INFECTION AND IMMUNITY, Aug. 2005, p. 4548–4559 Vol. 73, No. 80019-9567/05/$08.00�0 doi:10.1128/IAI.73.8.4548–4559.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Aspergillus Cyclooxygenase-Like Enzymes Are Associated withProstaglandin Production and Virulence

Dimitrios I. Tsitsigiannis,1†‡ Jin-Woo Bok,1† David Andes,1 Kristian Fog Nielsen,2Jens C. Frisvad,2 and Nancy P. Keller1*

University of Wisconsin—Madison, Madison, Wisconsin,1 and Center for Microbial Biotechnology,Technical University of Denmark, Lyngby, Denmark2

Received 17 December 2004/Returned for modification 7 February 2005/Accepted 3 March 2005

Oxylipins comprise a family of oxygenated fatty acid-derived signaling molecules that initiate criticalbiological activities in animals, plants, and fungi. Mammalian oxylipins, including the prostaglandins (PGs),mediate many immune and inflammation responses in animals. PG production by pathogenic microbes istheorized to play a role in pathogenesis. We have genetically characterized three Aspergillus genes, ppoA, ppoB,and ppoC, encoding fatty acid oxygenases similar in sequence to specific mammalian prostaglandin synthases,the cyclooxygenases. Enzyme-linked immunosorbent assay analysis showed that production of PG species isdecreased in both Aspergillus nidulans and A. fumigatus ppo mutants, implicating Ppo activity in generating PGs.The A. fumigatus triple-ppo-silenced mutant was hypervirulent in the invasive pulmonary aspergillosis murinemodel system and showed increased tolerance to H2O2 stress relative to that of the wild type. We propose thatPpo products, PG, and/or other oxylipins may serve as activators of mammalian immune responses contrib-uting to enhanced resistance to opportunistic fungi and as factors that modulate fungal development contrib-uting to resistance to host defenses.

Aspergillus spp. are opportunistic pathogens with a world-wide distribution. These organisms can produce a wide spec-trum of diseases in both plants and animals. Invasive pulmo-nary infection with the propensity to disseminate to other endorgans represents the most common and lethal disease state inhumans. Aspergillus spp. also are causal agents of fungal sinus-itis, asthma, and allergic alveolitis in nonimmunosuppressedpatients. The predominant pathogenic species is Aspergillusfumigatus, accounting for up to 90% of invasive human infec-tions (7, 37). However, several other Aspergillus spp. includingthe genetic model A. nidulans, can infect immunocompromisedpatients and exacerbate preexisting diseases (38, 52). The clin-ical significance of A. fumigatus and the importance of A.nidulans as a fungal model system justified prioritization forgenome sequencing, which was recently completed for bothspecies (14).

Aspergillus infections are commonly initiated by inhalation ofthe airborne asexual spores called conidia. In the case of A.fumigatus, the small size (3 to 5 �m) of conidia enables them toreach the pulmonary alveoli, the main site of infection. If thespores survive in the alveoli, e.g., in the absence of an adequatehost immune response, they germinate and propagate in vivo,leading to disseminated invasion by the fungus of other criticalorgans within the host. This phase of the disease, known asinvasive aspergillosis (IA), is severe and often fatal (35, 46)despite the use of antifungal drugs (21, 37). Part of the poor

prognosis in IA can be attributed to the lack of understandingof the mechanisms underlying Aspergillus pathogenesis. Thecurrent view is that A. fumigatus pathogenicity is dependent onthe production of ill-defined fungal proteins and toxins thatpromote mycelial growth in IA and on structural features ofthe conidia, e.g., pigmentation, that confer resistance to thehost’s antifungal mechanisms including phagocytosis of spores(3, 25, 37).

Recent studies have suggested a role for bioactive lipids,known as oxylipins, in impacting eukaryotic microbe-host in-teractions. Oxylipins encompass a large group of oxygenatedC18, C20, and C22 bioactive lipids derived from �3 (n-3) and �6(n-6) polyunsaturated fatty acids (22, 42). Eicosanoids com-prise a subclass of C20 oxylipins derived from dihomo-�-linole-nic acid, arachidonic acid (AA), and eicosapentaenoic acid,including the prostaglandins (PGs) and leukotrienes, which actas “local short-range hormones” in maintaining local ho-meostasis in a variety of tissues and cells (18). Eicosanoids arecritically involved in mammalian immune responses such asregulation of inflammation, pain, fever, and allergic responses,as well as regulation of the cardiovascular system, reproduc-tion, and renal function, and might play a role in carcinogen-esis (18, 54, 67). Mammalian prostaglandin synthases, and sub-sequent PG production, are activated by mechanical trauma orby specific growth factors, cytokines, and other abiotic or bioticstimuli, including pathogen invasion (18). A single eicosanoidcan have pleiotropic effects due to the existence of multiplereceptors for each lipid species. In turn, these receptors havedifferent effects on different cell types (18, 20). Host produc-tion of PGs upon infection is well documented (18); however,recent studies suggest that PG production by eukaryotic mi-crobes could be contributing to the infection process (23, 42,44). The potential link between pathogen eicosanoids and

* Corresponding author. Mailing address: Department of Plant Pa-thology, University of Wisconsin—Madison, Madison, WI 53706.Phone: (608) 262-9795. Fax: (608) 263-2626. E-mail: npk@plantpath.wisc.edu.

† D.I.T. and J.-W.B. contributed equally to this work.‡ Present address: The Sainsbury Laboratory, John Innes Centre,

Norwich Research Park, Colney Lane, Norwich NR4 7UH, UnitedKingdom.

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modulation of host immunity is intriguing and potentially atarget for future pharmaceuticals (42).

Initial efforts to elucidate an oxylipin biosynthetic pathway infungi stemmed from interest in deciphering potential oxylipin-driven cross-communication between plants and mycotoxi-genic aspergilli (8, 64). Studies of the genetic model A. nidu-lans resulted in the characterization of three dioxygenase-encoding genes, ppoA, ppoB, and ppoC, required forbiosynthesis of oleic and linoleic acid-derived oxylipins similarin structure to plant defense molecules and important in inte-grating asexual and sexual spore balance in A. nidulans (58–61). Because studies have established that oxylipin-generatingenzymes exhibit activity toward more than one substrate, andall three of these putative dioxygenases showed high homologyto mammalian cyclooxygenases (COX) (prostaglandin H syn-thases), we have here investigated the possibility that fungalPpo proteins could be involved in PG biosynthesis. We showthat both A. nidulans and A. fumigatus ppo genes contribute toPG production in these species. The A. fumigatus PG mutantshowed increased virulence in a murine model of pulmonaryaspergillosis and enhanced resistance to environmental stress.We suggest the possibility of oxylipins as cross talk bioactivelipids that induce host defense mechanisms important in re-tarding the development of pulmonary and invasive aspergil-losis.

MATERIALS AND METHODS

Strains, media, and culture conditions. A list of the A. nidulans and A.fumigatus strains generated for this study is shown in Table 1. All strains weregrown at 37°C and stored as glycerol stocks. Appropriate supplements corre-sponding to the auxotrophic markers were added to the media as required.Sexual crosses and fungal transformation through polyethylene glycol-mediatedfusion of protoplasts were conducted according to standard techniques for bothAspergillus species (4). RDIT64.2 (�ppoA �ppoB) is a recombinant strain result-ing from a cross between RDIT12.6 and TTMK2.60 (58), and RDIT74.8 (�ppoB�ppoC) is a recombinant strain resulting from a cross between RDIT58.3 andTTMK2.60. The A. nidulans �ppoA �ppoB �ppoC triple mutant was created bya sexual cross between RDIT54.13 and TTMK2.60 (58). All strains were main-tained on Aspergillus glucose minimal medium (GMM) {6.0 g NaNO3, 0.52 g

KCl, 0.52 g MgSO4 · 7H2O, 1.52 g KH2PO4, 1 ml trace elements [2.2 g ZnSO4 ·7H2O, 1.1 g H3BO3, 0.5 g MnCl2 · 4H2O, 0.5 g FeSO4 · 7H2O, 0.16 g CoCl2 ·5H2O, 0.16 g CuSO4 · 5H2O, 0.11 g (NH4)6Mo7O24 · 4H2O, 5.0 g Na4 EDTA in100 ml distilled H2O], 10 g glucose, 15.0 g agar, pH 6.5, in 1 liter distilled H2O}with appropriate supplements (27). Agar was not added for liquid medium.

Nucleic acid analysis. Standard methods were used for construction, mainte-nance, and isolation of recombinant plasmids (51). Fungal chromosomal DNAwas isolated and analyzed from lyophilized mycelia by using previously describedtechniques (65). Cultures for RNA extractions were grown by inoculating 30 mlof liquid GMM with 1 � 106 spores/ml of the appropriate strain before incuba-tion for 24 h, 48 h, or 72 h (under stationary conditions), followed by harvesting.Total RNA was extracted from lyophilized mycelia by using TRIzol reagent(Invitrogen Co.) according to the manufacturer’s recommendations. Approxi-mately 20 �g of total RNA was used for Northern blot analysis using a 1.2%agarose–1.5% formaldehyde gel transferred to a Hybond-XL membrane (Am-ersham Pharmacia Biotech). Expression studies for the different genes wereperformed with appropriate probes. Probes for A. fumigatus were generated fromgenomic DNA using the following primer combinations: ppoA2f (5�-TTCCCTGAATTCGTTTAGGGTAGC-3�) and ppoA2r (5�-GTTGAAAAGCTTGCAATGATCAACG-3�) for AfppoA, ppoB2f (5�-TACCCTGGAGCAATACCCACC-3�) and ppoB2r (5�-ACCGGCTACCCAGATCAAAGCA-3�) for AfppoB, andppoC2f (5�-ATCCAAGCGCACGTTCGCCG-3�) and ppoC2r (5�-TGAACTCCTTGCTGGCCTTTCC-3�) for AfppoC. Nucleotide sequences were analyzed andcompared using the Sequencher (Gene Codes Co., MI) and ClustalW (http://www.ebi.ac.uk/clustalw/) software programs.

Plasmid and strain construction. Aspergillus nidulans ppo genes were previ-ously cloned, sequenced, and disrupted by replacement with marker genes: ppoAwas replaced with metG, ppoB with pyroA, and ppoC with trpC (58, 59, 61).Briefly, disruption vectors were constructed by flanking the A. nidulans markergenes (metG, pyroA, and trpC) with �1-kb DNA fragments from upstream of thecorresponding ppo gene start codon and downstream of the corresponding ppostop codon. Double and triple ppo mutants were created by sexual crosses.

Aspergillus fumigatus AfppoA, AfppoB, and AfppoC genes were obtained fromthe TIGR database (http://www.tigr.org/tdb/e2k1/afu1/) based on a homologoussearch using A. nidulans ppoA, ppoB, and ppoC sequences. The polymeraseThermal ACE (Invitrogen Co.) was used for PCR amplifications. RNA interfer-ence (RNAi) technology was used to create an A. fumigatus vector that wouldsilence expression of all three ppo genes (AfppoA, AfppoB, and AfppoC) simul-taneously by sequentially arranging segments of each gene in both a forward anda reverse fashion in one plasmid to create an inverted-repeat transgene (IRT).Fragments (500 bp each) of AfppoA (AscI, BamHI-NdeI), AfppoB (NdeI-SphI), and AfppoC (SphI, NotI-NcoI) were amplified using primer combina-tions with the indicated restriction enzyme sites introduced. The primers usedwere PpoAf (5�-CTTCGGCGCGCCATGGATCCCGATAGAGGGCCTTGCCCATC-3�), PpoAr (5�-CCCTCATATGATTGTGGAAGACGCGAAAGA

TABLE 1. Aspergillus strains used in this study

Fungal strain Genotype Source

Aspergillus nidulans strainsTTMK2.60 biA1 �ppoB::pyroA pyroA4 metG1 veA1 trpC801 58RDIT54.13 �ppoC::trpC pyroA4 metG1 �ppoA::metG veA trpC801 58RDIT12.6 argB2 �ppoA::methG methG1 veA This studyRDIT58.3 pyroA4 �ppoC::trpC veA trpC801 This studyRDIT9.32 veA (wild type) 61RDIT12.9 metG1 �ppoA::metG veA 61RDIT59.1 pyroA4 �ppoB::pyroA veA 58RDIT58.12 �ppoC::trpC veA trpC801 59RDIT64.2 �ppoB::pyroA pyroA4 metG1 �ppoA::metG veA This studyRDIT74.8 �ppoB::pyro �ppoC::trpC pyroA4 veA trpC801 This studyRDIT54.7 �ppoC::trpC metG1 �ppoA::metG veA trpC801 59RDIT62.3 �ppoB::pyro �ppoC::trpC pyroA4 metG �ppoA::metG

veA trpC80158

Aspergillus fumigatus strainsAF293 Wild type Greg MayAF293.1 pyrG Greg MayTJW62.2 Afppo(IRT)::pyrG; pyrG This studyTJW62.5 Afppo(IRT)::pyrG; pyrG This studyTJW62.10 Afppo(IRT)::pyrG; pyrG This study

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GT-3�), PpoBf (5�-GGCTCATATGCGCGAAATATCCACCTGGTTT-3�),PpoBr (5�-TCAAGCATGCAAACCTGACGAACTGGGG-3�), PpoCf (5�-CCCAGCATGCACAAGACCTCTGGTTACTTGGA-3�), and PpoCr (5�-TAATCCATGGCGGCCGCAGGGTATCCAGCTGCGT-3�) (f, forward; r, re-verse). The ppoA PCR product was digested by NdeI, the ppoB PCR productby NdeI-SphI, and the ppoC PCR product by SphI. The three ppo fragmentswere ligated together, and the ligation mixture was used as a template toobtain a 1.5-kb PCR product (AfppoA-ppoB-ppoC) using the primer pairPpoAf–PpoCr. This 1.5-kb PCR product was initially digested with AscI andNcoI and ligated into the corresponding sites of pTMH44.2 (19) to generateplasmid pCEJ1. Digestion of pCEJ1 with BamHI and NotI yielded once morethe 1.5-kb triple-ppo PCR product, which was further ligated into the corre-sponding sites of pTMH44.2 (19) in a forward orientation to yield thepCEJ2.7 vector. Next, the 1.5-kb AscI-NcoI fragment was released frompCEJ1, allowing it to be placed in the AscI-NcoI site of pCEJ2.7 in a reverseorientation, to create the pCEJ2.7.4 vector. An internal �280-bp spacergreen fluorescent protein fragment separated the inverted repeats, and the A.nidulans gpdA promoter, which has been successfully used in different fungalsystems for high levels of transcription (48), drove the transgene. The As-pergillus parasiticus pyrG gene (pBZ5) (53) was inserted into an EcoRI site ofpCEJ2.7.4 to give pJW66.3 (Fig. 1). This final plasmid was used for transfor-mation of AF293.1 to silence expression of AfppoA, AfppoB, and AfppoC inA. fumigatus.

Prostaglandin analysis. A. nidulans and A. fumigatus wild-type and ppo mutantstrains were grown for 7 days at 37°C in RPMI medium (a defined mediumdevoid of fatty acids; Sigma Chemical Co.) with shaking at 300 rpm. After 7 daysthe cultures were incubated for an additional 2 h with 1 mM AA (CaymanChemicals, Ann Arbor, Mich.). Culture supernatants from both AA-fed andnon-AA-fed fungi were analyzed for prostaglandin production by using an en-zyme-linked immunosorbent assay (ELISA) kit (catalog no. 514012; CaymanChemicals) according to the manufacturer’s instructions. The antiserum used inthis assay exhibits high cross-reactivity for most PGs, allowing quantification ofPGE1, PGE2, PGF1, PGF2 (100% specificity), PGF3, PGD2, PGE3 (�50%specificity), and thromboxane B2 (TXB2; 5% specificity). It does not detectPGA, PGB1, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2, or misopristol. Thecultures without AA measure the endogenous production of PGs in the absenceof exogenous fatty acid substrates. Student’s t test and analysis of variance wereused to analyze the significance of differences between the experimental groupsusing the Statistical Analysis System (SAS Institute, Cary, NC).

Physiological studies. Conidium production studies for wild-type A. fumigatusand Afppo IRT mutant strains were performed on plates containing 30 ml ofsolid 1.5% GMM. Five milliliters of a top layer consisting of cool melted 0.7%agar–GMM containing 106 conidia of the appropriate strain was added to eachplate. Cultures were incubated in continuous dark at 37°C. A core with a diam-eter of 12.5 mm was removed from each plate after 2, 4, and 6 days and washomogenized for 1 min in 3 ml of sterile water supplemented with 0.01% Tween80 to release the spores. Spores were counted using a hemacytometer. Theexperiments were performed with four replicates. Radial growth tests wereperformed in triplicate with approximately 104 conidia centered on 30-ml GMMplates, and growth rates were recorded as colony diameter over time at fivetemperature regimes: 24°C, 28°C, 32°C, 37°C, and 42°C. For germination tests,strains were inoculated in minimal medium at 106 spores/ml and shaken for 24 hat 300 rpm and 37°C. Samples were examined at 2-h intervals, and the germi-nation rate was determined by counting 100 conidia. The mycelial weight oflyophilized tissue was assessed after 4 days of culture in liquid GMM. Physio-logical data were statistically compared by analysis of variance and Fisher’s least

significant difference using the Statistical Analysis System (SAS Institute, Cary,NC).

Bioassay of arachidonic acid and PGE2. The fatty acids used in this studyincluded arachidonic acid (20:4) and PGE2 obtained from Cayman ChemicalsCo. Amounts of 0.1 mg and 1 mg were dissolved in 50 �l of methanol and driedon 12.5-mm-diameter paper filter disks. A paper filter disk treated with methanolwas used as the solvent control. After drying, the fatty acid-containing disks andthe methanol-containing disks were laid on the agar surfaces of plates containing30 ml of solid 1.5% YGT medium (9) with a 5-ml top layer consisting of coolmelted 0.7% agar–YGT containing 105 conidia of the wild-type A. nidulans(RDIT9.32) or A. fumigatus (AF293) strain. The cultures were incubated underlight and dark conditions for 8 days.

Stress tolerance assays. Determination of tolerance levels against heat andoxidative stress was performed as previously described (10, 31). For the thermaltolerance assay, wild-type or Afppo IRT strain conidia were inoculated on solidGMM in triplicate (100 to 150 colonies per plate) and incubated at 37°C for 8 h.Cultures were transferred to 50°C for 3 or 4 h, and the plates were incubated foran additional 36 h at 37°C. Surviving colonies were counted. For the hydrogenperoxide conidial sensitivity assay, 1-ml conidial suspensions containing 105

spores were incubated with different hydrogen peroxide (H2O2) concentrations(0, 20, 40, 80, 150, and 250 mM) for 30 min at room temperature. Each sporesuspension was then diluted with sterile distilled water, and conidia were inoc-ulated on solid GMM. After incubation at 37°C for 36 h, colony numbers werecounted and calculated as a percentage of the control (10). For the assay ofhyphal sensitivity to H2O2, plates containing �50 30-h-grown colonies wereoverlaid with 10 ml of 0, 50, 100, and 200 mM H2O2 solutions. After a 10-minincubation at room temperature, the H2O2 solution was removed, and the plateswere washed twice with sterile distilled water and incubated further for 24 h at37°C. The number of colonies that survived was calculated as a percentage of thecontrol. All the experiments were performed in triplicate.

Animal model of Aspergillus infection. The virulence of isogenic wild-type andAfppo IRT strains was studied in a lung infection model, with the approval of theUniversity of Wisconsin animal care committee. Conidia were harvested byflooding of fungal colonies with 0.85% NaCl with Tween 80, enumerated with ahemacytometer, and adjusted to a final concentration of 6.5 log10 CFU/ml.Counts and the viability of the inocula were verified by duplicate serial plating onGMM plates. Six-week-old outbred Swiss ICR mice (Harlan Sprague-Dawley)weighing 24 to 27 g were immunosuppressed by intraperitoneal injection ofcyclophosphamide (100 mg/kg of body weight) on days 4, 1, and 3 and witha single dose of cortisone acetate (200 mg/kg). Mice were anesthetized viahalothane inhalation in a bell jar at day 0. Sedated mice (10 mice/fungal strain)were infected by nasal instillation of 50 �l of the inoculum (day 1) and monitoredthree times daily for 7 days postinfection. All surviving mice were sacrificed atday 7. The tissue fungal burden of a whole-lung homogenate was quantified byserial dilution and enumeration of CFU (CFU/2 lungs). The duration of survival(in days after inoculation) was recorded for each animal. Moribund animals weresacrificed and cumulative survival recorded. Survival and clearance of residualfungal burden in tissue (CFU/2 lungs) were used as the outcome variables toassess the relative virulence of isogenic strains.

Mouse lung metabolite analysis. Chloroform extracts from mouse lungs wereredissolved in 400 �l methanol (high-performance liquid chromatography grade)and loaded onto a Strata X (Phenomenex, Torrance, Ca) 60-mg SPE columnalready containing 3 ml water (Milli-Q). The SPE column had previously beensequentially activated with 2 ml methanol and 2 ml water. After the sample wasloaded, the column was washed with 1 ml water, and the sample was eluted with4 ml methanol-water (9:1) containing 0.5% formic acid (analytically pure). Thesamples were then evaporated in vacuo on a SpeedVac, redissolved twice in 50�l methanol, and filtered through a 4-mm-diameter 0.45-�m-pore-size polytet-rafluoroethylene (Teflon) syringe filter. Samples (3 �l) were then analyzed byliquid chromatography–high-resolution mass spectrometry (LC-HR-MS) on anAgilent 1100 LC system equipped with a UV photo diode array detector andcoupled to an LCT orthogonal time-of-flight mass spectrometer (Waters-Micro-mass, Manchester,United Kingdom) (40). Separation was performed on a Phe-nomenex (Torrance, CA) Luna II C18 (II) column (50 by 2 mm; inner diameter,3 �m) using a water-acetonitrile system at a flow rate of 0.3 ml/min, starting at15% acetonitrile, increasing the concentration linearly to 100% in 20 min, andholding at 100% for 5 min. The water was buffered with 10 mM ammoniumformate and 20 mM formic acid (both analytical grade) and the acetonitrile with20 mM formic acid. Gliotoxin and other metabolites were identified by compar-ison to reference standards (40) of known A. fumigatus metabolites (11, 12) andextracts from agar cultures, based on their retention time, UV spectra, andpositive electrospray spectra (ESI�). For secondary metabolites reported fromA. fumigatus (11, 12) but not available to us as standards, we used their known

FIG. 1. Plasmid construction to silence the three ppo genes simul-taneously in A. fumigatus. Segments (�500 bp) of each gene werealigned in both a forward and a reverse orientation in one plasmid tocreate the inverted-repeat transgene.

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[M�H]� ions as a basis for detecting them by selected ion chromatograms. Thesecondary metabolites not available as standards included gliotoxins E and G;gliotoxin acetate; dehydrogliotoxin; 5a,6-dehydrobisdethio-3,10a-bis(methylthio-)gliotoxin; pseurotins B, C, D, F2, and F1; TR-2; fumitremorgin C; fumiquina-zolines A to E; and tryptoquivalines A to H.

RESULTS

A. nidulans and A. fumigatus ppo genes encode cyclooxygen-ase-like enzymes. BLAST searches of the A. nidulans (http://www.broad.mit.edu/annotation/fungi/aspergillus/), and A. fumigatus(http://www.tigr.org/tdb/e2k1/afu1/) genome databases withthe biochemically characterized oxylipin-producing linoleatediol synthase (lds) gene from the filamentous fungus Gaeum-annomyces graminis (GenBank accession no. AF124979), horseCOX-2 (GenBank accession no. O19183), and human COX-1(GenBank accession no. P23219) revealed the presence ofthree genes named ppoA, ppoB, and ppoC in both aspergilli (A.fumigatus genes are distinguished by the “Af” prefix). Disrup-tion of ppoA (AY502073) (61), ppoC (AY613780) (59), andppoB (AY940146) (58) in A. nidulans led to strains defective inproducing monohydroxy linoleic and oleic acid-derived oxylip-ins. Protein domain searches against the PFAM database(http://pfam.wustl.edu) indicated that both A. nidulans Ppoproteins and A. fumigatus Ppo proteins AfPpoA (58.m07572),AfPpoB (67.m03008), and AfPpoC (59.m09493) have domainssimilar to those of animal heme peroxidases and cytochromeP450 oxygenases. Comparative sequence analysis (ClustalW)between the predicted Ppo amino acid sequences of the twospecies led to the phylogenetic tree shown in Fig. 2A and thecorresponding comparison values (Fig. 2B). AfPpoA showed48% and 47% identities with AfPpoB and AfPpoC, respec-tively, whereas AfPpoB and AfPpoC showed 56% identity.

The amino acid sequences of A. nidulans and A. fumigatusPpo proteins also revealed similarity with various mammalianCOX, the key enzymes in the production of prostaglandins in

vertebrates. COX exist as two isoforms (COX-1 and COX-2)and oxygenate arachidonic acid to the intermediate prostaglan-din PGH2 (54). PGH2 is converted to the biologically activeend product PGD2, PGE2, PGF2, PGI2, or TxA2 (thromboxaneA2) by other specific prostaglandin synthases (18, 54). Homol-ogy between A. nidulans and A. fumigatus Ppo and mammalianCOX amino acid sequences over the conserved catalytic do-mains ranged from 25% to 29% identity and 40% to 45%similarity for COX-2 paralogs (E values, 1024 to 1018) and25% to 26% identity and 38% to 40% similarity for COX-1paralogs (E values, 2 � 1018 to 5 � 1018).

A more detailed sequence alignment of the Aspergillus Pposequences with horse COX-2 and human COX-1 was per-formed with the ClustalW program to identify conserved func-tional motifs. The sequence similarity appeared to be restrictedto the catalytic domain of COX and was most striking along the-helices (predicted to be present in Ppo proteins by using thePredictProtein program, available at http://cubic.bioc.columbi-a.edu/predictprotein/) as shown in Fig. 3A. Structural homol-ogy shows that both the distal and proximal His heme ligandsand the important Tyr residue, which are required for enzymeactivity and are completely conserved within the COX family,aligned in context with identical amino acids of A. nidulansPpoC, A. nidulans PpoA, and A. fumigatus AfPpoA but notwith the other Aspergillus proteins. The core helix H2 harborsthe distal His heme ligand (consensus THXXFXT), and thecore helix H8 contains the proximal heme ligand and the im-portant Tyr residue (consensus EFNXXYXWH) of PGH syn-thases (54). In contrast to the structural conservation foundwithin the catalytic domain, the regions of COX falling outsideof this domain, that is, the epidermal growth factor-like do-main and the membrane-binding domain, do not seem to haveequivalent residues in the Ppo proteins. G. graminis Lds alsohas similar conserved regions in its sequence, as was previouslyreported (24); however, the biochemical involvement of Lds inPG biosynthesis has not been demonstrated.

RNAi silences expression of three ppo genes in A. fumigatus.To gain evidence of a role of AfPpo’s in fungal developmentand virulence, a gene-silencing approach was undertaken toinactivate the three genes simultaneously. The absence of asexual cycle in A. fumigatus renders the incorporation of manydeletions in the same individual a difficult task in this species.To evaluate the potential of RNAi technology as a means ofsilencing multiple genes in A. fumigatus, a vector was designedto simultaneously produce AfppoA, AfppoB, and AfppoC dou-ble-stranded RNA molecules in the fungal thallus (Fig. 1).Double-stranded RNA molecules are processed by RNAi ma-chinery to produce small interfering RNA (siRNA) moleculesthat trigger mRNA degradation of targeted genes (13, 19).This technology has been successfully demonstrated in both A.nidulans and A. fumigatus but only by silencing one or twogenes within each vector (19, 39).

The triple Afppo mutant (Afppo IRT) was created by trans-formation of strain 293.1 with pJW66. PCR and Southern blotanalysis of 85 transformants revealed the introduction of theIRT Afppo construct in five transformants. Macroscopically, allfive transformants were identical to each other and to the wildtype. Transformants TJW62.2, TJW62.5, and TJW62.10 andthe wild-type AF293 were selected for mouse virulence studies(see below); TJW62.2, TJW62.10, and AF293 were used for

FIG. 2. Phylogenetic analysis (A) and percentages of amino acididentity (B) of the A. nidulans and A. fumigatus Ppo proteins. Aminoacid sequences of the predicted Ppo proteins were aligned with theClustalW software program, and the tree diagram was created by theTreeView software program. Bar, 0.1 amino acid substitution per site.

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ELISA analysis (see below); and TJW62.2 was used for furtherphysiological and molecular analyses. As predicted, the A. fu-migatus transformants, which incorporated the Afppo IRTplasmid into their genome, displayed an Afppo silencing phe-notype, as monitored by expression studies of the three ppogenes. Figure 4 shows that in contrast to the wild type, all threegenes showed decreased expression in the Afppo IRT strain.Similar results were obtained for the other Afppo IRT strains(data not shown).

Phenotypic characterization of the Afppo IRT strain. Likethe �ppoA �ppoB �ppoC mutant of A. nidulans, the AfppoIRT mutant had no alterations in vegetative development orspore germination relative to the wild type in liquid GMM.However, in contrast to the A. nidulans �ppoA �ppoB �ppoCstrain, which demonstrated a significant reduction in asexualspore production and a significant increase in sexual sporeproduction (58), spore counts of the A. fumigatus triple ppomutant did not show any alterations in asexual spore produc-tion at 28°C and 37°C in GMM, the only conditions tested(data not shown). This might be due to the inability of A.fumigatus to form the sexual stage. Interestingly, radial growthexperiments on solid GMM with glucose as the sole carbonsource indicated that the Afppo IRT mutant grows 5 to 10%faster than the wild type at 24°C, 28°C, and 42°C but that thereis no difference at 37°C.

Ppo mediation of prostaglandin production in A. nidulansand A. fumigatus. One primary goal of this study was to deter-mine if any of the ppo genes could be involved in PG produc-tion. Recent reports indicate that several fungi can utilizeexogenous sources of arachidonic acid to produce a number ofdifferent eicosanoids (44, 45); however, no candidate enzymehas been uncovered. By following procedures used by otherlabs in establishing that 90% of fungal PGs are secreted (36,45), supernatants from Aspergillus strains grown in RPMI me-dium were examined for PG production. Dry weights of recov-

FIG. 3. Aspergillus Ppo proteins share similarity with mammalian COX. (A) Partial alignment of PpoC with human COX1 and horse COX2over the catalytic domain. The distal histidine (H) heme ligand (helix 2), the proximal H heme ligand (helix 8), and the important Tyr (Y) residue(helix 8) of COX enzymes are boxed. The alignment displays the degree of conservation among the polypeptides as observed in each column:asterisks, identical amino acids across the three proteins in the alignment; colons, conserved substitutions; periods, semiconserved substitutions.The multiple alignment analysis was carried out using ClustalW, and the prediction of helix domains was carried out using the PredictProteinprogram. (B) Partial ClustalW alignment of the A. nidulans and A. fumigatus Ppo predicted catalytic domain (boxes indicate the presence of distaland proximal H heme ligands and the Y residue in PpoA, PpoC, and AfPpoA polypeptides). Boldfaced letters indicate amino acids that areconserved among the sequences.

FIG. 4. RNAi technology successfully silenced the three ppo genesin A. fumigatus. Wild-type A. fumigatus and the Afppo IRT (TJW62.2)mutant were grown in stationary liquid GMM for 24 h, 48 h, and 72 hat 37°C. Ethidium bromide-stained rRNA is shown as a loading con-trol. The silenced Afppo IRT mutant showed decreased expression ofall three ppo genes at all time points.

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ered mycelium were identical, but there was a decrease inproduction of PGs in A. nidulans strains carrying a �ppoAallele (14%) and in all strains carrying the �ppoC allele (36%to 37%) (Fig. 5A) (P � 0.01). These data suggest that PpoCand PpoA are involved in PG production by A. nidulans. Ex-amination of two IRT Afppo mutants (TJW62.2 andTJW62.10) showed that both mutants had a 12% to 15% re-duction in PG biosynthesis (Fig. 5B) (P � 0.01). The amount

of PGs that was detected in non-AA-fed cultures of both as-pergilli was below the assay’s threshold of detection (40 pg/ml).

Increased virulence of Afppo IRT mutants in a murinemodel of pulmonary aspergillosis. The relative virulence of thewild type and three Afppo IRT mutants (TJW62.2, TJW62.5,and TJW62.10) was evaluated in a murine pulmonary infectionmodel where mice were monitored for survival on a daily basis(Fig. 6). The Afppo IRT strains caused an increased fatal

FIG. 5. A. nidulans and A. fumigatus ppo mutants demonstrated decreased levels of PG production. Shown are PG levels in culture supernatants(pg/ml) of 7-day cultures of wild-type A. nidulans (WT) and �ppo mutants as described in Table 1 (A) and wild-type A. fumigatus and two AfppoIRT transformants (TJW62.2 and TJW62.10) (B). Values are means of three replicates. Letters (a, b, c, and d) indicate statistical differences(P � 0.05).

FIG. 6. Silencing of the three Afppo genes in A. fumigatus led to hypervirulent strains. Virulence was studied in a murine lung infection model.Immunosuppressed Swiss ICR mice (Harlan Sprague-Dawley) were infected with wild-type AF293 or one of the Afppo IRT (TJW62.2, TJW62.5,TJW62.10) strains. Duration of survival (in days after inoculation) was recorded for each animal. The log rank test was used to perform pairwisecomparison of survival among the strain groups; P values for comparison of the survival of wild-type- and Afppo IRT-infected mice are 0.02 to�0.001.

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infection rate in the murine model (P, 0.02 to �0.001) relativeto the wild type. By day 3 postinoculation, mice infected withthe Afppo IRT strains began to die, with a 90 to 100% mor-tality rate reached by day 5 for TJW62.2 and TJW62.10 and a50% mortality rate by day 5 for TJW62.5. In contrast, the wildtype showed a 30% mortality rate at day 5. By day 6 none of themice treated with TJW62.2 or TJW62.10 survived, whereas50% of those infected with the wild type were still alive. Thevirulence experiment was repeated twice with similar results.From all mice culled, fungal colonies were recovered from thelungs, indicating that the pulmonary distress was due to as-pergillosis. No statistical differences were observed in the num-ber of CFU recovered from the lungs of wild-type- or AfppoIRT-infected mice sacrificed 3 days after inoculation (data notshown). The isolated fungal colonies from each group ofmouse lungs were confirmed as wild type or Afppo IRT bydiagnostic PCR and Southern blot analysis (data not shown).

Given the numerous studies implicating gliotoxin as a potentvirulence factor in aspergillosis (41), five lungs from sacrificedmice were extracted and analyzed for gliotoxin content 3 and 4days after infection with 107 wild-type (AF293) or Afppo IRT(TJW 62.2) conidia. LC-HR-MS (Fig. 7) showed no differencesin the quantities of gliotoxin detected between the wild typeand an Afppo IRT mutant. Gliotoxin was unambiguously de-tected at the same retention time in a reference standard, andboth HR-MS and UV spectra were correct. Noninfectedmouse lungs showed no detectable levels of gliotoxin (Fig. 7).No other gliotoxin analogs or secondary metabolites normallyproduced during in vitro culture of A. fumigatus (e.g., helvolicacid, fumigaclavines A to C, tryptoquivalines A to H,fumiquinazolines A to E, pseurotins A to F2, fumagillin, fumi-tremorgins A to C, verrucologen, or TR-2) could be detectedin the wild-type- or Afppo IRT-infected lung tissue. Most ofthese compounds ionize significantly better than gliotoxin and

FIG. 7. Chromatograms of chloroform extracts from murine lung extracts after infection with wild-type A. fumigatus or the triple ppo mutantTJW62.2. (A) Base peak (BPI) chromatograms showing no major differences from the control mouse. (B) Reconstructed ion chromatograms (m/z263.100 to 263.109) showing the major fragment ion of gliotoxin. By showing only this fragment, almost all noise from the many compoundsoriginating from the mouse lungs is filtered away and the gliotoxin peaks can clearly be detected at the same retention time as the referencestandard in the triple ppo mutant and the wild type, whereas only noise can be seen in the noninfected mouse lung (control). To the left, the ESI�

MS spectra for the triple ppo mutant (top), the wild type (middle), and a reference standard of gliotoxin (bottom) are shown.

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would thus have much lower detection limits (40), showing thatgliotoxin is the primary fungal metabolite produced in thelungs.

Increased stress sensitivity of the A. fumigatus triple ppomutant. Although we could not detect any difference in sporepigmentation and gliotoxin production (both implicated as vir-ulence factors) that could explain the increased virulence ofthe Afppo IRT mutant, we considered the possibility thatmechanisms of resistance to host defenses might be altered inthe Afppo IRT mutant. To test this hypothesis, the Afppo IRTmutant was subjected to oxidative and thermal stress. By fol-lowing procedures established for A. fumigatus and A. nidulans(30, 47), both conidia and hyphae were exposed to increasinglevels of H2O2, a treatment designed to mimic exposure to hostreactive oxygen species (ROS), typically activated duringpathogen ingress. Germination and survival rates of spores invarying concentrations of H2O2 were determined after 24 h at37°C. As shown in Fig. 8, conidia of the Afppo IRT mutantshowed a statistically significant 15-to-20% increase (P � 0.05)in survival at 20, 40, and 80 mM of H2O2 relative to the wildtype, which showed a steady decrease in oxidative tolerance.No colonies of either strain survived the 250 mM H2O2 treat-ment. In contrast to the increased resistance of conidia toH2O2 treatment, Afppo IRT hyphae were not more resistant toH2O2 than wild-type hyphae.

The resistance of the Afppo IRT mutant to thermal stresswas evaluated by treating germlings at 50°C for 0, 3, and 4 hand determining their ability to form viable colonies at 37°C.The wild type showed mean decreases of 10% and 93% incolony survival, whereas the Afppo IRT mutant showed 1% (P� 0.1) and 87% (P � 0.1) decreases, at 3 h and 4 h, respec-

tively. Based on these data, Ppo proteins and/or their products,among several other possible roles, might mediate signalingcascades that regulate heat and oxidative stress responses atdifferent developmental stages of the fungus.

Effects of arachidonic acid and PGE2 on Aspergillus physi-ology. Earlier studies in our lab showed that C18 unsaturatedfatty acids and their derived oxylipins induced developmentalchanges in A. nidulans including changes in sexual-to-asexualspore ratios and secondary metabolite production (8, 9). Sim-ilar studies have shown that PGs can induce developmentalchanges in Candida albicans (28, 43). We therefore thought itpossible that PGs and their progenitor, arachidonic acid, mightaffect Aspergillus physiological processes.

Wild-type A. fumigatus and A. nidulans cultures were exam-ined for reactions to PGE2 and arachidonic acid. The additionof PGE2 to A. nidulans cultures inhibited the formation ofconidia, in sharp contrast to arachidonic acid, which signifi-cantly induced conidiation (visualized as a green halo ofconidia around the disk) (Fig. 9). These results were reminis-cent of the differential reaction of A. nidulans to the plantoxylipin 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) and its precursor, linoleic acid, where the formerinduced conidial formation at 0.1 mg and the latter inhibitedconidiation at the same concentration (9). Neither PGE2 norarachidonic acid affected sporulation in A. fumigatus; however,they both inhibited the production of hyphal pigments (Fig. 9).

DISCUSSION

Oxylipin production is widespread among fungi, andprogress has recently been made in identification, regulation,

FIG. 8. Silencing of the three Afppo genes caused elevated oxidative resistance of A. fumigatus conidia. Bars show the relative percentages ofgermination of A. fumigatus conidia with different concentrations of H2O2 compared to 0 mM. Conidia of the A. fumigatus wild-type strain AF293(WT) and the triple-ppo-silenced Afppo IRT strain (TJW62.2) were incubated with varying concentrations (0 to 150 mM) of H2O2 as indicated.Values are means of three replicates. Error bars, standard errors.

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and cellular localization of the dioxygenases generating fungaloxylipins (17, 22, 33, 42, 58, 59, 61). In this study, we providethe first genetic evidence for fungi that Ppo proteins similar insequence to mammalian COX are involved in the productionof prostaglandins, a major group of oxylipins that regulateimmune responses in mammals. Silencing of the three ppogenes in the human pathogen A. fumigatus yielded hyperviru-lent strains in the mouse pulmonary model system. These stud-ies extend the involvement of oxylipins in aspergilli from con-trol of developmental and metabolic functions, such as sporebalance and fatty acid regulation (42, 58, 59, 61), to virulencein a host-pathogen interaction.

The types of oxylipins produced by fungi are numerous, andlikely many remain to be characterized. Although many studieshave focused on C18 oxylipins, several experiments have re-vealed that both pathogenic and nonpathogenic fungal speciesproduce detectable amounts of both C20 cyclooxygenase andlipoxygenase products (36, 44, 45). The arachidonic acid me-tabolites PGF2 and PGF2–lactone have been detected in anumber of environmental yeasts of the family Lipomycetaceae(Dipodascopsis, Lipomyces, Myxozyma, and Zygozyma) (22, 33,34, 55) as well as in Saccharomyces cerevisiae (33). The patho-genic yeasts Cryptococcus neoformans and Candida albicansand the filamentous fungus A. fumigatus produce both PGs andleukotrienes, and their amounts are significantly increased af-ter exogenous application of arachidonic acid (44, 45). Fur-thermore, COX inhibitors, including aspirin and other nonste-roidal anti-inflammatory drugs, inhibited hydroxyeicosa-tertraenoic acid, PGE2, and PGD2 production in severalmembers of the family Lipomycetaceae, supporting the viewthat a COX-like enzyme is active in these fungi (5, 32, 36).

However, no candidate protein has been identified in fungiprior to this report.

The structural similarity between Ppo proteins and COX ledus to investigate the possibility that these enzymes could beinvolved in PG production. COX possesses two enzymatic ac-tivities, a cyclooxygenase that catalyzes the oxygenation ofpolyunsaturated substrates, such as arachidonic acid, to formprostaglandin G2 (PGG2) and a peroxidase that can use avariety of electron donors to reduce PGG2 to form prostaglan-din H2 (PGH2) (54). The amino acid sequences of Ppo pro-teins are predicted to contain both an oxygenase and a perox-idase domain (58, 59, 61). Further detailed comparison of theamino acid sequences of all six Ppo proteins with human andhorse COX revealed that three of the proteins, A. nidulansPpoC and PpoA and A. fumigatus PpoA, contained the con-served catalytic residues found in COX -helices, which in-cluded the proximal and distal heme ligands and the criticaltyrosine residue of PGH synthases (24, 54). Considering thecorrelation of loss of PG activity in the respective A. nidulansppoC and ppoA mutants (Fig. 5) and presence of catalyticresidues, it is tempting to speculate that these amino acids areindicators of possible PG activity in fungi. Investigation ofsingle A. fumigatus Ppo mutants may shed further light on theviability of this observation.

Through sexual genetics, we were able to analyze every pos-sible combination of ppo mutant background in A. nidulans.The consistent decrease in PG production in all combinationsof the �ppoC allele suggests a major role for this enzyme in PGbiosynthesis (Fig. 5A). The slight increase in production ofPGs in �ppoB and �ppoA �ppoB strains correlated with anupregulation in the expression level of ppoC (58, 59), furtherimplicating PpoC in the synthesis of PGs. Previous biochemicaldata has shown that PpoC is also likely involved in the pro-duction of oleic acid-derived oxylipins (59). The ability of afatty acid oxygenase to utilize different fatty acids as substratesis not uncommon. For instance, the G. graminis Lds can oxy-genate oleic, -linolenic, and ricinoleic acids (56).

In contrast to the ease of combining alleles in A. nidulans,disrupting three genes in the asexual fungus A. fumigatus in-volves considerable effort and has not been reported to date.To address this issue, we attempted to silence all three Afppogenes by using one vector. As demonstrated by Northern blot(Fig. 4) and PG (Fig. 5B) analyses, this approach was success-ful. RNAi has emerged as an effective method for silencinggene expression in many eukaryotes and has recently beenused successfully in genome-wide functional tests (2, 29); webelieve this method will greatly help in further genomic studiesof A. fumigatus. Caveats with this method are (i) the potentialof off-target effects by the siRNAs that could jeopardize cor-rect interpretation of gene function and (ii) the fact that sometranscripts are still produced and not completely eliminated,unlike the situation in traditional gene replacement, as illus-trated in Fig. 4. The fact that PG reduction in the Afppo IRTstrain is not as pronounced as in the A. nidulans triple mutantcould well be a reflection of this incomplete transcript suppres-sion. Additionally, the presence of alternative enzymes orpathways for PG biosynthesis is expected, since both the A.nidulans triple mutant and the Afppo IRT strain still producedsubstantial amounts of PGs. It is also possible that levels ofother eicosanoids are decreased in the ppo mutants, since our

FIG. 9. Prostaglandin PGE2 and AA affect A. nidulans and A. fu-migatus development. Wild-type RDIT9.32 and AF293 cultures weretreated with filter paper disks containing the solvent control (metha-nol), 0.1 mg of AA or PGE2, and 1.0 mg of AA or PGE2. PGE2 at 1.0mg inhibited asexual sporulation in A. nidulans RDIT9.32 (40-h cul-ture in the light); in contrast, 1.0 mg AA induced asexual sporulationin A. nidulans RDIT9.32 (24-h culture in the light). All the concentra-tions tested for either PGE2 or AA inhibit pigment biosynthesis in A.fumigatus (40-h culture in the dark).

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detection method was limited to specific PGs and did not coverthe full array of known eicosanoids.

Despite a decrease of only 12% in PG production as mea-sured by ELISA (Fig. 5B), the Afppo IRT strains displayed asignificant increase in virulence in the murine pulmonarymodel (Fig. 6). To our knowledge this is the first report of ahypervirulent A. fumigatus mutant. Since a unique character-istic of mammalian PGs is their potency at very low (nanomo-lar) concentrations and their very short half-lives—they areproduced de novo and act near the site of their synthesis—wespeculate that the small decrease in the endogenous fungal PGlevels can lead to a significant increase in the virulence of A.fumigatus (18). Two determinants implicated as virulence fac-tors in A. fumigatus include spore pigmentation (6, 57) andgliotoxin production (41). Loss of pigmentation results in in-creased phagocytosis, whereas gliotoxin, a secondary metabo-lite produced in tissues of mice following development of in-vasive aspergillosis (15), is a potent immunomodulating agentand an inducer of apoptotic cell death in a number of celltypes (62). Microscopic examination of Afppo IRT sporesdid not reveal any differences in pigmentation compared tothe wild type. Chemical analysis indicated that gliotoxinproduction in mouse lungs by Afppo IRT strains was notaltered from that by the wild type (Fig. 7). These factors,therefore, do not seem to play a role in the increased viru-lence of the Afppo IRT strains.

Physiological examination of one of the Afppo IRT strainsdid, however, indicate increased resistance to environmentalstress as observed by H2O2 treatment (Fig. 8). H2O2 treatmentof fungal propagules is an indirect method of measuring theputative resistance of the pathogen to host ROS, a major host

antimicrobial effector system also active against Aspergillusconidia (63). ROS production in mammals occurs during thecourse of neutrophil and macrophage activation and is impli-cated in the defense against fungal pathogens (1, 49, 50). In-creased resistance to a ROS-mounted defense could protect apathogen and render it more virulent.

Although the hypothesis is not examined in this study, wealso propose that part of the increased virulence of the AfppoIRT strains is due to changes in host physiology. A plausibleexplanation is that the Ppo-generated PGs enhance host de-fense mechanisms, perhaps through initiation of inflammationresponses involved in recruiting phagocytic cells (16, 66). PGsand eicosanoids in general regulate both proinflammatory andanti-inflammatory responses of the immune system. A singlePG molecule can have pleiotropic effects due to the existenceof numerous receptors for each lipid species, and in turn, thesereceptors can elicit different responses on different cell types(18, 20). A decrease in PG signaling might lead to a decreaseor slower response time in these host-mounted defenses.Finally, considering the detectable macroscopic reaction ofAspergillus spp. to exogenously applied arachidonic acid andPGE2, we propose that Aspergillus (and other eukaryoticpathogens) may share similar oxylipin signaling pathways withhost cells. PGs are transported out of cells and interact withcell surface receptors linked to G proteins to initiate appro-priate signaling pathways in mammalian cells (26). Our cen-tral hypothesis is that fungal oxylipins, similarly to the en-dogenous mammalian PGs, have the potential to mediateinterkingdom signaling via a cross talk communication inwhich the pathogen triggers the host defense immune re-sponses at the site of infection by binding to mammalian G

FIG. 10. Hypothetical model depicting Aspergillus-host oxylipin signaling. Ppo enzymes are putative cyclooxygenase-like enzymes generatingdifferent oxylipin species, including prostaglandins. We propose that oxylipins either directly regulate developmental and virulence pathways in thefungal cell or exit the cell through the mediation of specific transporters. The oxylipins outside the cell act as autocrine or paracrine ligands thatbind and sensitize G protein-coupled receptors, activating downstream signaling cascades. This signaling activation triggers the upregulation of themammalian immune system to retard the fungal pathogenenesis. Ppo, psi-producing oxygenases; LTs, leukotrienes; LOX, lipoxygenase.

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protein receptors; this process may result in the retardationof pathogenesis (Fig. 10).

ACKNOWLEDGMENTS

This work was funded by NSF MCB-0196233 to N.P.K. and a No-vartis (Syngenta) Crop Protection Graduate Fellowship to D.I.T.

We thank Courtney Jahn for experimental help and Thomas Ham-mond for providing the plasmid vector pTMH44.2. Genomic data forA. fumigatus were provided by The Institute for Genomic Research(www.tigr.org/tdb/e2k1/afu1) and The Wellcome Trust, Sanger Insti-tute (www.sanger.ac.uk/Projects/A_fumigatus); genomic data for A.nidulans were provided by The Broad Institute (www.broad.mit.edu/annotation/fungi/aspergillus/). Coordination of analyses of these datawas enabled by an international collaboration involving more than 50institutions from 10 countries and coordinated from Manchester,United Kingdom (www.cadre.man.ac.uk and www.aspergillus.man.ac.uk).

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