WO2010032230A1

WO2010032230A1 - Non-auxotrophic selection marker

Info

Publication number: WO2010032230A1
Application number: PCT/IE2008/000091
Authority: WO
Inventors: Stephen Carberry; John Martin Doyle; Hubertus Haas; Kevin Kavanagh; Markus Schrettl
Original assignee: National University Of Ireland, Maynooth
Priority date: 2008-09-22
Filing date: 2008-09-22
Publication date: 2010-03-25
Also published as: WO2010032230A9

Abstract

A non-auxotrophic selection marker located in a gene cluster encoding a metabolite for an organism, such as a fungus, lacking said marker is provided. The selection marker gene can be used to detect and confirm the introduction of foreign genes without the use of exogenous drug-resistant genes to facilitate transformant selection. The selection marker gene can be applied to detect the presence of fungal infection and predict the likelihood of the development of disease or allergic response at an early stage so that medical intervention may be sought.

Description

Description A selection marker

Technical Field

This invention relates to a selection marker system used to detect and confirm the introduction of foreign genes without the use of exogenous drug-resistance genes to facilitate transformant selection.

Background Art

Genetic modification of filamentous fungi for the improved production of food additives, industrial enzymes or pharmaceuticals is an on-going requirement of the biotechnological industry (Archer, D.B and Dyer, P.S (2004) Current Opinion in Microbiology 7:499-504).

Antibiotic (cephalosporin)-producing fungi such as Acremonium chrγsogenum are continually subjected to strain improvement, with a concomitant requirement for new selection markers, to increase product yield and decrease the level of unwanted side-products (Rodriguez-Saiz, M et al (2004) FEMS Microbiol Lett. 235:43-49).

In addition to technical difficulties associated with transformation of fungi, the actual detection and confirmation of gene insertion (i.e., genetic transformation) is problematic. Only a limited number of auxotrophic mutants (e.g., the pyrimidine biosynthesis gene, pyrG (Weidner, G et al (1998) Curr. Genet. 33:378-385) or argB required for arginine biosynthesis (Xue, T et al (2004) Arch Microbiol. 182:346-353) are available and are of restricted use due to spontaneous reversion issues.

More frequently, genes encoding drug resistance (e.g., hygromycin B (hph), phleomycin (JPhleό) and pyrithiamine (ptrA)) are included in transformation vectors to allow for selection of transformants. However, the introduction of drug resistance characteristics into genetically modified organisms for food or biopharmaceutical use is problematic and manufacturers are continually seeking dominant selection markers which facilitate detection of transformants that do not result in simultaneous introduction of non- natural drug resistance which requires the continuous presence of the selection drug during manufacture (Basch, J et al (2004) J. Ind. Microbiol. Biotechnol. 31:531-539).

Currently, fungal transformation (for either gene deletion or insertion of new genes) involves the generation of fungal protoplasts to allow uptake of exogenous DNA. The exogenous DNA may be added as a single PCR amplicon containing the marker gene flanked by regions homologous to the gene of interest (i.e., the gene which is to be deleted) (Reeves, E. P etal (2006) FEBS J. 273:3038-3053).

Alternatively, the exogenous DNA may be added as a bipartite amplicon whereby the two flanking regions are present on separate PCR fragments along with overlapping fragments of the relevant marker gene (Schrettl, M et al (2007) PIoS Pathog. 3:1195-1207). The bipartite approach increases the likelihood of specific gene deletion and reduces the possibility of detecting ectopic integrants (i.e., insertion of transforming DNA away from the region of interest).

There are two current strategies used for determination of fungal transformation. One method involves the complementation of an auxotrophic mutation using a functional copy of the relevant gene as part of the transformation vector or PCR construct. However, auxotrophic mutants can spontaneously revert to wild-type thereby producing "false- positive" transformants. In addition positional effects can interfere with expression to result in additional phenotypes.

An alternate method uses drug resistance genes to confer drug tolerance on the transformed fungal species and thereby allows for selection of transformants.

Drug resistance genes are primarily of non-fungal origin and the introduction of non-species DNA is either prohibited or results in extensive validation to ensure biosafety. The introduction of drug resistance genes into fungal strains for the production of human biopharmaceuticals is effectively prohibited by Regulatory Authorities.

In addition, the introduction of drug resistance genes into non- native species may result in uncontrolled gene transfer and unwanted occurrence of drug resistance in pathogenic species. Aspergillus fumigatus is responsible for approximately 4% of all hospital-based deaths in Europe (Brookman, J.L. and Denning, D. W. (2000) Curr. Opin. Microbiol. 3: 468-474). The organism is an opportunistic fungal pathogen of immunocompromised patients and is the commonest etiological agent of invasive aspergillosis (IA) (Brakhage A.A. and Langfelder, K. (2002) Ann. Rev. Microbiol. 56: 433-455). IA results in significant mortality (as high as 60-90%) and is therefore the leading worldwide cause of death due to fungal infection. IA is a major cause of illness and death among bone marrow and solid organ transplant and leukaemia patients, in addition to those with pre-existing pulmonary malfunction. Aspergillosis accounts for at least 3,500 deaths per annum in the USA (Kontoyiannis, D.P. and Bodey, G.P. (2002) Eur J Clin Microbiol Infect Dis 21: 161-172) and there is consequently an urgent requirement for the development of new antifungal drugs.

Gliotoxin has been shown to play a significant role in mediating the virulence of Aspergillus fumigatus (Cramer, R.A. et al (2006) Eukaryot Cell 5: 972-980); Stack, D et al (2007) Microbiology 153: 1297-1306). Gliotoxin is an epipolythiodioxopiperazine (ETP)-type toxin (326 Da) containing an essential disulphide bridge.

The cytotoxic activity of gliotoxin is generally mediated by direct inactivation of essential protein thiols (Waring, P. et al (1995) Biochem. Pharmacol. 49:1195-1201), NFicβ inhibition, and by inhibition of the respiratory burst in neutrophils by disrupting NADPH oxidase assembly (Tsunawaki, S. et al. (2004) Infect Immun. 72:3373-3382), thereby facilitating in vivo fungal dissemination. In A. fumigatus, enzymatic machinery responsible for gliotoxin biosynthesis, and metabolism is encoded by a multi-gene cluster which is coordinately-expressed during gliotoxin biosynthesis. Identification of the gliotoxin biosynthetic cluster by Gardiner et al. ((2004) MoI Microbiol. 53:1307-1318) and biochemical verification by Cramer et al. ((2006) supra); Kupfahl et al. ((2006) MoI Microbiol 62:292-302), have shown that at least 12 genes are involved in gliotoxin metabolism as illustrated in Fig. 1 which is taken from Gardiner D M, Howlett BJ. (2005) FEMS Microbiol Lett. 248:241-248.

Bioinformatic analysis of gene function has predicted the putative functions of several of the cluster genes. The multi-gene cluster includes gliA, a transporter, gliC, a cytochrome P450 oxidoreductase, gliF, a cytochrome P450 oxidoreductase, gliG, a putative glutathione S transferase, glil, an aminocyclopropane carboxylate synthase, glU, a dipeptidase, gliK, a gliotoxin biosynthesis protein, gliM, O-methyl transferase, gliN, methyl transferase, gliP, a bimodular non-ribosomal peptide synthetase (NRPS), gliT, a putative thioredoxin reductase and gliZ, a zinc finger transcription factor.

However, even though putative functions have been proposed for the genes within the cluster, no biochemical evidence exists for many of the functions, except gliP, gliA and gliZ. Moreover, the functional identification of these genes does not illuminate the potential functions of the remaining ones. In mammalian cells it has been demonstrated that the oxidized form of gliotoxin is actively concentrated in a glutathione-dependent manner and that it then exists within the cell almost exclusively in the reduced form which causes cell damage (Bernardo, P.H et al (2003) J. Biol Chem. 278:46549-55). As glutathione levels fall due to apoptosis, the oxidized form of gliotoxin then effluxes from the cell where the cytocidal effects of gliotoxin are perpetuated in a pseudocatalytic manner. Conversely, it has been shown that gliotoxin may substitute for 2-cys peroxiredoxin activity in HeLa cells by accepting electrons from NADPH via the thioredoxin reductase— thioredoxin redox system to reduce H₂O₂ to H₂O. In this way, nanomolar levels of gliotoxin may actually protect against intracellular oxidative stress (Choi, H. S et al (2007) Biochem. Biophys. Res. Commun. 359:523-8).

The gliT gene has been annotated in silico as a putative thioredoxin reductase (Fox, E.M. and Howlett, B. J. (2008) Mycol Res 112: 162-169; 33) and recent bioinformatic studies suggest that gliT, and putative orthologues, are present in many gliotoxin- or ETP- encoding gene clusters across a wide range of fungal species (Fox, E.M. and Howlett, B. J.(2008) supra; Patron N. J. et al (2007) BMC Evol Biol 7:174).

A thioredoxin system in Aspergillus nidulans has recently been described whereby a thioredoxin mutant exhibited decreased growth, impaired reproductive function and altered catalase activity (Thon, M. et al (2007) J Biol Chem. 282:27259-27269). A thioredoxin reductase (termed AnTrxR) was identified which functions to regenerate reduced thioredoxin in A. nidulans. BLAST analysis indicates minimal sequence identity between GIiT and AnTrxR.

Functional analysis of fungal genes, especially the successive transformation of the same organism, depends on efficient selection markers. However, considerable limitations exist to the widespread application of many of the existing selection markers employed to date in the transformation of fungi. There is, therefore, a commercial and scientific need for effective dominant and or non-auxotrophic selection markers for the transformation of fungi.

In addition, the high cost of gliotoxin is a result of the low yield of gliotoxin during fermentation. If this low yield is due to gliotoxin auto-toxicity, a previously unobserved phenomenon, then there is a need for a method of overcoming gliotoxin auto-toxicity to increase gliotoxin yields. This would be of commercial benefit to gliotoxin manufacturers.

There is also a need for a diagnostic test to detect the presence of the fungal infections and predict the likelihood of the development of Aspergillosis related disease or a fungal-induced allergic response at an early stage so that medical intervention may be sought.

Disclosure of the Invention

Accordingly, the invention provides a non-auxotrophic selection marker for an organism lacking said marker. The invention provides a method for determining the transformation of an organism based on effective dominant selection markers.

An advantage of the invention is the identification of successful transformant selection without the use of exogenous drug-resistance genes.

The invention improves on the capability to detect and confirm the introduction of foreign genes using a reliable technique.

Another advantage of the present invention is that the invention does not introduce non-natural drug resistance and therefore does not require a continuous presence of a selection drug during manufacture. This is particularly important for genetically modified transformants for biotechnological use, where regulatory approval of the use of drug- resistance genes is required to ensure biosafety.

A further advantage of the present invention is that a non- auxotrophic selection marker is used that will not spontaneously revert to wild-type, thereby preventing 'false-positive' transformants.

Preferably, the organism is an eukaryotic organism.

It will be appreciated that eukaryotic organisms include animals, plants and fungi.

Preferably, the organism is a fungus. Further, preferably, the organism is a filamentous fungus.

According to one embodiment of the invention, the selection marker is located in a gene cluster encoding a fungal metabolite.

The invention utilises a gene located within a gene cluster that encodes a metabolite, that is essential for the functioning, maintenance or protection of the fungi.

Preferably, the fungal metabolite is a mycotoxin.

Further, preferably, the mycotoxin is an epipolythiodioxopiperazine.

Still further, preferably, the mycotoxin is gliotoxin.

According to one embodiment of the invention, the non- auxotrophic selection marker is gliToτ a homologue thereof.

It will be appreciated that gliT, encoding a putative thioredoxin reductase, is located in the gliotoxin biosynthetic gene cluster and is involved in protection against gliotoxin toxicity in the organism. However, prior to the findings described herein, the function of this gene was unknown.

An advantage of the invention is that transformants that contain the gliT gene exhibit protection against gliotoxin toxicity when grown in the presence of gliotoxin. According to another embodiment of the invention, the non- auxotrophic selection marker is an orthologue oigliT.

The non-auxotrophic selection marker can be a gene that has evolved directly from the same ancestral locus as gliT.

Preferably, the filamentous fungus is selected from Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.

According to a preferred embodiment of the invention, the filamentous fungus is Aspergillus fumigatus.

Preferably, the gliT is used as a selection marker for transformation in Aspergillus fumigatus.

The deletion of the gliT gene encoding a putative thioredoxin reductase in Aspergillus fumigatus results in a mutant, AgHT. The mutant Δg&Thas no resistance, and is highly sensitive, to the presence of gliotoxin and growth is prevented in the presence of exogenous gliotoxin.

Further, preferably, the selection is carried out in the presence of a selection medium containing gliotoxin.

The invention allows for effective transformation and subsequent mutant selection of fungi. If transformation is successful then the mutant fungal protoplast AgHT uptakes exogenous DNA, including the gliT gene and gliotoxin resistance is conferred to the protoplast. Subsequent mutant selection in the presence of gliotoxin ensures only successful transformants are present.

According to one embodiment of the invention the gliTov homologue or orthologue thereof is used as a selection marker for transformation in a non-gHT encoding organism.

The invention allows for gliotoxin resistance to be conferred to naϊve organsims that do not contain the gliT gene, thereby allowing transformation and mutant selection in these organisms.

An advantage of the invention is that transformation and mutant selection is not limited to gliT encoding organisms.

Preferably, the non-gHT encoding organism is Aspergillus nidulans.

Aspergillus nidulans does not produce gliotoxin, and does not encode an orthologue or functional homologue of gliT. The organism is sensitive to exogenously added gliotoxin and does not grow in the presence of gliotoxin.

An advantage of the invention is that Aspergillus nidulans and other non-gHT encoding organisms can be protected against gliotoxin by the introduction of the gliT gene into the organism. Thus, allowing transformation selection in these organisms.

Further, preferably, the non-auxotrophic selection marker is a dominant marker. Still further, preferably, the selection is carried out in the presence of a selection medium containing gliotoxin.

If transformation is successful then the exogenous DNA, including the gliT gene is incorporated into the cell and gliotoxin resistance is conferred on the organism. Subsequent mutant selection in the presence of gliotoxin ensures only successful transformants containing the gliT gene are present.

According to one embodiment of the invention, the invention provides a method for increasing the production of a fungal protein by a fungus capable of expressing said protein, which method comprises incorporating additional copies of a gene coding for the protein in said fungus.

An advantage of the invention is that elevating the gene copy number of a particular gene increases the expression of the protein that it encodes.

Preferably, the fungal protein is GliT protein.

Further preferably, the GliT protein is a gliotoxin reductase.

By "gliotoxin reductase" herein is meant an enzyme which is capable of reducing or cleaving the disulphide bridge present in gliotoxin.

Still further, preferably, an overproduction of GHT leads to an increased yield of gliotoxin. The engineering of extra copies of the gliT gene into commercial gliotoxin-producing fungi increases GIiT production, which overcomes autotoxicity and increases gliotoxin yields.

This aspect of the invention is of benefit to gliotoxin manufacturers due to the current high cost of gliotoxin resulting from gliotoxin auto-toxicity limiting the yield of gliotoxin during fermentation.

An advantage of this invention is that gliotoxin yields are improved without the introduction of foreign DNA or drug resistance. Thus, there are no regulatory objections to strain modifications.

According to a further embodiment of the invention, there is provided a method for predicting the likelihood of infection of a subject by Aspergillus, which comprises detecting an antibody to GIiT in a body fluid from said subject.

The invention provides a method for determining the likelihood of infection of a subject by Aspergillus species, based on the detection of an antibody to the GIiT protein. The antibodies detected are highly specific to the GIiT protein. Thus, the method according to the invention greatly facilitates the early diagnosis and treatment of infection by Aspergillus. .

The body fluid can be, for example, whole blood, plasma, serum, salvia, urine, tears, lymph and cerebrospinal fluid. An advantage of the method according to the invention is that it enables rapid and non-invasive screening for infection of a subject by Aspergillus.

Preferably, the detection of an antibody to GIiT can be used to predict the likelihood of the development of invasive aspergillosis, saprophytic aspergillosis and/or allergic bronchopulmonary aspergillosis.

An advantage of the present invention is that it permits one to diagnose, or aid in the diagnosis of, invasive aspergillosis, saprophytic aspergillosis and/or allergic bronchopulmonary aspergillosis based on the detection of an antibody to the GIiT protein.

According to one embodiment of the invention, the invention provides a method for predicting the likelihood of the onset of a fungal- induced allergic response in a subject, which comprises detecting an antibody to a protein expressed by a gene in the gliotoxin gene cluster in a body fluid from said subject.

The method according to the invention facilitates the noninvasive detection a fiingal-induced allergic response in a subject and serves as a prognostic method to determine the likelihood of the development of a fungal-induced allergic response in a subject.

The method according to the invention also improves on the capability to diagnose, detect and monitor fungal-induced allergic response in a subject using a reliable and non-invasive technique. Preferably, the antibody is IgG or IgE.

The detection of the protein in accordance with the invention can be solely by immunoassay.

A particular requirement of the method according to the invention is protein with the requisite affinity and specificity for its antibody targets, present in biological fluids.

Preferably, the expressed protein is GIiT or GIiG.

The detection of antibodies directed towards GIiT or GIiG indicates the presence of, or past exposure to, a fungus and can be used to predict the likelihood of the development of a fungal-induced allergic response in a subject.

According to one embodiment of the invention, the GIiT is recombinant GIiT.

According to another embodiment of the invention the GIiG is recombinant GIiG.

According to a further embodiment of the invention, there is provided a mutant fungal strain which lacks the gHTgene or a homologue or orthologue thereof.

A sample of the deleted fungal strain according to the invention was deposited at the CABI Bioscience UK Centre (Egham, Surrey, UK) on August 15, 2008 and accorded the accession number IMI CC 396691 and the name Aspergillus fumigatus AgHT.

The invention provides Aspergillus fumigatus AgHT strain IMI CC 396691.

A sample of the complemented fungal strain according to the invention was deposited at the CABI Bioscience UK Centre (Egham, Surrey, UK) on August 15, 2008 and accorded the accession number IMI CC 396692 and the name Aspergillus fumigatus AgHT**⁰.

The invention also provides Aspergillus fumigatus AgHT**⁰ strain IMI CC 396692.

A sample of the fungal strain, which was generated by transfer of gliT to Aspergillus nidulans, according to the invention was deposited at the CABI Bioscience UK Centre (Egham, Surrey, UK) on August 15, 2008 and accorded the accession number IMI CC 396693 and the name Aspergillus nidulans TRAN⁶¹¹⁷.

The invention further provides Aspergillus nidulans TRAN^{8 lT} strain IMI CC 396693.

The mutant fungal strain lacks the gliT gene and is therefore sensitive, or not resistant to gliotoxin. This forms the basis of an effective transformation and subsequent mutant selection model. A successful transformation that incorporates the gliT gene and additional exogenous DNA into the fungal protoplast restores its resistance to gliotoxin. Subsequent exposure to gliotoxin allows the selection of the successfully transformed protoplasts from non-successful transformants.

Preferably, the fungus is selected from Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.

Brief Description of the Drawings

Fig.l shows the organisation of the gliotoxin gene cluster in A.fumigatus,'

Fig. 2 shows the generation of mutant A. fumigatus AgHT (left) and mutant complementation strain gliT* (right) as described in Examples 2 and 3;

Fig. 3 is an image of Southern analysis of AgHT mutant versus wild-type DNA as described in Example 2;

Fig. 4 is an image of a Northern blot analysis of the induction of gliotoxin gene cluster expression as described in Examples 2 and 4;

Fig. 5 is a graph of % survival versus time (h) as described in

Example 2. In Fig. 5 — ■ — denotes wild-type; Φ denotes AgHT;

Fig. 6. is a photograph of protoplasts of A.fumigatus wild-type and AgHT strain on plates as described in Example 3;

Fig. 7. is an image of a Southern blot of gliT complementation, single and multiple gliT integrants identified in two distinct transformants as described in Examples 3 and 9; Fig. 8 is a bar graph illustrating wild-type and AgHT strain growth in the presence of gliotoxin and reduced glutathione (GSH) as described in Example 3, 'filled' area denotes wild-type, 'empty' area denotes AgHT ;

Fig. 9 is a bar graph illustrating phenotypic analysis of A. fumigatus wild-type and AgHT strains in the presence of gliotoxin as described in Example 3, 'filled' area denotes wild-type, 'empty' area denotes AgHT;

Fig. 10 is an image of 2D-PAGE analysis of GIiT expression following addition of exogenous gliotoxin (GT) to A. fumigatus cultures as described in Example 4.

Fig. 11 is an image of SDS-PAGE analysis of (insoluble) recombinant GIiT produced in inclusion bodies in E. coli as described in Example 5;

Fig. 12 is an image of SDS-PAGE analysis of recombinant GIiG expression and solubility as described in Example 5;

Fig. 13 is an image of a Western blot analysis of recombinant GIiG expression and solubility as described in Example 5;

Fig. 14 is a bar graph illustrating distribution of human IgG reactivity to recombinant GIiT as described in Example 6;

Fig. 15 is an image of a Western blot analysis of human IgG reactivity to GIiT as described in Example 6; Fig. 16 is a bar graph illustrating distribution of human IgG reactivity to recombinant GIiG as described in Example 6;

Fig. 17 is an image of SDS-PAGE of native GIiT in protein extracts from A. fumigatus 46645 (wild type and AgHT) and strain 26933 as described in Example 7;

Fig. 18 is an image of ECL detection of native GIiT using immunoafϊinity purified human IgG (isolated using recombinant GIiT- Sepharose media) in A. fumigatus 46645 and strain 26933 as described in Example 7;

Fig. 19 is an image of a Western blot analysis of protein precipitates following ammonium sulphate addition (0, 20, 50 and 80 % (w/v), respectively) to GIiT lysate supernatant as described in Example 9;

Fig. 20 is a graph of Absorbance (A280 nm and A454 nm) versus elution volume (ml) for a Q-Sepharose ion-exchange fractionation of GIiT dialysate (post-ammonium sulphate precipitation) as described in Example 9;

Fig. 21 is an image of SDS-PAGE analysis of Q-Sepharose ion- exchange chromatography (IEX) fractions as described in Example 9;

Fig. 22 is an image of Western blot analysis of Q-Sepharose

IEX fractions as described in Example 9; Fig. 23 is a graph of Enzymatic activity (U/ml) versus Gliotoxin concentration (μM) for determination of gliotoxin reductase activity in GliT-containing fractions by NADPH oxidation as described in Example 9;

Fig. 24 is an image of Northern analysis of gliT expression in three Aspergillus nidulans transformants (AnP^llT) (Arfi^llT = A. nidulans TRAN^⁷'⁷), A. nidulans wild-type (AnWT) and Aspergillus fumigatus wild-type (AfWT) as described in Example 10; and

Fig. 25 is a photograph of growth assays of A. nidulans wild- type (An WT) and transformants as described in Example 10 (An?''⁷ = A. nidulans TRAN⁸*⁷).

Modes for Carrying out the Invention

The invention will be further illustrated by the following Examples.

Example 1

A. fumigatus growth conditions

A number of A. fumigatus strains used in the present and subsequent Examples are shown in Table f. Theses strains were grown at 37 ⁰C in Aspergillus minimal media (AMM). AMM contained 1% (w/v) glucose as carbon-source, 5 mM ammonium tartrate as nitrogen- source, and trace elements as described in Pontecorvo, G. et a (1953) Adv. Genet. 5:141-238.

Table 1.

* Mutant complementation strain AgIiT**⁰ carried out by introducing gliT gene only to AgIiT . ** Haas, H. etal., (1999) J. Biol. Chem. 274:4613-4619).

Liquid cultures were performed with 200 ml AMM in 500 ml Erlenmeyer flasks inoculated with 10 conidia. For growth assays, 10 conidia of the respective strains were point inoculated on AMM plates, containing the indicated supplements and incubated for 48 h at 37 ⁰C.

TOPO TA cloning system (Invitrogen) and TOPlO E. coli cells (F-mcrA A(mrr-hsdRMS-mcrBQ ψS0lacZAM\5 AlacXIΛ rec Al araDl39 galU galK A(ara-leu)7697 rpsL (Str^R) endAl nupG) were used for general plasmid DNA propagation and A. fumigatus genomic DNA was purified using a ZR Fungal/Bacterial DNA Kit (Zymoresearch).

Example 2

Deletion oigliT in A. fumigatus

A AgHT mutant was generated by homologous transformation of A. fumigatus using the bipartite marker technique described in Nielsen, M.L. et al. ((2006) Fungal Genet Biol 43:54-64) with modifications. A. fumigatus strain ATCC46645 was co-transformed with two DNA constructs, each containing an incomplete fragment of a pyrithiamine resistance gene (ptrA) fused to 1.2 kb, and 1.3 kb oigliT flanking sequences, respectively.

These marker fragments shared a 557-bp overlap within the ptrA cassette, which served as a potential recombination site during transformation. During transformation, homologous integration of each fragment into the genome flanking gliT allows recombination of the ptrA fragments and generation of the intact resistance gene at the site of recombination.

Two rounds of PCR generated each fragment. First, each flanking region was amplified from ATCC46645 genomic DNA using primer ogliTl and ogliT4 for flanking region A (1.3 kb), and ogliT2 and ogliT-3 for flanking region B (1.2 kb) as shown in Table 2. Subsequent to gel-purification, the fragments were digested with Spel and Hindϊll, respectively. Υh&ptrA selection marker was released from plasmid pSK275 by digestion with Spel and HincRlI, and ligated with the two flanking regions A and B described above.

Table 2.

Add-on restriction enzyme sites are underlined.

For generation of AgIiT, two overlapping fragments were amplified from the ligation products using primers ogliT-5 and optrA-2 for fragment C (2.6 kb) and primers ogliT-6 and optrA-1 for fragment D (2.2 kb). Subsequently ATCC46645 was transformed simultaneously with the overlapping fragments C and D. In the generated mutant allele of AgliT-ptrA the deleted region comprises amino acids 1 - 325 oigliT.

The DNA constructs were generated by PCR and characterised by DNA sequence analysis which confirmed the replacement of gliT by overlapping /?fo4 regions on intact 5' and 3' flanking regions as shown in Fig. 2.

The solid box in Fig. 2 represents the gliT gene andptrA represents the pyrithiamine resistance gene. The arrow indicates the transcriptional orientation and the crosses represent recombination sites. The dashed line represents the plasmid backbone of plasmid pgliT as described in Example 3, and the restriction enzymes are as follows Aαtll, A; HindlU, H; Nαrl, N; Spel, S; andXbαl, X. Double arrows along with the predicted size indicate expected fragments in Southern analysis.

Following protoplast transformation, Southern analysis was used to screen for gliT (negative) andptrA (positive) colonies and transfoπnants were found to lack the gliT gene as shown in Fig. 3. Subsequent Northern analysis as described in Example 4, shown in Fig. 4 confirmed that gliT expression was absent in AgHT, compared to A. fumigαtus WT.

Deletion of g/zTfrom A. fumigαtus does not alter fungal virulence in the Gαlleriα mellonellα insect larval model and was demonstrated as follows. A.fumigatus strains ATCC46645 (wild-type) and AgHT were cultivated on MEA agar plates at 37 ⁰C. After 72 h, conidia were harvested with Phosphate Buffered Saline-Tween 80 (0.1% (w/v) (PBST), washed three times in PBST and re-suspended in 1 ml of PBS.

Conidia density was determined using a haemocytometer and Galleria mellonella larvae, in groups often, were inoculated with 10⁷ conidia in a final volume of 20 μl into the last left pro-leg. For gliotoxin treatment, larvae were challenged with 3 μg/ml gliotoxin, 2 h after the conidial infection. Control experiments used PBS only and gliotoxin injections.

Mortality rates were recorded up to 72 h post-infection. Mortality was assessed based on lack of movement and melanisation of the cuticula (Renwick, J. et al (2006) Mycopathologia 161, 377-384). As shown in Fig. 5, a deletion oigliT in A. fumigatus did not lead to altered virulence in this fungus. Also, melanisation appeared to be wild-type like. Controls injections did not influence survival of larvae (data not shown).

AgHT protoplasts were unable to grow in the presence of gliotoxin (5 μg/ml), exogenous gliotoxin had no effect on WT growth and AgIiT protoplasts grew and regenerated mycelia perfectly in the absence of gliotoxin as shown in Fig. 6. Example 3

Reconstitution of a functional gliT in A. fumigatus

For reconstitution of the AgIiT strain with a functional gliT copy, a 3.2 kb PCR fragment, amplified using primers ogliT-5 and ogliT-6, was subcloned into pCR2.1-TOPO (Invitrogen). The resulting 7.1 kb pgliT was linearised with Aatll and used to transform A. fumigatus AgIiT protoplasts as shown in Fig. 2.

Taking advantage of the decreased resistance of the AgHT mutant to exogenous added gliotoxin. AgHT protoplasts were . transformed with gliTanά screened for wild-type resistance to gliotoxin for genetic complementation as shown in Fig. 2. Positive deletion- and reconstituted- strains were screened by Southern analysis according to Sambrook, J. et αl (1989) (Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press) as shown in Fig. 7 and hybridisation probes were generated using primers ogliT-5 and ogliT-4 to detect Afarl-digested fragments. The strain with multiple integrants (Fig. 7) is termed A. fumigatus Aglif^ (DVQ CC 396692) and was used for GliT purification as described in Example 9.

To analyse gliotoxin production, A. fumigatus wild-type (WT) and mutant strains were grown up to 72 h in A. fumigatus minimal medium.

Supernatants were chloroform extracted overnight and fractions were lyophilized to complete dryness. Samples were resolubilised in MeOH and analysed using a reversed phase HPLC as described in Reeves, E.P. et al (2004) Mycopathologia 158:73-79. Gliotoxin, as determined by absorbance at 254 nm and identical retention times (14.39 min), was evident in both WT and A.fumigatus AgIiT extracts.

AgHT protoplasts were unable to grow in the presence of gliotoxin (5 μg/ml), exogenous gliotoxin had no effect on WT growth and AgIiT protoplasts grew and regenerated mycelia perfectly in the absence of gliotoxin as shown in Fig. 6.

The AgIiT strain grew at identical rates to WT. Conidia

(n =100) of A. fumigatus wild-type and Δg/zTwere plated onto media in the presence of gliotoxin (5 μg/ml) and in the presence of gliotoxin (5 μg/ml) and reduced glutathione(GSH). The number of visible (growing) colonies were counted after 48 hr incubation at 37°C. Fig. 8 shows the number of colonies plotted as the percentage of growing colonies compared to AMM control plates.

It was also observed that addition of reduced glutathione (GSH) to test plates completely abolished the cytotoxic effects of exogenous gliotoxin, by chemically cleaving the disulphide bond of gliotoxin, which indicates that the oxidized form of gliotoxin is imported into A. fumigatus as shown in Fig. 8.

Subsequent phenotypic analysis of A. fumigatus ATCC46645 (WT) and AgIiT conidia demonstrated that gliotoxin (5 μg/ml) significantly inhibited AgHT growth on minimal medium (MM) and completely inhibited AgHT growth on both MM and Sabouraud medium (gliotoxin, 10 μg/ml) as shown in Fig. 9. These results clearly indicate that AgHT is highly sensitive to exogenous gliotoxin.

Consequently, mutant complementation was carried out by introducing gliT only (no antibiotic resistance gene) to complement AgHT with selection in the presence of gliotoxin (10 μg/ml). Transformants, which had recovered resistance to exogenous gliotoxin, were confirmed by Southern analysis to have an intact and functional copy of gliT present as shown in Fig. 7.

This result confirms that gliT confers resistance to gliotoxin in A.fumigatns and means that AgHT mutants have significant potential for future functional genomic studies involving A. fumigatus since gene deletions in this strain are selectable by g/zTreintroduction, with selection in the presence of gliotoxin.

Example 4

Gliotoxin induces expression of gliT in A. fumigatus WT and the gliotoxin gene cluster in A. fumigatus and AsIiT

Elevated GliT (protein) levels were detected following exposure of gliotoxin. Here, A. fumigatus ATCC 26933 (1 x 10⁵ cfu / ml) was grown for 24 h in Sabouraud media (25 ml cultures), at 37 ⁰C with shaking at 200 rpm. After 24 h, gliotoxin (700 μg) dissolved in methanol was added to the cultures. After 4 h incubation, mycelia were harvested, filtered under pressure, washed with PBS and resuspended in lysis buffer (100 mM Tris-HCl, 50 mM NaCl, 20 mM EDTA, 10% (v/v) Glycerol, 30 mM DTT, 1 mM phenylmethylsulphonyl fluoride (PMSF) and 1 μg/ml pepstatin A pH 7.5; 3 ml of lysis buffer per gram of mycelia). Lysis was accomplished by grinding in liquid N₂ followed by brief sonication on ice. Mycelial lysates were centrifuged (10000 x g; 30 min) to remove cell debris and the subsequent supernants analysed by 2D-PAGE and MALTI-ToF MS following trichloroacetic acid (TCA)/acetone precipitation as described in Carberry S et al. (2006) Biochem Biophys Res Comm. 341(4): 1096-1104. Fig. 10 illustrates the threefold upregulation of GIiT expression in A. fumigatus following 3 h exposure to exogenous gliotoxin.

For Northern analysis, total RNA was isolated using TRI reagent (Sigma- Aldrich) and Northern analysis was performed according to Sambrook et al. (1989) supra. Hybridisation probes were generated by PCR using primer ogliA-1 and ogliA-2 for gliA, ogliG-7 and ogliG-8 for gliG, ogliT-7 and ogliT-8 for gliT, and ogliZ-1 and ogliZ-2 for gliZ (for sequences see Table 2).

gliZ, A and G encode the cluster transcriptional regulator, gliotoxin transporter and a putative glutathione s-transferase, respectively.

Northern analysis showed that expression of these three genes plus gliT, from the gliotoxin gene cluster, was induced in A. fumigatus ATCC44645 within 3 h following gliotoxin (5 μg/ml) addition at 21 h. However, expression of gliT was significantly up-regulated relative to the other genes tested as shown in Fig. 4.

Lanes 1, 2 and 3 of Fig. 4 correspond to A.fumigαtus RNA extracts from 21 h Aspergillus Minimum Medium (AMM), 21 h AMM, shifted to gliotoxin (5 μg/ml) for 3 h and 24 h AMM, respectively. The expression of four cluster genes (lane 2) in the gliotoxin gene cluster was induced by gliotoxin addition (5 μg/ml) in A.fumigαtus ATCC46645. Expression of gliT was significantly up-regulated relative to all others tested. No gliT expression was detectable in the AgHT mutant whereas the expression of all other genes was identical to the wild-type.

No gliT expression was detectable in the AgHT mutant whereas the expression of all other genes was identical to the WT, including the continued absence of expression at 24 h in the absence of added gliotoxin as shown in Fig. 4. RNA loading per track was equivalent as judged by rRNA visualisation.

These observations are in complete accordance with the proteomic data as shown in Fig. 10 which demonstrated a threefold upregulation of GliT expression in A.fumigαtus following 3 h exposure to exogenous gliotoxin as shown in Fig. 4. Moreover, they demonstrate that gliT expression is regulated independently, or differentially, relative to other components in the gliotoxin gene cluster. Example 5 Cloning and expression of gliT and gliG

The gliT said gliG sequences, respectively, were amplified from cDNA using primers as shown in Table 3 incorporating terminal Hindlll and Xhol sites to facilitate downstream cloning. PCR products were separately cloned into the pCR2.1 cloning vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

10

Table 3

GHT and gliG were subsequently individually cloned into the 15 pProEX-Htb expression vector (Invitrogen). Ligations were performed using Quickstick ligase (Bioline, London, UK) according to the manufacturer's instructions. Specifically, following purification, restriction digests were carried out on pProEX™ Htb expression vectors, gliG and gliT TCR products using the appropriate restriction enzymes 20 Hindlll and Xhol. Analysis of restriction digests by electrophoresis on 1% (w/v) agarose gels stained with ethidium bromide identified bands for each gene of the correct product size and purity. Purified products were ligated and ligation reactions were used to transform DH5α™ (gliG) and BL21 DE3 (gHT) competent E.coli cells by standard protocols.

Expression of GIiT was induced by the addition of isopropyl β-D-thiogalactoside (IPTG; to 0.6 mM) and monitored by SDS-PAGE and Western blot analysis.

The recombinant protein was present in cell lysate pellet, indicating insolubility, and was purified using differential extraction, with a yield of approximately 25 mg per gram of E. coli shown in Fig. 11. SDS-PAGE analysis confirmed a molecular mass of 38 kDa for recombinant GHT. SDS-PAGE analysis of (insoluble) recombinant GIiT as shown in Fig. 11 produced in inclusion bodies in E.coli. Lane 1 and 2 contain GIiT in the presence ( 1 ) and absence (2) of dithiothreitol reductant. Lane 3 contains molecular mass marker. GIiT is dimeric in the absence of reductant.

For GIiT purification, cells were lysed with lysozyme (90 μg/ml) and sodium deoxycholate (0.04 %(w/v)), in the presence of protease inhibitors (1 μg/ml leupeptin and pepstatin, respectively and 1 mM PMSF). Cell debris was removed by centrifugation at 10,000 g for 10 min and N-terminal (His)₆-tagged recombinant protein was purified from the supernatant by Ni-NTA chromatography (Qiagen, West Sussex, UK) by elution with 250 mM imidazole in 50 mM sodium phosphate/300 mM NaCl. Purified GIiT was dialysed (twice; once overnight, and once for 4 h) against 5OmM sodium carbonate pH 9.4 containing 0.02% (w/v) sodium azide for storage at 4⁰C. Protein concentrations were determined using the Bradford method as described in Tilburn, J. et al. ((2005) Genetics 171 :393-401) with bovine serum albumin as a standard.

Purified recombinant GIiT was analysed by MALDI ToF MS and peptides (following tryptic digestion) were identified corresponding to the theoretical amino acid sequence of the protein whereby 20% sequence coverage was observed. No activity was recoverable and refolding attempts proved unsuccessful.

To detect recombinant protein expression of GIiG in recombinant E.coli, induction of protein expression with IPTG followed by SDS-PAGE and Western blot analysis was used. Small scale expression cultures were induced and grown for 3 h. Both SDS-PAGE and Western blot analysis showed the presence of recombinant protein for GHG at the correct molecular mass and recombinant GIiG (Figs. 12 and 13). Bradford protein assay was used to quantify recombinant protein expression. Optimal expression was observed after 3 h growth and resulted in the production of 11.7 mg/ml of GIiG. Example 6

Anti-GliT and GIiG Human Ig Detection by ELISA

Recombinant GIiT was immobilized on Nunc Maxisorb microwells (0.5 μg/well; 5 μg/ml in 50 mM sodium carbonate buffer pH 9.4), followed by blocking with 1% (w/v) BSA and 10% (w/v) sucrose in 50 mM sodium carbonate buffer pH 9.4.

Human serum specimens, diluted 1/100 in l%(w/v) BSA in PBST, were added to microwells, in duplicate, and incubated for 1 h. After washing, anti-human IgG -horseradish peroxidase conjugate was added for 1 h.

Antibody reactivity was subsequently visualized, after removal of unbound antibody-enzyme conjugate, by tetramethylbenzidine addition with absorbance detection at 450/630 nm.

The immunoreactivity (detection of IgG[anti-GliT]) in human sera towards GIiT was examined by coating purified recombinant GIiT onto microwells. Surprisingly, ELISA analysis demonstrated that 100% of human sera (42/42) tested exhibited significant reactivity against recombinant GIiT as shown in Fig. 14. This result was confirmed by Western analysis of selected sera against the protein as shown in Fig. 15.

Fig. 14 shows that there was strong anti-GHT IgG reactivity evident in all specimens tested whereby all sera showed an absorbance 450/630 nm of greater than 1.0. A significant proportion (~25 %) of the specimens tested had an absorbance value greater than 3 which indicates extremely strong anti-GHT IgG reactivity in those specimens. No negative specimens were detected which indicates generalised IgG reactivity against A. fumigatus GHT.

ELISAs were carried out to confirm anti-GHT IgG reactivity and to determine anti-GHT IgE reactivity. Sera (n = 5) were (i) diluted in l%(w/v) BSA in PBST or (H) pre-adsorbed with anti-human IgG prior to analysis for both IgG and IgE against GHT.

Each non-adsorbed and adsorbed specimen (final dilution 1/100) was then added to GHT-coated microwells for either IgG (using anti-human IgG-HRP conjugate) or IgE (using rabbit IgG [anti-human IgE] and anti-rabbit IgG-HRP conjugate) detection. Final antibody/conjugate dilutions are given in Table 4.

Anti-GHT IgG reactivity was evident for all 5 non-adsorbed specimens. However following IgG depletion this reactivity was significantly reduced which confirms specificity of human IgG binding to GHT. Anti-GHT IgE reactivity is also present in all sera, especially following removal of competing human IgG.

Anti-rabbit IgG-HRP conjugate bound to microwells in the absence of anti-IgE antibody (i.e., no α IgE* column) but this background reactivity (0.371-0.692) was significantly below specific IgE reactivity against GIiT (0.952-1.386). Thus, both IgG and IgE directed against A. fumigatus GIiT are present in human sera.

Table 4

Fig. 15 shows a Western blot analysis of human IgG reactivity to GIiT. Lanes 1 to 11 contain recombinant GIiT probed with different serum samples diluted 1/100 in PBST. Serum samples 1, 8 and 11 reveal no or very weak reactivity to GIiT whereas all other serum samples tested show definite IgG reactivity to GIiT.

An ELISA was carried out to detect anti-GHG IgG reactivity.

Recombinant GIiG (5 μg/ml) was coated on 96 well microtitre plates and probed with human sera (1:100). Twenty human serum specimens were tested for anti-GHG IgG reactivity. Anti-human IgG-HRP was added to the plate to detect human IgG antibodies against GIiG. The results shown in Fig. 16 indicate that all 20 samples had IgG reactivity meaning that all 20 individuals had raised an immune response against GIiG. Absorbance (A 450/630 nm) values of above 1.5 were observed for 100 % of the specimens tested. Half of all specimens tested exhibited absorbance values greater than 2.5 which indicates that high levels of IgG anti-GliG are present. Increased absorbance values indicate increased IgG reactivity. No negative specimens (no IgG reactivity evident) were detected.

Example 7

Immunoaffinity Purification of Human IgG (anti-GliT)

Serum specimens containing high titer IgG [anti-GliT] were pooled, diluted 1 in 4 in PBS, and applied to a GliT-Sepharose affinity column (2 ml), prepared as per manufacturer's instructions.

After removal of unbound proteins by PBS washing, immobilised IgG [anti-GliT] was eluted using 100 mM glycine pH 2.8, followed by immediate neutralisation using 1 M Trizma base.

Resultant immunoaffinity purified (IAP) IgG [anti-GliT] was used to detect native GIiT by Western analysis as shown in Fig. 17 (SDS-PAGE) and Fig. 18 (Western blot analysis). In Figs. 17 and Fig. 18 lane 1 contains A.fumigαtus 26933, lane 2 contains 46645 (WT), lane 3 contains AgHT, lane 4 contains recombinant GHT (1 μg) and lane M contains molecular mass marker. GIiT was not detected in A.fumigatus AgHT (Fig. 18). This immunoproteomic strategy provides for a source of IgG [anti-GHT] for immunodetection of native GIiT as shown in Fig. 18.

Example 8

Generation of murine antisera

Recombinant GIiT (50 μg of GIiT immunisation /?er mouse, followed by two boosters of 25 μg GIiT per mouse), from Example 5, was immunised into mice (n = 2) by commercial arrangement (Harlan, UK) and anti-GHT reactivity was determined by Western blot analysis. Post- seroconversion, murine antisera was collected and used to detect GIiT in A.fumigatus as discussed in Example 9.

Example 9

Purification of native GIiT from A. fumigatus AsWf^ and Identification of GIiT as Gliotoxin Reductase.

Purification of GIiT protein was undertaken from the A. fumigatus transformant A.fumigatus Aglit^ (IMI CC 396692) which was shown by Southern analysis to contain multiple copies of gliT as shown in Fig. 7. This reconstituted A. fumigatus Δglii ^c strain was grown for 48 h at 37 ⁰C in 400ml (x 2) of AMM media. The mycelia were extracted (43.5g wet weight), frozen with liquid nitrogen and crushed before being resuspended in lysis buffer (100 mM Tris-HCl, 50 mM NaCl, 10 mM dithiothreitol; 20 mM EDTA and 10% (v/v) glycerol (protease inhibitors PMSF (1 mM), Pepstatin (1 μg/ml) and TLCK (1 μg/ml) were also added); (176 ml).

The protein extract was then sonicated repeatedly on ice before being centrifuged at 12,000 g (4⁰C) for 20min. The protein concentration of the GIiT lysate was 256 μg/ml.

An aliquot of the GIiT lysate (ImI) was ammonium sulphate precipitated and the pellets resuspended and analysed by SDS-PAGE and Western blot to identify the optimal ammonium sulphate concentration for GIiT precipitation.

Subsequently, the entire GIiT lysate was ammonium sulphate precipitated and the 50% pellet was resolubilised in 20 mM Bis-Tris propane pH 6.7. This solution was dialysed three times against 50 vols of the same buffer at 4°C.

The dialysate (100ml) was then centrifuged at 12,000 g for 20 min and filtered (0.45 μm) to remove particulates. A Q-Sepharose Ion- Exchange (IEX) column (volume: 4 ml) was equilibrated with 20 mM Bis-Tris propane pH 6.7 before the dialysate was loaded onto the column at lml/min. The column was then washed with 20 mM Bis-Tris propane pH 6.7 before the sample was eluted using an NaCl gradient (0.5 M final). Absorbance detection was at 280nm and 454nm (A 454 nm is characteristic of flavin adenine dinucleotide (FAD) containing enzymes such as thioredoxin reductases).

Fractions from Q-Sepharose IEX were subjected to SDS- PAGE, Western blot and activity analysis for GIiT. GIiT activity towards gliotoxin was assessed by incubating 0-12 μM gliotoxin in the presence 100 mM potassium phosphate/10 mM EDTA, 0.05% (w/v) bovine serum albumin pH 7.5 and 0.2 mM NADPH, with and without GIiT for 3 min at 25 ⁰C.

Oxidation of NADPH via A_A340nH1 decrease was determined spectrophotometiically. Additionally, GIiT was immunoprecipitated from solution and residual supernatant activity determined to assess GIiT contribution to NADPH oxidation, in the presence of gliotoxin.

Western analysis using murine antisera [anti-GHT] revealed that 50% ammonium sulphate precipitated native GIiT as shown in Fig. 19. GIiT presence in the lysate supernatant was evident at '0%'. No GIiT was evident at either 20 or 80 % (w/v) ammonium sulphate but 50% (w/v) resulted in optimal GIiT precipitation.

Ion-exchange chromatography was used to partially purify GIiT as shown in Fig. 20. Here it can be seen that GIiT elutes from the Q-Sepharose column at approximately 250 mM NaCl. Importantly, fractions subsequently shown by immunological as shown in Figs. 21 and 22, and activity analysis as shown in Fig. 23, to contain GIiT also exhibited absorbance at 454nm which is characteristic of flavin adenine dinucleotide (FAD) containing enzymes like oxidoreductases.

Figs. 21 and 22 illustrate the presence of GIiT in column fractions following Q-Sepharose chromatography. Immunoreactive bands, at the expected molecular mass of GIiT are clearly evident in fractions 25-28.

Following confirmation of GIiT presence as shown in Figs. 21 and 22, the activity of GHT towards gliotoxin was evaluated. It can be seen in Table 5 and Fig. 23 that effectively no GIiT activity, as measured by NADPH oxidation (ΔA 340 nm/ 180 s), was detectable in the absence of gliotoxin. Neither did NADPH oxidation occur in the presence of gliotoxin but absence of GIiT.

Table 5

However, addition of gliotoxin between 3-9 μM final concentration, revealed GIiT activity whereby reduction of the gliotoxin disulphide bridge was mediated by GIiT enzymatic activity using NADPH as a source of reducing equivalents. As an additional confirmation of GIiT activity, the enzyme was immunoprecipitated followed by centrifugation and the residual activity determined in the supernatant. Removal of GIiT from pooled column fractions by immunoprecipitation resulted in a 46% diminution GIiT enzymatic activity as shown in Fig. 23, and further confirms that gliotoxin is reduced by GIiT in the presence of NADPH.

Example 10

Transformation of a gliT- naive organism (Aspergillus nidulans) with gliT and subsequent acquisition of resistance to exogeneous gliotoxin.

A construct containing the constitutive øfe^promoter (Spellig, T. et al, (1996) MoI. Gen. Genet. 252:503-509), the gliT coding sequence and its terminator was used to introduce Aspergillus fumigatus g/zTectopically in A. nidulans with phleomycin (Punt, PJ. and Van den Hondel, CA. (1992) Methods Enzymol. 216:447-457) as a positive control selectable marker by homologous recombination in A. nidulans.

To obtain this gliT encoding construct, gliT was amplified with oligonucleotides ogliT-Bglll and oghT-Notl (for oligo sequences see Table 2) using A. fumigatus genomic DNA as a template. The resulting PCR fragment was cloned into pCR2.1-TOPO (Invitrogen) to obtain pgliT-BgHVNotl. Subsequently, a 0.9 kb fragment containing the otef promoter was released from plasmid pUCG-H (Langfelder, K. et al, (2001) Infect Immun. 69:6411-8) via Kpnl/BamHL digest and ligated to KpnVBamHl digested pgliT-Bglll/Notl to receive potefgliT. Subsequent, potefgliT was co-transformed with the phleomycin containing plasmid ρAN8-l (Punt, PJ., Mattern, I.E. and van den Hondel, C. A.M. J. J. (1988) Fungal Genet. Newsl. 35:25-30) into A. nidulans TRAN (Haas, H. et al (1999) J Biol Chem. 274:4613-4619). Transformation was carried out as described above (gliT) and phleomycin resistant transformants were further selected to screen for integration (via Southern analysis) and expression (via Northern analysis) of A. fumigatus gliT in A. nidulans.

It is clear from Fig. 24 that gliT is not expressed in A. nidulans WT but that, following transformation and protoplast selection on phleomycin, transformants have been identified which express A. fumigatus-deήved gliT. This confirms that gliT has been inserted into A. nidulans (termed Aspergillus nidulans TRAN⁸"¹ (IMI CC 396693) and that it is expressed at the mRNA level.

Fig. 25 illustrates that A. nidulans transformants which encode gliT grow normally on AMM plates, as does A. nidulans WT. However, neither A. nidulans WT or a transformant containing a non-expressed copy of gliT (transformant An^g//714 of Fig. 25) grow in the presence of gliotoxin (50 μg/ml). Significantly, both A. nidulans transformants which express functional gliT (transformants An^g/'^r 8 and An^g/'^r 9 of Fig. 25), can grow in the presence of gliotoxin (50 μg/ml). This confirms that gliT can be used as a selection marker for detecting transformation of gliT-naive organisms.

National University of Ireland Maynooth

Department of Biology

Maynooth

Co. Kildare

Ireland

3 September 2008

NOTIFICATION OF ACCEPTANCE OF A DEPOSIT FOR THE PURPOSES

OF PATENT PROCEDURES

Microorganism: Aspergillus fumigatus ΔgliT IMI CC Number: 396691

Strain Number: # 1

The above designated microorganism, received on 14 August 2008 was accepted for deposit for patent purposes on 15 August 2008 in accordance with the terms and conditions set out in the Application form signed by you 25 July 2008, a copy of which should have been retained by you.

Yours faithfully

" ^" . . . " ^{■ ■ ■} . - ^{• ■} ' ^; i/i i I i

D. Joan Keliey ^' T W⁽J 91^ bxeculive Director Bioservices

National University of Ireland Maynooth

Department of Biology

Maynooth

Co. Kildare

Ireland

3 September 2008

NOTIFICATIONOFACCEPTANCE OFADEPOSITFORTHE PURPOSES

OF PATEMTPROCEDURES

Microorganism: Aspergillus fumigaiυs hgll _:-TMC IMI CC Number: 396692

Strain Number: # 2

The above designated microorganism, received on 14 August 2008 was accepted for deposit for patent purposes on 15 August 2008 in accordance with the terms and conditions set out in the Application form signed by you 25 July 200B₁ 3 copy of which should have been retained by you.

V ours faithfully

' r

( ,

Joan Kellβy ΪW2Q ΘTY ^^'

Executive Director Bioservices

National University of Ireland Maymooth

Department of Biology

Maynooth

Co. Kildare

Ireland

3 September 2008

NOTIFICATION OF ACCEPTANCE OF A DEPOSIT FOR THE PURPOSES

OF PATENT PROCEDURES

Microorganism: Aspergillus nidυlans TRAN^a'^iT IMI CC Number: 396693

Strain Number: # 3

Yours faithfully

/ Ct"Ji biW;^<1

Dr Joan Kelley _f VWuG 9TY I

Executive Director Bioservices

Claims

Claims: -

1. A non-auxotrophic selection marker for an organism lacking said marker.

2. A non-auxotrophic selection marker according to Claim 1, wherein the organism is an eukaryotic organism.

3. A non-auxotrophic selection marker according to Claim 2, wherein the organism is a fungus.

4. A non-auxotrophic selection marker according to Claim 3, wherein the organism is a filamentous fungus.

5. A non-auxotrophic selection marker according to any preceding claim, wherein the selection marker is located in a gene cluster encoding a fungal metabolite.

6. A non-auxotrophic selection marker according to Claim 5, wherein the fungal metabolite is a mycotoxin.

7. A non-auxotrophic selection marker according to Claim 6, wherein the mycotoxin is an epipolythiodioxopiperazine.

8. A non-auxotrophic selection marker according to Claim 7, wherein the mycotoxin is gliotoxin.

9. A non-auxotrophic selection marker according to Claim 8, which is gliT or a homologue thereof.

10. A non-auxotrophic selection marker according to Claim 9, which is an orthologue oϊgliT.

11. A non-auxotrophic selection marker according to any one of claims 4-10, wherein the filamentous fungus is selected from Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.

12. A non-auxotrophic selection marker according to Claim 11, wherein the filamentous fungus is Aspergillus fumigatus.

13. A non-auxotrophic selection marker according to any one of Claims 9-12, wherein the gliT is used as a selection marker for transformation in Aspergillus fumigatus.

14. A non-auxotrophic selection marker according to Claim 13, wherein the selection is carried out in the presence of a selection medium containing gliotoxin.

15. A non-auxotrophic selection marker according to Claim 9 or 10, wherein the gliT or homologue or orthologue thereof is used as a selection marker for transformation in a non-gliT encoding organism.

16. A non-auxotrophic selection marker according to Claim 15, wherein the non-gliT encoding organism is Aspergillus nidulans.

17. A non-auxotrophic selection marker according to Claim 15 or 16, which is a dominant marker.

18. A non-auxotrophic selection marker according to any one of claims 15-17, wherein the selection is carried out in the presence of a selection medium containing gliotoxin.

19. A method for increasing the production of a fungal protein by a fungus capable of expressing said protein, which method comprises incorporating additional copies of a gene coding for the protein in said fungus.

20. A method according to Claim 19, wherein the fungal protein is GIiT protein.

21. A method according to Claim 20, wherein the GIiT protein is a gliotoxin reductase.

22. A method according to Claim 20 or 21 , wherein an overproduction of GIiT leads to an increased yield of gliotoxin.

23. A method for predicting the likelihood of infection of a subject by Aspergillus, which comprises detecting an antibody to GHT in a body fluid from said subject.

24. A method according to Claim 23, wherein the detection of an antibody to GIiT can be used to predict the likelihood of the development of invasive aspergillosis, saprophytic aspergillosis and/or allergic bronchopulmonary aspergillosis.

25. A method for predicting the likelihood of the onset of a fungal-induced allergic response in a subject, which comprises detecting an antibody to a protein expressed by a gene in the gliotoxin gene cluster in a body fluid from said subject.

26. A method according to Claim 25, wherein the antibody is IgG or IgE.

27. A method according to Claim 25 or 26, wherein the expressed protein is GIiT or GIiG.

28. A method according to Claim 27, wherein the GIiT is recombinant GIiT.

29. A method according to Claim 27, wherein the GIiG is recombinant GHG.

30. A mutant fungal strain which lacks the gliT gene or a homologue or orthologue thereof.

31. A mutant strain according to Claim 30, wherein the fungus is selected from Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.

32. Aspergillus fumigatus AgHT strain IMI CC 396691.

33. Aspergillus fumigatus Aglif*⁰ strain IMI CC 396692.

34. Aspergillus nidulans TRAN⁸"⁷ strain IMI CC 396693.