatmeal) 25.0°C, or Cornmeal agar (Difco 0386), 26.0 C; Phytophthora capsici cultures are maintained on on V-8 agar at 24°C; Pyricularia oryzae cultures are maintained on rice polish agar at 24°C under white fluorescent lights (12hr artificial day), and were subcultured every 7 - 14 days by the transfer of my
  • 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 attempts 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 IPTG (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 is prepared as described in 4.2.1 above, then digested with the appropriate restriction enzyme and run on 0.8 %> agarose gel. The gel is then submerged in 250 mM HC1 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 PLC DNA or by PCR of an appropriate region.
  • a suitable probe can be generated from PLC-A by PCR using the primer pair SEQ TD Nos. 60 and 61 and the methods give in 4.2.2.
  • l ⁇ 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 to 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, 300mM 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, 300mM 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 (lOOmM 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 lOOmM 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.
  • 20 mL washing buffer 1 %> w/v blocking reagent for nucleic acid hybridisation, Roche, dissolved in lOOmM maleic acid, 150 mM NaCl, pH 7
  • 20 mL blocking solution (1 %> w/v blocking reagent for nucleic acid hybridisation, Roche, dissolved in lOOmM maleic acid, 150 mM Na
  • 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 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 PLC 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 PLC Expression during infection of wax moth larvae (Galleria melonell ⁇ ) 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, unpublished observations). We show that A. fumigatus PLC-A, PLC-B and PLC-C are induced during infection of wax moth larvae. 5.1 Growth and infection of wax-moth larvae
  • 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 (250mg +/- 50mg) 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 transferred to a sterile Petri dish.
  • Larvea injected with lOul PBS/Tween only larvae injected with lOul 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 Asyergillus fumigatus-m ⁇ ected 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 SAB 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, stirred for lh and autoclaved for lh) and cooled with liquid nitrogen.
  • Ground sample was transferred 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 (06/2001, Pages 75-78, http://www.qiagen.com/literature/handbooks/ rna/rnamini/1016272HBRNY_062001WW.pdf).
  • step 3 600 ⁇ l 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 RW1; Optional step 9a was carried out twice; At step 10, 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 11, the elution step was repeated to give a total volume of 60 ⁇ l RNA.
  • 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 ⁇ l RNase-free DNase
  • 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.
  • 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 transferred 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 using primers that amplify the ⁇ -tubulin gene (SEQ LD Nos. 62 and 63). 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 ⁇ l 2x ReddyMix PCR mastermix (ABIgene) 1 ⁇ l each primer (5 pmol) template gDNA (1.5-4 ⁇ g /ml) nuclease-free water to give a final volume of 25 ⁇ l
  • PLC-A SEQ LD No. 23 PLC-AF2 (1732) + SEQ LD No. 24 PLC-AR2 (2216).
  • the primer pair SEQ LD No. 21 (1822) and SEQ LD No. 22 (2334) may also be used.
  • PLC-B SEQ LD No. 31 PLC-B-F2 (1525) and SEQ LD No. 32 PLC-B-R2
  • SEQ LD No. 4 The primer pair SEQ ' LD No. 29 (1840) and SEQ LD No. 30 (2337) may also be used.
  • PLC-C SEQ LD No. 39
  • PLC-C-F2 (1143)
  • SEQ LD No. 40 PLC-C-R2
  • SEQ LD No. 7 The primer pair SEQ LD No. 37 (1274) and SEQ LD No. 38 (1769) may also be used.
  • a no-template control was set up with water instead of cDNA.
  • Step 2 95°C - 1 min Step 3, 55°C - 1.5 min
  • FIG. 3 there is shown expression data of PLC-A, PLC-B and PLC-C in wax moth larvae which have been infected with A. fumigatus.
  • PLC-A, PLC-B and PLC- C were all seen to be expressed by A. fumigatus growing in culture.
  • mice were infected with Aspergillus fumigatus and organs harvested as follows. Thirteen male CD1 mice were injected with the immunosuppressant cyclophosphamide (0.025 g/ml; 200 mg/kg) TV 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. This was repeated at 24 and 48 hours after infection. RNA was prepared from mouse tissues as described for wax moth larvae above (5.2 and 5.3).
  • 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 5-10 ⁇ l aliquots at -20°C.
  • PLC-A SEQ LD. No. 23 PLC-AF2 (1732) + SEQ LD. No. 24 PLC-AR2 (2216). Numbers in brackets refer to 5' base of oligonucleotide with reference to SEQ LD No. 1.
  • PLC-B SEQ LD No. 31 PLC-B-F2 (1525) and SEQ LD No. 32 PLC-B-R2 (2012). Numbers in brackets refer to 5' base of oligonucleotide with reference to SEQ LD No. 4.
  • PLC-C SEQ LD No. 39 PLC-C-F2 (1143) and SEQ LD No. 40 PLC-C-R2 (1643). Numbers in brackets refer to 5' base of oligonucleotide with reference to SEQ LD No. 7.
  • primer set i.e. for each gene of interest
  • cDNA sample the following reactions are set up: 12.5 ⁇ l 2x ReddyMix PCR mastermix (ABgene), 1.5 ⁇ l cDNA (50 ng/ ⁇ l), 1.5 ⁇ l of each of the primer pair (at 10 pm/ ⁇ l) Water to a final volume of 25 ⁇ l
  • a no-template control is set up with water instead of cDNA.
  • Step l 95°C - 5 min Step 2, 95°C - 1 min
  • Step 6 8°C - hold 39 cycles of steps 2, 3 and 4 are carried out.
  • Reaction products are then run on agarose gels and visualised by staining with ethidium bromide.
  • Phospholipase assay Phospholipase C was assayed in the supematants from ftmgal cultures using the colorimetric assay of Kurioka and Matsuda (1976, Anal Biochem 75, 281-9). This assay is based on the specific hydrolysis of the colorimetric substrate p- nitrophenylphosphorylcholine (pNPPC, Sigma N5879) by phospholipase C. Phospholipase C cleaves pNPPC at the phosphate bond to release p-nitrophenol and phosphorylcholine. The rate of reaction can then be followed by monitoring the accumulation of the coloured p-nitrophenol with time.
  • pNPPC p- nitrophenylphosphorylcholine
  • A. fumigatus strain Af293 cultures were grown on Vogel's salt with 15 w/v glucose; Vogel's salt with 0.5%> w/v soy bean phospholipids; and phosphate-free Vogel's salt with 0.5% w/v soy bean phospholipids, and supematants harvested. Glucose cultures were grown for 24 hours. Cultures containing phospholipid should be grown for up to 48 hours.
  • the PLC assay was performed by mixing 800 ⁇ l of substrate 10 mM pNPPC in 0.25 M Tris-HCl, 60% (w/v) sorbitol pH 7.2, with 200 ⁇ l of filter sterilised culture supernatant in a 2 ml cuvette. Reaction mixtures were incubated at 37°C, and the absorbance was read at 1 h intervals at 410 or 485 nm, and plotted against time for each test sample. Calibration curves were plotted by comparing the absorbance of a known amount of control PLC with the unknown sample. Clostridium per ⁇ ngens or Bacillus cereus PLC were used as positive controls.
  • a microplate assay was also used and consisted of 200 ⁇ l of substrate/buffer with 50 ⁇ l of culture supernatant or positive control PLC. Serial dilutions were performed across a 96 well microplate. The results are shown in Table VI below.
  • the methods described herein can be used for screening recombinant PLCs for inhibitors.
  • 20 ⁇ l recombinant PLC, produced as described in Example 9 are added to 10 ⁇ l test compound and 20 ⁇ l substrate in 96 or 384 well plates. Positive and negative substrate and compound controls are included as well as a no-enzyme control. All samples are assayed in duplicate.
  • the colorimetric substrate described above can be used in the assay, although other colorimetric substrates could also be envisaged. Plates are incubated at 37°C and absorbances read at 1 hr intervals. This procedure would identify compounds which inhibited the cleavage of substrate by the PLC.
  • Recombinant PLC-A described in Example 9, was assayed as above with the following modifications: To each well of a 96-well plate was added; 150 ⁇ l of Tris/sorbitol buffer (with 20 mM CaCl 2 ), 40 ⁇ l of substrate pNPPC (20mM), and 2 ⁇ g or 10 ⁇ g of purified protein. OD measurements were taken at 405nm. Plates were incubated at 37°C and read at 405 nm. Results are shown in Table VII and indicate that recombinant PLC-A cleaved pNPPC and was therefore functional.
  • Screening for PLC-A inhibitors can be carried out using the above assay, or assays based upon it, by including inhibitor substances in the assay and determining the extent to which the cleavage of pNCCP is reduced.
  • Other substrates which may be more suitable for assaying, can be determined by substituting them for pNCCP and determining whether they are cleaved more than pNCCP, and/or are cleaved more rapidly.
  • Example 8 Method for detecting fungal infection
  • the PLC sequences described in the invention may be exploited to diagnose fungal infections.
  • Samples from patients potentially carrying an infection with A. fumigatus, A. nidulans, or A. niger or of rice leaves or stem potentially infected with M. grisea, or other organisms are processed to extract DNA using the DNAeasy Tissue kit or QIAamp DNA Blood Mini kit(Quiagen, Crawley, UK), although other DNA preparation methods are available and suitable.
  • DNA DNAeasy Tissue kit or QIAamp DNA Blood Mini kit(Quiagen, Crawley, UK), although other DNA preparation methods are available and suitable.
  • Reaction mix 12.5 ⁇ l 2x ReddyMix PCR mastermix (ABgene)
  • PLC-B SEQ LD No. 66 (1592) + SEQ LD No. 67 (1855). Numbers in brackets refer to position of 5' in SEQ LD No. 4.
  • PLC-C SEQ LD No. 68 (1935) + SEQ LD No. 69 (2145). Numbers in brackets refer to position of 5' in SEQ LD No. 7.
  • A. nidulans SEQ LD No. 70 (160) + SEQ LD No. 71 (306). Numbers in brackets refer to position of 5' in SEQ LD No. 10.
  • M. grisea SEQ LD No. 74 (234) + SEQ LD No. 75 (353). Numbers in brackets refer to position of 5' in SEQ LD No. 15.
  • Appropriate controls include; (i) template DNA but no primers; primers but no template (negative controls); (ii) cDNA encoding fungal PLC or DNA from cultured fungi instead of patient DNA (positive control).
  • PCR products can be 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, A. niger or M. grisea, or other organisms can be detected by means of antibodies raised against the fungal PLCs.
  • One suitable means is the use of a capture ELISA.
  • microtitre plates are coated with a monoclonal antibody raised against the fungal PLC. Then the plates are incubated with diluted patient samples, or appropriate protein extracts of samples (particularly if the samples are biopsies). Plates are then incubated with a polyclonal antibody (again against the PLCs). Finally, binding of the second antibody is 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.
  • A. fumigatus cDNA was prepared as described in Example 3.1.1.
  • PCR is carried out using the cDNA above to generate polynucleotides encoding PLC- A, PLC-B and PLC-C.
  • the appropriate primer pairs are given here, to correspond with A+B pairs in the method below.
  • Primers with SEQ ID Nos. 76, 78, and 80 include the sequence CACCATG at the 5' end to enable cloning into a pETlOl/D-TOPO vector (Invitrogen). PCR reactions are carried out using the following reaction mixture and conditions:
  • PCR products are purified using Qiagen' s QIAquick PCR Purification Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, Westshire, RHIO 9AX, UK) according to the manufacturer's instructions.
  • the purified PCR products are examined on agarose gels.
  • the cDNA fragments are then cloned in to the pETlOl/D-TOPO vector
  • the plasmids are then transformed in to TOP 10 chemically competent E. coli cells and plated on to a prewarmed ampicillin (+) 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 Plasmid 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 BL21 Star (DE3) One Shot E. coli cells and grown overnight at 37°C. Protein expression is then induced by the addition of isopropylthio- ⁇ -galactosidase (LPTG) and detected by SDS PAGE and western blotting using an anti-His antibody. Purification of the recombinant protein is then performed by metal affinity chromatography.
  • Alternative expression systems can be used for expression in bacteria, such as the glutathione S-transferase or mannose-binding fusion-protein system.
  • PCR products were purified using Qiagen's QIAquick PCR Purification Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, Westshire, RHIO 9 AX, UK) according to the manufacturers instructions.
  • the purified PCR products were examined on agarose gels. cDNA fragments were then cloned in to the pET30 Xa/LIC vector (Novagen), transformed into Nova Blue chemically competent E. coli cells, and plated on to a prewarmed kanamycin (+) selection plate. After an overnight incubation at 37° C, kanamycin-resistant colonies were selected and grown up in LB medium (30 ⁇ g/ml kanamycin). Plasmid DNA was isolated using the Plasmid Mini Kit (Qiagen). Confirmation of the presence and conect orientation of the inserts was determined by restriction analysis with Ncol and sequencing of the construct.
  • Purified plasmid DNA which had been confirmed to be of the conect sequence and orientation, was transformed into chemically competent BL21 Star (DE3) One Shot E. coli cells and grown overnight at 30° C. 5 ml of an over-night culture were used to innoculate 100 ml of LB, 30 ⁇ g/ml kanamycin, and the cultures incubated at 30° C, 220 rpm until the cell density reached an optical density of 0.6 (approximately 3 hours). Expression of the recombinant protein was then induced with LPTG (ImM) for 4 hours.
  • LPTG LPTG
  • Bacteria were harvested by centrifugation at 4500 rpm for 10 minutes and the pellets lysed in lysis buffer (10 ml Bugbuster (Novagen), 10 ⁇ l Benzonase (Novagen), 0.4 ⁇ l lysozyme (Novagen) and 100 ⁇ l IM imadazole for 20 minutes at room temperature. Cells were then spun down at 16000g for 20' at 4° C and the supernatant, containing soluble recombinant protein, removed to a clean tube. Supernatant was 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.
  • lysis buffer 10 ml Bugbuster (Novagen), 10 ⁇ l Benzonase (Novagen), 0.4 ⁇ l lysozyme (Novagen) and 100 ⁇ l IM imadazole for 20 minutes at room temperature. Cells were then spun down at 16
  • Protein-resin mix was then poured into a column, washed twice in 4 ml of wash buffer (2.5 ml IM phosphate buffer pH8 , 6.25 ml 4M NaCl, 1 ml IM Imidazole pH8, 0.5 ml 10% Tween 20; made up to 50 ml in n.H 2 O) and then eluted successively in 1 ml (El), 2 ml (E2), and 1 ml (E3) fractions with elution buffer (250 ⁇ l IM Phosphate Buffer pH8, 625 ⁇ l 4M NaCl, 1.25 ml IM imidazole pH8, 50 ⁇ l 10% Tween 20, made up to 5 ml in n.H 2 O).
  • wash buffer 2.5 ml IM phosphate buffer pH8 , 6.25 ml 4M NaCl, 1 ml IM Imidazole pH8, 0.5 ml 10% Tween 20; made up to 50 ml
  • Figure 5 shows the elution fractions, containing PLC-A, run on an SDS-PAGE gel and stained with coomassie. Fractions containing purified recombinant protein were desalted using a PD10 column (Amersham Pharmacia) equilibrated in 20 mM Tris.HCl pH 8.0. Protein fractions may be analysed by Western blotting using an S-tag HRP conjugate (Novagen).
  • Recombinant fragments of other PLCs can be generated using primer pairs designed against conesponding regions of the genes.
  • Example 10 Production of an antibody
  • Antibodies against the fungal PLCs will be of considerable use as diagnostic reagents (see example 4 above).
  • As an immunogen recombinant domains are used (as described in example 9).
  • synthetic polypeptides encoding regions either unique to the individual PLCs, or likely to provide cross-reactivity within a set of PLCs, a set of species, or a range of genera 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 serum 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 constructs or whole cell lysates and extracts as targets. Negative controls, such as other PLCs, different constructs or different species are also employed to test specificity and/or to determine the range of species and/or genus cross-reactivity.
  • Example 11 Production of fungi with PLC genes functionally disabled.
  • a BAC (bacterial artificial chromosome) clone library containing the A. fumigatus genome, partially digested with BamHJ 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 incubated 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 (as illustrated in Figure 4, the sequence of which is SEQ LD No. 82 which is an engineered plasmid), 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 the 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 KC1 ) and shaking for 2 h at 80 rpm in 30 ° C .
  • the protoplasts are washed with 0.6 M KC1 and then with STC (Sorbitol, Tris, CaC12).
  • 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 was unsuccessful surviving. In this case, the transformation is carried 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

L'invention concerne un polynucléotide comprenant un gène PLC isolé d'un organisme sélectionné de manière indépendante d'un groupe comprenant Aspergillus; Blumeria; Candida; Cryptococcus; Encephalitozoon; Fusarium; Leptosphaeria; Magnaporthe; Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Trichophyton; et Ustilago.. L'invention concerne également un polypeptide PLC isolé d'un organisme sélectionné de manière indépendante d'un groupe de genres comprenant Aspergillus; Blumeria; Candida; Cryptococcus; Encephalitozoon; Fusarium; Leptosphaeria; Magnaporthe; Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Trichophyton; et Ustilago.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006223119A (ja) * 2005-02-15 2006-08-31 Sankyo Lifetech Co Ltd 新規ホスホリパーゼｃ
WO2007105264A1 (fr) * 2006-03-10 2007-09-20 Mitsubishi-Kagaku Foods Corporation Nouvelle phospholipase c
JP2010252815A (ja) * 2010-08-20 2010-11-11 Mitsubishi-Kagaku Foods Corp 新規ホスホリパーゼｃ
US8241876B2 (en) 2008-01-07 2012-08-14 Bunge Oils, Inc. Generation of triacylglycerols from gums
WO2012164088A1 (fr) * 2011-06-03 2012-12-06 Universite Montpellier 2 Sciences Et Techniques Procédé de diagnostic de la tuberculose active
US8460905B2 (en) 2007-09-11 2013-06-11 Bunge Oils, Inc. Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases with reduced reaction time
US8956853B2 (en) 2007-01-30 2015-02-17 Bunge Oils, Inc. Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases
US11170872B2 (en) 2019-11-05 2021-11-09 Apeel Technology, Inc. Prediction of latent infection in plant products

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* Cited by examiner, † Cited by third party
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JPH08266213A (ja) * 1995-03-31 1996-10-15 Kyowa Hakko Kogyo Co Ltd パン生地の製造法
US6146869A (en) * 1999-10-21 2000-11-14 Novo Nordisk Biotech, Inc. Polypeptides having phospholipase B activity and nucleic acids encoding same

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006223119A (ja) * 2005-02-15 2006-08-31 Sankyo Lifetech Co Ltd 新規ホスホリパーゼｃ
WO2007105264A1 (fr) * 2006-03-10 2007-09-20 Mitsubishi-Kagaku Foods Corporation Nouvelle phospholipase c
US7993874B2 (en) 2006-03-10 2011-08-09 Mitsubishi-Kagaku Foods Corporation Phospholipase C enzyme(s)
US8956853B2 (en) 2007-01-30 2015-02-17 Bunge Oils, Inc. Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases
US8460905B2 (en) 2007-09-11 2013-06-11 Bunge Oils, Inc. Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases with reduced reaction time
US8241876B2 (en) 2008-01-07 2012-08-14 Bunge Oils, Inc. Generation of triacylglycerols from gums
US8541211B2 (en) 2008-01-07 2013-09-24 Bunge Oils, Inc. Generation of triacylglycerols
JP2010252815A (ja) * 2010-08-20 2010-11-11 Mitsubishi-Kagaku Foods Corp 新規ホスホリパーゼｃ
WO2012164088A1 (fr) * 2011-06-03 2012-12-06 Universite Montpellier 2 Sciences Et Techniques Procédé de diagnostic de la tuberculose active
US9274110B2 (en) 2011-06-03 2016-03-01 Universite Montpellier 2 Sciences Et Technique Diagnosis method of active tuberculosis
US11170872B2 (en) 2019-11-05 2021-11-09 Apeel Technology, Inc. Prediction of latent infection in plant products
US12380965B2 (en) 2019-11-05 2025-08-05 Apeel Technology, Inc. Prediction of infection in plant products

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