Aspergillus Cyclooxygenase-Like Enzymes Are Associated with Prostaglandin Production and Virulence
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感染与免疫杂志 2005年第8期
University of Wisconsin—Madison, Madison, Wisconsin
Center for Microbial Biotechnology, Technical University of Denmark, Lyngby, Denmark
ABSTRACT
Oxylipins comprise a family of oxygenated fatty acid-derived signaling molecules that initiate critical biological 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 is theorized 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 is decreased 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 murine model system and showed increased tolerance to H2O2 stress relative to that of the wild type. We propose that Ppo products, PG, and/or other oxylipins may serve as activators of mammalian immune responses contributing to enhanced resistance to opportunistic fungi and as factors that modulate fungal development contributing to resistance to host defenses.
INTRODUCTION
Aspergillus spp. are opportunistic pathogens with a worldwide distribution. These organisms can produce a wide spectrum of diseases in both plants and animals. Invasive pulmonary infection with the propensity to disseminate to other end organs represents the most common and lethal disease state in humans. Aspergillus spp. also are causal agents of fungal sinusitis, asthma, and allergic alveolitis in nonimmunosuppressed patients. The predominant pathogenic species is Aspergillus fumigatus, accounting for up to 90% of invasive human infections (7, 37). However, several other Aspergillus spp. including the genetic model A. nidulans, can infect immunocompromised patients and exacerbate preexisting diseases (38, 52). The clinical significance of A. fumigatus and the importance of A. nidulans as a fungal model system justified prioritization for genome sequencing, which was recently completed for both species (14).
Aspergillus infections are commonly initiated by inhalation of the airborne asexual spores called conidia. In the case of A. fumigatus, the small size (3 to 5 μm) of conidia enables them to reach the pulmonary alveoli, the main site of infection. If the spores survive in the alveoli, e.g., in the absence of an adequate host immune response, they germinate and propagate in vivo, leading to disseminated invasion by the fungus of other critical organs within the host. This phase of the disease, known as invasive 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 understanding of the mechanisms underlying Aspergillus pathogenesis. The current view is that A. fumigatus pathogenicity is dependent on the production of ill-defined fungal proteins and toxins that promote mycelial growth in IA and on structural features of the conidia, e.g., pigmentation, that confer resistance to the host'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 interactions. Oxylipins encompass a large group of oxygenated C18, C20, and C22 bioactive lipids derived from 3 (n-3) and 6 (n-6) polyunsaturated fatty acids (22, 42). Eicosanoids comprise a subclass of C20 oxylipins derived from dihomo--linolenic acid, arachidonic acid (AA), and eicosapentaenoic acid, including the prostaglandins (PGs) and leukotrienes, which act as "local short-range hormones" in maintaining local homeostasis in a variety of tissues and cells (18). Eicosanoids are critically involved in mammalian immune responses such as regulation of inflammation, pain, fever, and allergic responses, as well as regulation of the cardiovascular system, reproduction, and renal function, and might play a role in carcinogenesis (18, 54, 67). Mammalian prostaglandin synthases, and subsequent PG production, are activated by mechanical trauma or by specific growth factors, cytokines, and other abiotic or biotic stimuli, including pathogen invasion (18). A single eicosanoid can have pleiotropic effects due to the existence of multiple receptors for each lipid species. In turn, these receptors have different effects on different cell types (18, 20). Host production of PGs upon infection is well documented (18); however, recent studies suggest that PG production by eukaryotic microbes could be contributing to the infection process (23, 42, 44). The potential link between pathogen eicosanoids and modulation of host immunity is intriguing and potentially a target for future pharmaceuticals (42).
Initial efforts to elucidate an oxylipin biosynthetic pathway in fungi stemmed from interest in deciphering potential oxylipin-driven cross-communication between plants and mycotoxigenic aspergilli (8, 64). Studies of the genetic model A. nidulans resulted in the characterization of three dioxygenase-encoding genes, ppoA, ppoB, and ppoC, required for biosynthesis of oleic and linoleic acid-derived oxylipins similar in structure to plant defense molecules and important in integrating asexual and sexual spore balance in A. nidulans (58-61). Because studies have established that oxylipin-generating enzymes exhibit activity toward more than one substrate, and all three of these putative dioxygenases showed high homology to mammalian cyclooxygenases (COX) (prostaglandin H synthases), we have here investigated the possibility that fungal Ppo proteins could be involved in PG biosynthesis. We show that both A. nidulans and A. fumigatus ppo genes contribute to PG production in these species. The A. fumigatus PG mutant showed increased virulence in a murine model of pulmonary aspergillosis and enhanced resistance to environmental stress. We suggest the possibility of oxylipins as cross talk bioactive lipids that induce host defense mechanisms important in retarding the development of pulmonary and invasive aspergillosis.
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 were grown at 37°C and stored as glycerol stocks. Appropriate supplements corresponding to the auxotrophic markers were added to the media as required. Sexual crosses and fungal transformation through polyethylene glycol-mediated fusion of protoplasts were conducted according to standard techniques for both Aspergillus species (4). RDIT64.2 (ppoA ppoB) is a recombinant strain resulting 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 and TTMK2.60. The A. nidulans ppoA ppoB ppoC triple mutant was created by a sexual cross between RDIT54.13 and TTMK2.60 (58). All strains were maintained 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 in 100 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, maintenance, and isolation of recombinant plasmids (51). Fungal chromosomal DNA was isolated and analyzed from lyophilized mycelia by using previously described techniques (65). Cultures for RNA extractions were grown by inoculating 30 ml of liquid GMM with 1 x 106 spores/ml of the appropriate strain before incubation 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. Approximately 20 μg of total RNA was used for Northern blot analysis using a 1.2% agarose-1.5% formaldehyde gel transferred to a Hybond-XL membrane (Amersham Pharmacia Biotech). Expression studies for the different genes were performed with appropriate probes. Probes for A. fumigatus were generated from genomic 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, and ppoC2f (5'-ATCCAAGCGCACGTTCGCCG-3') and ppoC2r (5'-TGAACTCCTTGCTGGCCTTTCC-3') for AfppoC. Nucleotide sequences were analyzed and compared 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 previously cloned, sequenced, and disrupted by replacement with marker genes: ppoA was replaced with metG, ppoB with pyroA, and ppoC with trpC (58, 59, 61). Briefly, disruption vectors were constructed by flanking the A. nidulans marker genes (metG, pyroA, and trpC) with 1-kb DNA fragments from upstream of the corresponding ppo gene start codon and downstream of the corresponding ppo stop codon. Double and triple ppo mutants were created by sexual crosses.
Aspergillus fumigatus AfppoA, AfppoB, and AfppoC genes were obtained from the TIGR database (http://www.tigr.org/tdb/e2k1/afu1/) based on a homologous search using A. nidulans ppoA, ppoB, and ppoC sequences. The polymerase Thermal ACE (Invitrogen Co.) was used for PCR amplifications. RNA interference (RNAi) technology was used to create an A. fumigatus vector that would silence expression of all three ppo genes (AfppoA, AfppoB, and AfppoC) simultaneously by sequentially arranging segments of each gene in both a forward and a 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 combinations with the indicated restriction enzyme sites introduced. The primers used were PpoAf (5'-CTTCGGCGCGCCATGGATCCCGATAGAGGGCCTTGCCCATC-3'), PpoAr (5'-CCCTCATATGATTGTGGAAGACGCGAAAGAGT-3'), PpoBf (5'-GGCTCATATGCGCGAAATATCCACCTGGTTT-3'), PpoBr (5'-TCAAGCATGCAAACCTGACGAACTGGGG-3'), PpoCf (5'-CCCAGCATGCACAAGACCTCTGGTTACTTGGA-3'), and PpoCr (5'-TAATCCATGGCGGCCGCAGGGTATCCAGCTGCGT-3') (f, forward; r, reverse). The ppoA PCR product was digested by NdeI, the ppoB PCR product by NdeI-SphI, and the ppoC PCR product by SphI. The three ppo fragments were ligated together, and the ligation mixture was used as a template to obtain a 1.5-kb PCR product (AfppoA-ppoB-ppoC) using the primer pair PpoAf-PpoCr. This 1.5-kb PCR product was initially digested with AscI and NcoI and ligated into the corresponding sites of pTMH44.2 (19) to generate plasmid pCEJ1. Digestion of pCEJ1 with BamHI and NotI yielded once more the 1.5-kb triple-ppo PCR product, which was further ligated into the corresponding sites of pTMH44.2 (19) in a forward orientation to yield the pCEJ2.7 vector. Next, the 1.5-kb AscI-NcoI fragment was released from pCEJ1, allowing it to be placed in the AscI-NcoI site of pCEJ2.7 in a reverse orientation, to create the pCEJ2.7.4 vector. An internal 280-bp spacer green fluorescent protein fragment separated the inverted repeats, and the A. nidulans gpdA promoter, which has been successfully used in different fungal systems for high levels of transcription (48), drove the transgene. The Aspergillus parasiticus pyrG gene (pBZ5) (53) was inserted into an EcoRI site of pCEJ2.7.4 to give pJW66.3 (Fig. 1). This final plasmid was used for transformation of AF293.1 to silence expression of AfppoA, AfppoB, and AfppoC in A. fumigatus.
Prostaglandin analysis. A. nidulans and A. fumigatus wild-type and ppo mutant strains were grown for 7 days at 37°C in RPMI medium (a defined medium devoid of fatty acids; Sigma Chemical Co.) with shaking at 300 rpm. After 7 days the cultures were incubated for an additional 2 h with 1 mM AA (Cayman Chemicals, Ann Arbor, Mich.). Culture supernatants from both AA-fed and non-AA-fed fungi were analyzed for prostaglandin production by using an enzyme-linked immunosorbent assay (ELISA) kit (catalog no. 514012; Cayman Chemicals) according to the manufacturer's instructions. The antiserum used in this assay exhibits high cross-reactivity for most PGs, allowing quantification of PGE1, PGE2, PGF1, PGF2 (100% specificity), PGF3, PGD2, PGE3 (<50% specificity), and thromboxane B2 (TXB2; 5% specificity). It does not detect PGA, PGB1, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2, or misopristol. The cultures without AA measure the endogenous production of PGs in the absence of exogenous fatty acid substrates. Student's t test and analysis of variance were used to analyze the significance of differences between the experimental groups using the Statistical Analysis System (SAS Institute, Cary, NC).
Physiological studies. Conidium production studies for wild-type A. fumigatus and Afppo IRT mutant strains were performed on plates containing 30 ml of solid 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 each plate. Cultures were incubated in continuous dark at 37°C. A core with a diameter of 12.5 mm was removed from each plate after 2, 4, and 6 days and was homogenized for 1 min in 3 ml of sterile water supplemented with 0.01% Tween 80 to release the spores. Spores were counted using a hemacytometer. The experiments were performed with four replicates. Radial growth tests were performed in triplicate with approximately 104 conidia centered on 30-ml GMM plates, and growth rates were recorded as colony diameter over time at five temperature 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 h at 300 rpm and 37°C. Samples were examined at 2-h intervals, and the germination rate was determined by counting 100 conidia. The mycelial weight of lyophilized tissue was assessed after 4 days of culture in liquid GMM. Physiological 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 study included arachidonic acid (20:4) and PGE2 obtained from Cayman Chemicals Co. Amounts of 0.1 mg and 1 mg were dissolved in 50 μl of methanol and dried on 12.5-mm-diameter paper filter disks. A paper filter disk treated with methanol was used as the solvent control. After drying, the fatty acid-containing disks and the methanol-containing disks were laid on the agar surfaces of plates containing 30 ml of solid 1.5% YGT medium (9) with a 5-ml top layer consisting of cool melted 0.7% agar-YGT containing 105 conidia of the wild-type A. nidulans (RDIT9.32) or A. fumigatus (AF293) strain. The cultures were incubated under light and dark conditions for 8 days.
Stress tolerance assays. Determination of tolerance levels against heat and oxidative stress was performed as previously described (10, 31). For the thermal tolerance assay, wild-type or Afppo IRT strain conidia were inoculated on solid GMM 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 for an additional 36 h at 37°C. Surviving colonies were counted. For the hydrogen peroxide 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 spore suspension was then diluted with sterile distilled water, and conidia were inoculated on solid GMM. After incubation at 37°C for 36 h, colony numbers were counted and calculated as a percentage of the control (10). For the assay of hyphal sensitivity to H2O2, plates containing 50 30-h-grown colonies were overlaid with 10 ml of 0, 50, 100, and 200 mM H2O2 solutions. After a 10-min incubation at room temperature, the H2O2 solution was removed, and the plates were washed twice with sterile distilled water and incubated further for 24 h at 37°C. The number of colonies that survived was calculated as a percentage of the control. All the experiments were performed in triplicate.
Animal model of Aspergillus infection. The virulence of isogenic wild-type and Afppo IRT strains was studied in a lung infection model, with the approval of the University of Wisconsin animal care committee. Conidia were harvested by flooding of fungal colonies with 0.85% NaCl with Tween 80, enumerated with a hemacytometer, 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 on GMM plates. Six-week-old outbred Swiss ICR mice (Harlan Sprague-Dawley) weighing 24 to 27 g were immunosuppressed by intraperitoneal injection of cyclophosphamide (100 mg/kg of body weight) on days –4, –1, and 3 and with a single dose of cortisone acetate (200 mg/kg). Mice were anesthetized via halothane inhalation in a bell jar at day 0. Sedated mice (10 mice/fungal strain) were infected by nasal instillation of 50 μl of the inoculum (day 1) and monitored three times daily for 7 days postinfection. All surviving mice were sacrificed at day 7. The tissue fungal burden of a whole-lung homogenate was quantified by serial dilution and enumeration of CFU (CFU/2 lungs). The duration of survival (in days after inoculation) was recorded for each animal. Moribund animals were sacrificed and cumulative survival recorded. Survival and clearance of residual fungal burden in tissue (CFU/2 lungs) were used as the outcome variables to assess the relative virulence of isogenic strains.
Mouse lung metabolite analysis. Chloroform extracts from mouse lungs were redissolved in 400 μl methanol (high-performance liquid chromatography grade) and loaded onto a Strata X (Phenomenex, Torrance, Ca) 60-mg SPE column already containing 3 ml water (Milli-Q). The SPE column had previously been sequentially activated with 2 ml methanol and 2 ml water. After the sample was loaded, the column was washed with 1 ml water, and the sample was eluted with 4 ml methanol-water (9:1) containing 0.5% formic acid (analytically pure). The samples were then evaporated in vacuo on a SpeedVac, redissolved twice in 50 μl methanol, and filtered through a 4-mm-diameter 0.45-μm-pore-size polytetrafluoroethylene (Teflon) syringe filter. Samples (3 μl) were then analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HR-MS) on an Agilent 1100 LC system equipped with a UV photo diode array detector and coupled to an LCT orthogonal time-of-flight mass spectrometer (Waters-Micromass, Manchester,United Kingdom) (40). Separation was performed on a Phenomenex (Torrance, CA) Luna II C18 (II) column (50 by 2 mm; inner diameter, 3 μm) using a water-acetonitrile system at a flow rate of 0.3 ml/min, starting at 15% acetonitrile, increasing the concentration linearly to 100% in 20 min, and holding at 100% for 5 min. The water was buffered with 10 mM ammonium formate and 20 mM formic acid (both analytical grade) and the acetonitrile with 20 mM formic acid. Gliotoxin and other metabolites were identified by comparison to reference standards (40) of known A. fumigatus metabolites (11, 12) and extracts from agar cultures, based on their retention time, UV spectra, and positive electrospray spectra (ESI+). For secondary metabolites reported from A. fumigatus (11, 12) but not available to us as standards, we used their known [M+H]+ ions as a basis for detecting them by selected ion chromatograms. The secondary 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; fumiquinazolines A to E; and tryptoquivalines A to H.
RESULTS
A. nidulans and A. fumigatus ppo genes encode cyclooxygenase-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 with the biochemically characterized oxylipin-producing linoleate diol synthase (lds) gene from the filamentous fungus Gaeumannomyces graminis (GenBank accession no. AF124979), horse COX-2 (GenBank accession no. O19183), and human COX-1 (GenBank accession no. P23219) revealed the presence of three genes named ppoA, ppoB, and ppoC in both aspergilli (A. fumigatus genes are distinguished by the "Af" prefix). Disruption of ppoA (AY502073) (61), ppoC (AY613780) (59), and ppoB (AY940146) (58) in A. nidulans led to strains defective in producing monohydroxy linoleic and oleic acid-derived oxylipins. Protein domain searches against the PFAM database (http://pfam.wustl.edu) indicated that both A. nidulans Ppo proteins and A. fumigatus Ppo proteins AfPpoA (58.m07572), AfPpoB (67.m03008), and AfPpoC (59.m09493) have domains similar to those of animal heme peroxidases and cytochrome P450 oxygenases. Comparative sequence analysis (ClustalW) between the predicted Ppo amino acid sequences of the two species led to the phylogenetic tree shown in Fig. 2A and the corresponding comparison values (Fig. 2B). AfPpoA showed 48% and 47% identities with AfPpoB and AfPpoC, respectively, whereas AfPpoB and AfPpoC showed 56% identity.
The amino acid sequences of A. nidulans and A. fumigatus Ppo proteins also revealed similarity with various mammalian COX, 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 prostaglandin PGH2 (54). PGH2 is converted to the biologically active end product PGD2, PGE2, PGF2, PGI2, or TxA2 (thromboxane A2) by other specific prostaglandin synthases (18, 54). Homology between A. nidulans and A. fumigatus Ppo and mammalian COX amino acid sequences over the conserved catalytic domains ranged from 25% to 29% identity and 40% to 45% similarity for COX-2 paralogs (E values, 10–24 to 10–18) and 25% to 26% identity and 38% to 40% similarity for COX-1 paralogs (E values, 2 x 10–18 to 5 x 10–18).
A more detailed sequence alignment of the Aspergillus Ppo sequences with horse COX-2 and human COX-1 was performed with the ClustalW program to identify conserved functional motifs. The sequence similarity appeared to be restricted to the catalytic domain of COX and was most striking along the -helices (predicted to be present in Ppo proteins by using the PredictProtein program, available at http://cubic.bioc.columbia.edu/predictprotein/) as shown in Fig. 3A. Structural homology shows that both the distal and proximal His heme ligands and the important Tyr residue, which are required for enzyme activity and are completely conserved within the COX family, aligned in context with identical amino acids of A. nidulans PpoC, A. nidulans PpoA, and A. fumigatus AfPpoA but not with the other Aspergillus proteins. The core helix H2 harbors the distal His heme ligand (consensus THXXFXT), and the core helix H8 contains the proximal heme ligand and the important Tyr residue (consensus EFNXXYXWH) of PGH synthases (54). In contrast to the structural conservation found within the catalytic domain, the regions of COX falling outside of this domain, that is, the epidermal growth factor-like domain and the membrane-binding domain, do not seem to have equivalent residues in the Ppo proteins. G. graminis Lds also has similar conserved regions in its sequence, as was previously reported (24); however, the biochemical involvement of Lds in PG 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 development and virulence, a gene-silencing approach was undertaken to inactivate the three genes simultaneously. The absence of a sexual cycle in A. fumigatus renders the incorporation of many deletions in the same individual a difficult task in this species. To evaluate the potential of RNAi technology as a means of silencing multiple genes in A. fumigatus, a vector was designed to simultaneously produce AfppoA, AfppoB, and AfppoC double-stranded RNA molecules in the fungal thallus (Fig. 1). Double-stranded RNA molecules are processed by RNAi machinery to produce small interfering RNA (siRNA) molecules that 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 two genes within each vector (19, 39).
The triple Afppo mutant (Afppo IRT) was created by transformation of strain 293.1 with pJW66. PCR and Southern blot analysis of 85 transformants revealed the introduction of the IRT Afppo construct in five transformants. Macroscopically, all five transformants were identical to each other and to the wild type. Transformants TJW62.2, TJW62.5, and TJW62.10 and the wild-type AF293 were selected for mouse virulence studies (see below); TJW62.2, TJW62.10, and AF293 were used for ELISA analysis (see below); and TJW62.2 was used for further physiological and molecular analyses. As predicted, the A. fumigatus transformants, which incorporated the Afppo IRT plasmid into their genome, displayed an Afppo silencing phenotype, as monitored by expression studies of the three ppo genes. Figure 4 shows that in contrast to the wild type, all three genes 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. Like the ppoA ppoB ppoC mutant of A. nidulans, the Afppo IRT mutant had no alterations in vegetative development or spore germination relative to the wild type in liquid GMM. However, in contrast to the A. nidulans ppoA ppoB ppoC strain, which demonstrated a significant reduction in asexual spore production and a significant increase in sexual spore production (58), spore counts of the A. fumigatus triple ppo mutant did not show any alterations in asexual spore production 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 growth experiments on solid GMM with glucose as the sole carbon source 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 there is no difference at 37°C.
Ppo mediation of prostaglandin production in A. nidulans and A. fumigatus. One primary goal of this study was to determine if any of the ppo genes could be involved in PG production. Recent reports indicate that several fungi can utilize exogenous sources of arachidonic acid to produce a number of different eicosanoids (44, 45); however, no candidate enzyme has been uncovered. By following procedures used by other labs in establishing that 90% of fungal PGs are secreted (36, 45), supernatants from Aspergillus strains grown in RPMI medium were examined for PG production. Dry weights of recovered mycelium were identical, but there was a decrease in production of PGs in A. nidulans strains carrying a ppoA allele (14%) and in all strains carrying the ppoC allele (36% to 37%) (Fig. 5A) (P < 0.01). These data suggest that PpoC and PpoA are involved in PG production by A. nidulans. Examination of two IRT Afppo mutants (TJW62.2 and TJW62.10) showed that both mutants had a 12% to 15% reduction in PG biosynthesis (Fig. 5B) (P < 0.01). The amount of PGs that was detected in non-AA-fed cultures of both aspergilli was below the assay's threshold of detection (40 pg/ml).
Increased virulence of Afppo IRT mutants in a murine model of pulmonary aspergillosis. The relative virulence of the wild type and three Afppo IRT mutants (TJW62.2, TJW62.5, and TJW62.10) was evaluated in a murine pulmonary infection model where mice were monitored for survival on a daily basis (Fig. 6). The Afppo IRT strains caused an increased fatal infection rate in the murine model (P, 0.02 to >0.001) relative to the wild type. By day 3 postinoculation, mice infected with the Afppo IRT strains began to die, with a 90 to 100% mortality rate reached by day 5 for TJW62.2 and TJW62.10 and a 50% mortality rate by day 5 for TJW62.5. In contrast, the wild type showed a 30% mortality rate at day 5. By day 6 none of the mice treated with TJW62.2 or TJW62.10 survived, whereas 50% of those infected with the wild type were still alive. The virulence experiment was repeated twice with similar results. From all mice culled, fungal colonies were recovered from the lungs, indicating that the pulmonary distress was due to aspergillosis. No statistical differences were observed in the number of CFU recovered from the lungs of wild-type- or Afppo IRT-infected mice sacrificed 3 days after inoculation (data not shown). The isolated fungal colonies from each group of mouse lungs were confirmed as wild type or Afppo IRT by diagnostic PCR and Southern blot analysis (data not shown).
Given the numerous studies implicating gliotoxin as a potent virulence factor in aspergillosis (41), five lungs from sacrificed mice were extracted and analyzed for gliotoxin content 3 and 4 days after infection with 107 wild-type (AF293) or Afppo IRT (TJW 62.2) conidia. LC-HR-MS (Fig. 7) showed no differences in the quantities of gliotoxin detected between the wild type and an Afppo IRT mutant. Gliotoxin was unambiguously detected at the same retention time in a reference standard, and both HR-MS and UV spectra were correct. Noninfected mouse lungs showed no detectable levels of gliotoxin (Fig. 7). No other gliotoxin analogs or secondary metabolites normally produced during in vitro culture of A. fumigatus (e.g., helvolic acid, fumigaclavines A to C, tryptoquivalines A to H, fumiquinazolines A to E, pseurotins A to F2, fumagillin, fumitremorgins A to C, verrucologen, or TR-2) could be detected in the wild-type- or Afppo IRT-infected lung tissue. Most of these compounds ionize significantly better than gliotoxin and would thus have much lower detection limits (40), showing that gliotoxin is the primary fungal metabolite produced in the lungs.
Increased stress sensitivity of the A. fumigatus triple ppo mutant. Although we could not detect any difference in spore pigmentation and gliotoxin production (both implicated as virulence factors) that could explain the increased virulence of the Afppo IRT mutant, we considered the possibility that mechanisms of resistance to host defenses might be altered in the Afppo IRT mutant. To test this hypothesis, the Afppo IRT mutant was subjected to oxidative and thermal stress. By following procedures established for A. fumigatus and A. nidulans (30, 47), both conidia and hyphae were exposed to increasing levels of H2O2, a treatment designed to mimic exposure to host reactive oxygen species (ROS), typically activated during pathogen ingress. Germination and survival rates of spores in varying concentrations of H2O2 were determined after 24 h at 37°C. As shown in Fig. 8, conidia of the Afppo IRT mutant showed a statistically significant 15-to-20% increase (P < 0.05) in survival at 20, 40, and 80 mM of H2O2 relative to the wild type, which showed a steady decrease in oxidative tolerance. No colonies of either strain survived the 250 mM H2O2 treatment. In contrast to the increased resistance of conidia to H2O2 treatment, Afppo IRT hyphae were not more resistant to H2O2 than wild-type hyphae.
The resistance of the Afppo IRT mutant to thermal stress was evaluated by treating germlings at 50°C for 0, 3, and 4 h and determining their ability to form viable colonies at 37°C. The wild type showed mean decreases of 10% and 93% in colony survival, whereas the Afppo IRT mutant showed 1% (P < 0.1) and 87% (P < 0.1) decreases, at 3 h and 4 h, respectively. Based on these data, Ppo proteins and/or their products, among several other possible roles, might mediate signaling cascades that regulate heat and oxidative stress responses at different developmental stages of the fungus.
Effects of arachidonic acid and PGE2 on Aspergillus physiology. Earlier studies in our lab showed that C18 unsaturated fatty acids and their derived oxylipins induced developmental changes in A. nidulans including changes in sexual-to-asexual spore ratios and secondary metabolite production (8, 9). Similar studies have shown that PGs can induce developmental changes in Candida albicans (28, 43). We therefore thought it possible that PGs and their progenitor, arachidonic acid, might affect Aspergillus physiological processes.
Wild-type A. fumigatus and A. nidulans cultures were examined for reactions to PGE2 and arachidonic acid. The addition of PGE2 to A. nidulans cultures inhibited the formation of conidia, in sharp contrast to arachidonic acid, which significantly induced conidiation (visualized as a green halo of conidia around the disk) (Fig. 9). These results were reminiscent of the differential reaction of A. nidulans to the plant oxylipin 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) and its precursor, linoleic acid, where the former induced conidial formation at 0.1 mg and the latter inhibited conidiation at the same concentration (9). Neither PGE2 nor arachidonic acid affected sporulation in A. fumigatus; however, they both inhibited the production of hyphal pigments (Fig. 9).
DISCUSSION
Oxylipin production is widespread among fungi, and progress has recently been made in identification, regulation, and cellular localization of the dioxygenases generating fungal oxylipins (17, 22, 33, 42, 58, 59, 61). In this study, we provide the first genetic evidence for fungi that Ppo proteins similar in sequence to mammalian COX are involved in the production of prostaglandins, a major group of oxylipins that regulate immune responses in mammals. Silencing of the three ppo genes in the human pathogen A. fumigatus yielded hypervirulent strains in the mouse pulmonary model system. These studies extend the involvement of oxylipins in aspergilli from control of developmental and metabolic functions, such as spore balance and fatty acid regulation (42, 58, 59, 61), to virulence in a host-pathogen interaction.
The types of oxylipins produced by fungi are numerous, and likely many remain to be characterized. Although many studies have focused on C18 oxylipins, several experiments have revealed that both pathogenic and nonpathogenic fungal species produce detectable amounts of both C20 cyclooxygenase and lipoxygenase products (36, 44, 45). The arachidonic acid metabolites PGF2 and PGF2-lactone have been detected in a number of environmental yeasts of the family Lipomycetaceae (Dipodascopsis, Lipomyces, Myxozyma, and Zygozyma) (22, 33, 34, 55) as well as in Saccharomyces cerevisiae (33). The pathogenic yeasts Cryptococcus neoformans and Candida albicans and the filamentous fungus A. fumigatus produce both PGs and leukotrienes, and their amounts are significantly increased after exogenous application of arachidonic acid (44, 45). Furthermore, COX inhibitors, including aspirin and other nonsteroidal anti-inflammatory drugs, inhibited hydroxyeicosa-tertraenoic acid, PGE2, and PGD2 production in several members of the family Lipomycetaceae, supporting the view that a COX-like enzyme is active in these fungi (5, 32, 36). However, no candidate protein has been identified in fungi prior to this report.
The structural similarity between Ppo proteins and COX led us to investigate the possibility that these enzymes could be involved in PG production. COX possesses two enzymatic activities, a cyclooxygenase that catalyzes the oxygenation of polyunsaturated substrates, such as arachidonic acid, to form prostaglandin G2 (PGG2) and a peroxidase that can use a variety of electron donors to reduce PGG2 to form prostaglandin H2 (PGH2) (54). The amino acid sequences of Ppo proteins are predicted to contain both an oxygenase and a peroxidase domain (58, 59, 61). Further detailed comparison of the amino acid sequences of all six Ppo proteins with human and horse COX revealed that three of the proteins, A. nidulans PpoC and PpoA and A. fumigatus PpoA, contained the conserved catalytic residues found in COX -helices, which included the proximal and distal heme ligands and the critical tyrosine residue of PGH synthases (24, 54). Considering the correlation of loss of PG activity in the respective A. nidulans ppoC and ppoA mutants (Fig. 5) and presence of catalytic residues, it is tempting to speculate that these amino acids are indicators of possible PG activity in fungi. Investigation of single A. fumigatus Ppo mutants may shed further light on the viability of this observation.
Through sexual genetics, we were able to analyze every possible combination of ppo mutant background in A. nidulans. The consistent decrease in PG production in all combinations of the ppoC allele suggests a major role for this enzyme in PG biosynthesis (Fig. 5A). The slight increase in production of PGs in ppoB and ppoA ppoB strains correlated with an upregulation in the expression level of ppoC (58, 59), further implicating PpoC in the synthesis of PGs. Previous biochemical data has shown that PpoC is also likely involved in the production of oleic acid-derived oxylipins (59). The ability of a fatty acid oxygenase to utilize different fatty acids as substrates is not uncommon. For instance, the G. graminis Lds can oxygenate 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 involves considerable effort and has not been reported to date. To address this issue, we attempted to silence all three Afppo genes by using one vector. As demonstrated by Northern blot (Fig. 4) and PG (Fig. 5B) analyses, this approach was successful. RNAi has emerged as an effective method for silencing gene expression in many eukaryotes and has recently been used successfully in genome-wide functional tests (2, 29); we believe this method will greatly help in further genomic studies of A. fumigatus. Caveats with this method are (i) the potential of off-target effects by the siRNAs that could jeopardize correct interpretation of gene function and (ii) the fact that some transcripts are still produced and not completely eliminated, unlike the situation in traditional gene replacement, as illustrated in Fig. 4. The fact that PG reduction in the Afppo IRT strain is not as pronounced as in the A. nidulans triple mutant could well be a reflection of this incomplete transcript suppression. Additionally, the presence of alternative enzymes or pathways for PG biosynthesis is expected, since both the A. nidulans triple mutant and the Afppo IRT strain still produced substantial amounts of PGs. It is also possible that levels of other eicosanoids are decreased in the ppo mutants, since our detection method was limited to specific PGs and did not cover the full array of known eicosanoids.
Despite a decrease of only 12% in PG production as measured by ELISA (Fig. 5B), the Afppo IRT strains displayed a significant increase in virulence in the murine pulmonary model (Fig. 6). To our knowledge this is the first report of a hypervirulent A. fumigatus mutant. Since a unique characteristic of mammalian PGs is their potency at very low (nanomolar) concentrations and their very short half-lives—they are produced de novo and act near the site of their synthesis—we speculate that the small decrease in the endogenous fungal PG levels can lead to a significant increase in the virulence of A. fumigatus (18). Two determinants implicated as virulence factors in A. fumigatus include spore pigmentation (6, 57) and gliotoxin production (41). Loss of pigmentation results in increased phagocytosis, whereas gliotoxin, a secondary metabolite produced in tissues of mice following development of invasive aspergillosis (15), is a potent immunomodulating agent and an inducer of apoptotic cell death in a number of cell types (62). Microscopic examination of Afppo IRT spores did not reveal any differences in pigmentation compared to the wild type. Chemical analysis indicated that gliotoxin production in mouse lungs by Afppo IRT strains was not altered from that by the wild type (Fig. 7). These factors, therefore, do not seem to play a role in the increased virulence of the Afppo IRT strains.
Physiological examination of one of the Afppo IRT strains did, however, indicate increased resistance to environmental stress as observed by H2O2 treatment (Fig. 8). H2O2 treatment of fungal propagules is an indirect method of measuring the putative resistance of the pathogen to host ROS, a major host antimicrobial effector system also active against Aspergillus conidia (63). ROS production in mammals occurs during the course of neutrophil and macrophage activation and is implicated in the defense against fungal pathogens (1, 49, 50). Increased resistance to a ROS-mounted defense could protect a pathogen and render it more virulent.
Although the hypothesis is not examined in this study, we also propose that part of the increased virulence of the Afppo IRT strains is due to changes in host physiology. A plausible explanation is that the Ppo-generated PGs enhance host defense mechanisms, perhaps through initiation of inflammation responses involved in recruiting phagocytic cells (16, 66). PGs and eicosanoids in general regulate both proinflammatory and anti-inflammatory responses of the immune system. A single PG molecule can have pleiotropic effects due to the existence of numerous receptors for each lipid species, and in turn, these receptors can elicit different responses on different cell types (18, 20). A decrease in PG signaling might lead to a decrease or slower response time in these host-mounted defenses. Finally, considering the detectable macroscopic reaction of Aspergillus spp. to exogenously applied arachidonic acid and PGE2, we propose that Aspergillus (and other eukaryotic pathogens) may share similar oxylipin signaling pathways with host cells. PGs are transported out of cells and interact with cell surface receptors linked to G proteins to initiate appropriate signaling pathways in mammalian cells (26). Our central hypothesis is that fungal oxylipins, similarly to the endogenous mammalian PGs, have the potential to mediate interkingdom signaling via a cross talk communication in which the pathogen triggers the host defense immune responses at the site of infection by binding to mammalian G protein receptors; this process may result in the retardation of pathogenesis (Fig. 10).
ACKNOWLEDGMENTS
This work was funded by NSF MCB-0196233 to N.P.K. and a Novartis (Syngenta) Crop Protection Graduate Fellowship to D.I.T.
We thank Courtney Jahn for experimental help and Thomas Hammond for providing the plasmid vector pTMH44.2. Genomic data for A. fumigatus were provided by The Institute for Genomic Research (www.tigr.org/tdb/e2k1/afu1) and The Wellcome Trust, Sanger Institute (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 data was enabled by an international collaboration involving more than 50 institutions from 10 countries and coordinated from Manchester, United Kingdom (www.cadre.man.ac.uk and www.aspergillus.man.ac.uk).
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, United Kingdom.
REFERENCES
1. Aratani, Y., F. Kura, H. Watanabe, H. Akagawa, Y. Takano, K. Suzuki, M. C. Dinauer, N. Maeda, and H. Koyama. 2002. Relative contributions of myeloperoxidase and NADPH-oxidase to the early host defense against pulmonary infections with Candida albicans and Aspergillus fumigatus. Med. Mycol. 40:557-563.
2. Ashrafi, K., F. Y. Chang, J. L. Watts, A. G. Fraser, R. S. Kamath, J. Ahringer, and G. Ruvkun. 2003. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421:268-272.
3. Bertout, S., C. Badoc, M. Mallie, J. Giaimis, and J. M. Bastide. 2002. Spore diffusate isolated from some strains of Aspergillus fumigatus inhibits phagocytosis by murine alveolar macrophages. FEMS Immunol. Med. Microbiol. 33:101-106.
4. Bok, J. W., and N. P. Keller. 2004. LaeA, a regulator of secondary metabolism in Aspergillus spp. Eukaryot. Cell 3:527-535.
5. Botha, A., J. L. Kock, and S. Nigam. 1997. The production of eicosanoid precursors by mucoralean fungi. Adv. Exp. Med. Biol. 433:227-229.
6. Brakhage, A. A., K. Langfelder, G. Wanner, A. Schmidt, and B. Jahn. 1999. Pigment biosynthesis and virulence. Contrib. Microbiol. 2:205-215.
7. Brookman, J. L., and D. W. Denning. 2000. Molecular genetics in Aspergillus fumigatus. Curr. Opin. Microbiol. 3:468-474.
8. Burow, G. B., T. C. Nesbitt, J. Dunlap, and N. P. Keller. 1997. Seed lipoxygenase products modulate Aspergillus mycotoxin biosynthesis. Mol. Plant-Microbe Interact. 10:380-387.
9. Calvo, A. M., L. L. Hinze, H. W. Gardner, and N. P. Keller. 1999. Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp. Appl. Environ. Microbiol. 65:3668-3673.
10. Chang, Y. C., B. H. Segal, S. M. Holland, G. F. Miller, and K. J. Kwon-Chung. 1998. Virulence of catalase-deficient Aspergillus nidulans in p47phox–/– mice. Implications for fungal pathogenicity and host defense in chronic granulomatous disease. J. Clin. Investig. 101:1843-1850.
11. Cole, R. J., B. B. Jarvis, and M. A. Schweikert. 2003. Handbook of secondary fungal metabolites, vol. III. Academic Press, Amsterdam, The Netherlands.
12. Cole, R. J., and M. A. Schweikert. 2003. Handbook of secondary fungal metabolites, vol. I and II. Academic Press, Amsterdam, The Netherlands.
13. Denli, A. M., and G. J. Hannon. 2003. RNAi: an ever-growing puzzle. Trends Biochem. Sci. 28:196-201.
14. Denning, D. W., M. J. Anderson, G. Turner, J. P. Latge, and J. W. Bennett. 2002. Sequencing the Aspergillus fumigatus genome. Lancet Infect. Dis. 2:251-253.
15. Eichner, R. D., P. Waring, A. M. Geue, A. W. Braithwaite, and A. Mullbacher. 1988. Gliotoxin causes oxidative damage to plasmid and cellular DNA. J. Biol. Chem. 263:3772-3777.
16. Filler, S. G., B. O. Ibe, A. S. Ibrahim, M. A. Ghannoum, J. U. Raj, and J. E. Edwards, Jr. 1994. Mechanisms by which Candida albicans induces endothelial cell prostaglandin synthesis. Infect. Immun. 62:1064-1069.
17. Fox, S. R., A. Akpinar, A. A. Prabhune, J. Friend, and C. Ratledge. 2000. The biosynthesis of oxylipins of linoleic and arachidonic acids by the sewage fungus Leptomitus lacteus, including the identification of 8R-hydroxy-9Z,12Z-octadecadienoic acid. Lipids 35:23-30.
18. Funk, C. D. 2001. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294:1871-1875.
19. Hammond, T. M., and N. P. Keller. 2005. RNA silencing in Aspergillus nidulans is independent of RNA dependent RNA polymerases. Genetics 169:607-617.
20. Hatae, N., Y. Sugimoto, and A. Ichikawa. 2002. Prostaglandin receptors: advances in the study of EP3 receptor signaling. J. Biochem. 131:781-784.
21. Herbrecht, R. 2002. Improving the outcome of invasive aspergillosis: new diagnostic tools and new therapeutic strategies. Ann. Hematol. 81(Suppl. 2):S52-S53.
22. Herman, R. P. 1998. Oxylipin production and action in fungi and related organisms, p. 115-130. In A. F. Rowley, H. Kuhn, and T. Schewe (ed.), Eicosanoids and related compounds in plants and animals. Princeton University Press, Princeton, N.J.
23. Herman, R. P., and C. A. Herman. 1985. Prostaglandins or prostaglandin like substances are implicated in normal growth and development in oomycetes. Prostaglandins 29:819-830.
24. Hornsten, L., C. Su, A. E. Osbourn, P. Garosi, U. Hellman, C. Wernstedt, and E. H. Oliw. 1999. Cloning of linoleate diol synthase reveals homology with prostaglandin H synthases. J. Biol. Chem. 274:28219-28224.
25. Jahn, B., F. Boukhallouk, J. Lotz, K. Langfelder, G. Wanner, and A. A. Brakhage. 2000. Interaction of human phagocytes with pigmentless Aspergillus conidia. Infect. Immun. 68:3736-3739.
26. Jump, D. B. 2002. Dietary polyunsaturated fatty acids and regulation of gene transcription. Curr. Opin. Lipidol. 13:155-164.
27. Kafer, E. 1977. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv. Genet. 19:33-131.
28. Kalo-Klein, A., and S. S. Witkin. 1990. Prostaglandin E2 enhances and gamma interferon inhibits germ tube formation in Candida albicans. Infect. Immun. 58:260-262.
29. Kamath, R. S., A. G. Fraser, Y. Dong, G. Poulin, R. Durbin, M. Gotta, A. Kanapin, N. Le Bot, S. Moreno, M. Sohrmann, D. P. Welchman, P. Zipperlen, and J. Ahringer. 2003. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421:231-237.
30. Kawasaki, L., and J. Aguirre. 2001. Multiple catalase genes are differentially regulated in Aspergillus nidulans. J. Bacteriol. 183:1434-1440.
31. Kawasaki, L., D. Wysong, R. Diamond, and J. Aguirre. 1997. Two divergent catalase genes are differentially regulated during Aspergillus nidulans development and oxidative stress. J. Bacteriol. 179:3284-3292.
32. Kock, J. L., D. J. Coetzee, M. S. van Dyk, M. Truscott, A. Botha, and O. P. Augustyn. 1992. Evidence for, and taxonomic value of, an arachidonic acid cascade in the Lipomycetaceae. Antonie Leeuwenhoek 62:251-259.
33. Kock, J. L., C. J. Strauss, C. H. Pohl, and S. Nigam. 2003. The distribution of 3-hydroxy oxylipins in fungi. Prostaglandins Other Lipid Mediat. 71:85-96.
34. Kock, J. L., P. Venter, D. Linke, T. Schewe, and S. Nigam. 1998. Biological dynamics and distribution of 3-hydroxy fatty acids in the yeast Dipodascopsis uninucleata as investigated by immunofluorescence microscopy. Evidence for a putative regulatory role in the sexual reproductive cycle. FEBS Lett. 427:345-348.
35. Kontoyiannis, D. P., and G. P. Bodey. 2002. Invasive aspergillosis in 2002: an update. Eur. J. Clin. Microbiol. Infect. Dis. 21:161-172.
36. Lamacka, M., and J. Sajbidor. 1998. The content of prostaglandins and their precursors in Mortierella and Cunninghamella species. Lett. Appl. Microbiol. 26:224-226.
37. Latge, J. P. 1999. Aspergillus fumigatus and aspergillosis. Clin. Microbiol. Rev. 12:310-350.
38. Marr, K. A., T. Patterson, and D. Denning. 2002. Aspergillosis. Pathogenesis, clinical manifestations, and therapy. Infect. Dis. Clin. N. Am. 16:875-894.
39. Mouyna, I., C. Henry, T. L. Doering, and J. P. Latge. 2004. Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol. Lett. 237:317-324.
40. Nielsen, K. F., and J. Smedsgaard. 2003. Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology. J. Chromatogr. A 1002:111-136.
41. Nieminen, S. M., J. Maki-Paakkanen, M. R. Hirvonen, M. Roponen, and A. von Wright. 2002. Genotoxicity of gliotoxin, a secondary metabolite of Aspergillus fumigatus, in a battery of short-term test systems. Mutat. Res. 520:161-170.
42. Noverr, M. C., J. R. Erb-Downward, and G. B. Huffnagle. 2003. Production of eicosanoids and other oxylipins by pathogenic eukaryotic microbes. Clin. Microbiol. Rev. 16:517-533.
43. Noverr, M. C., and G. B. Huffnagle. 2004. Regulation of Candida albicans morphogenesis by fatty acid metabolites. Infect. Immun. 72:6206-6210.
44. Noverr, M. C., S. M. Phare, G. B. Toews, M. J. Coffey, and G. B. Huffnagle. 2001. Pathogenic yeasts Cryptococcus neoformans and Candida albicans produce immunomodulatory prostaglandins. Infect. Immun. 69:2957-2963.
45. Noverr, M. C., G. B. Toews, and G. B. Huffnagle. 2002. Production of prostaglandins and leukotrienes by pathogenic fungi. Infect. Immun. 70:400-402.
46. Oren, I., and N. Goldstein. 2002. Invasive pulmonary aspergillosis. Curr. Opin. Pulm. Med. 8:195-200.
47. Paris, S., D. Wysong, J. P. Debeaupuis, K. Shibuya, B. Philippe, R. D. Diamond, and J. P. Latge. 2003. Catalases of Aspergillus fumigatus. Infect. Immun. 71:3551-3562.
48. Punt, P. J., N. D. Zegers, M. Busscher, P. H. Pouwels, and C. A. van den Hondel. 1991. Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene. J. Biotechnol. 17:19-33.
49. Roilides, E., C. A. Lyman, T. Sein, R. Petraitiene, and T. J. Walsh. 2003. Macrophage colony-stimulating factor enhances phagocytosis and oxidative burst of mononuclear phagocytes against Penicillium marneffei conidia. FEMS Immunol. Med. Microbiol. 36:19-26.
50. Roilides, E., K. Uhlig, D. Venzon, P. A. Pizzo, and T. J. Walsh. 1993. Enhancement of oxidative response and damage caused by human neutrophils to Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and gamma interferon. Infect. Immun. 61:1185-1193.
51. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
52. Segal, B. H., E. S. DeCarlo, K. J. Kwon-Chung, H. L. Malech, J. I. Gallin, and S. M. Holland. 1998. Aspergillus nidulans infection in chronic granulomatous disease. Medicine (Baltimore) 77:345-354.
53. Skory, C. D., P. K. Chang, J. Cary, and J. E. Linz. 1992. Isolation and characterization of a gene from Aspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis. Appl. Environ. Microbiol. 58:3527-3537.
54. Smith, W. L., D. L. DeWitt, and R. M. Garavito. 2000. Cyclooxygenases: structural, cellular, and molecular biology. Annu. Rev. Biochem. 69:145-182.
55. Strauss, T., A. Botha, J. L. Kock, I. Paul, D. P. Smith, D. Linke, T. Schewe, and S. Nigam. 2000. Mapping the distribution of 3-hydroxylipins in the Mucorales using immunofluorescence microscopy. Antonie Leeuwenhoek 78:39-42.
56. Su, C., and E. H. Oliw. 1996. Purification and characterization of linoleate 8-dioxygenase from the fungus Gaeumannomyces graminis as a novel hemoprotein. J. Biol. Chem. 271:14112-14118.
57. Tsai, H. F., Y. C. Chang, R. G. Washburn, M. H. Wheeler, and K. J. Kwon-Chung. 1998. The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence. J. Bacteriol. 180:3031-3038.
58. Tsitsigiannis, D. I., T. M. Kowieski, R. Zarnowski, and N. P. Keller. 2005. Three putative oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans. Microbiology 151:1809-1821.
59. Tsitsigiannis, D. I., T. M. Kowieski, R. Zarnowski, and N. P. Keller. 2004. Endogenous lipogenic regulators of spore balance in Aspergillus nidulans. Eukaryot. Cell 3:1398-1411.
60. Tsitsigiannis, D. I., R. A. Wilson, and N. P. Keller. 2002. Lipid mediated signaling in the Aspergillus/seed interaction, p. 186-191. In S. A. Leong, C. Allen, and E. W. Triplet (ed.), Biology of plant-microbe interactions, vol. 3. International Society for Plant-Microbe Interactions, St. Paul, Minn.
61. Tsitsigiannis, D. I., R. Zarnowski, and N. P. Keller. 2004. The lipid body protein, PpoA, coordinates sexual and asexual sporulation in Aspergillus nidulans. J. Biol. Chem. 279:11344-11353.
62. Waring, P., T. Khan, and A. Sjaarda. 1997. Apoptosis induced by gliotoxin is preceded by phosphorylation of histone H3 and enhanced sensitivity of chromatin to nuclease digestion. J. Biol. Chem. 272:17929-17936.
63. Washburn, R. G., J. I. Gallin, and J. E. Bennett. 1987. Oxidative killing of Aspergillus fumigatus proceeds by parallel myeloperoxidase-dependent and -independent pathways. Infect. Immun. 55:2088-2092.
64. Wilson, R. A., H. W. Gardner, and N. P. Keller. 2001. Cultivar-dependent expression of a maize lipoxygenase responsive to seed infesting fungi. Mol. Plant-Microbe Interact. 14:980-987.
65. Yelton, M. M., J. E. Hamer, and W. E. Timberlake. 1984. Transformation of Aspergillus nidulans by using a trpC plasmid. Proc. Natl. Acad. Sci. USA 81:1470-1474.
66. Yoshikai, Y. 2001. Roles of prostaglandins and leukotrienes in acute inflammation caused by bacterial infection. Curr. Opin. Infect. Dis. 14:257-263.
67. Zha, S., V. Yegnasubramanian, W. G. Nelson, W. B. Isaacs, and A. M. De Marzo. 2004. Cyclooxygenases in cancer: progress and perspective. Cancer Lett. 215:1-20.(Dimitrios I. Tsitsigianni)
Center for Microbial Biotechnology, Technical University of Denmark, Lyngby, Denmark
ABSTRACT
Oxylipins comprise a family of oxygenated fatty acid-derived signaling molecules that initiate critical biological 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 is theorized 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 is decreased 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 murine model system and showed increased tolerance to H2O2 stress relative to that of the wild type. We propose that Ppo products, PG, and/or other oxylipins may serve as activators of mammalian immune responses contributing to enhanced resistance to opportunistic fungi and as factors that modulate fungal development contributing to resistance to host defenses.
INTRODUCTION
Aspergillus spp. are opportunistic pathogens with a worldwide distribution. These organisms can produce a wide spectrum of diseases in both plants and animals. Invasive pulmonary infection with the propensity to disseminate to other end organs represents the most common and lethal disease state in humans. Aspergillus spp. also are causal agents of fungal sinusitis, asthma, and allergic alveolitis in nonimmunosuppressed patients. The predominant pathogenic species is Aspergillus fumigatus, accounting for up to 90% of invasive human infections (7, 37). However, several other Aspergillus spp. including the genetic model A. nidulans, can infect immunocompromised patients and exacerbate preexisting diseases (38, 52). The clinical significance of A. fumigatus and the importance of A. nidulans as a fungal model system justified prioritization for genome sequencing, which was recently completed for both species (14).
Aspergillus infections are commonly initiated by inhalation of the airborne asexual spores called conidia. In the case of A. fumigatus, the small size (3 to 5 μm) of conidia enables them to reach the pulmonary alveoli, the main site of infection. If the spores survive in the alveoli, e.g., in the absence of an adequate host immune response, they germinate and propagate in vivo, leading to disseminated invasion by the fungus of other critical organs within the host. This phase of the disease, known as invasive 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 understanding of the mechanisms underlying Aspergillus pathogenesis. The current view is that A. fumigatus pathogenicity is dependent on the production of ill-defined fungal proteins and toxins that promote mycelial growth in IA and on structural features of the conidia, e.g., pigmentation, that confer resistance to the host'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 interactions. Oxylipins encompass a large group of oxygenated C18, C20, and C22 bioactive lipids derived from 3 (n-3) and 6 (n-6) polyunsaturated fatty acids (22, 42). Eicosanoids comprise a subclass of C20 oxylipins derived from dihomo--linolenic acid, arachidonic acid (AA), and eicosapentaenoic acid, including the prostaglandins (PGs) and leukotrienes, which act as "local short-range hormones" in maintaining local homeostasis in a variety of tissues and cells (18). Eicosanoids are critically involved in mammalian immune responses such as regulation of inflammation, pain, fever, and allergic responses, as well as regulation of the cardiovascular system, reproduction, and renal function, and might play a role in carcinogenesis (18, 54, 67). Mammalian prostaglandin synthases, and subsequent PG production, are activated by mechanical trauma or by specific growth factors, cytokines, and other abiotic or biotic stimuli, including pathogen invasion (18). A single eicosanoid can have pleiotropic effects due to the existence of multiple receptors for each lipid species. In turn, these receptors have different effects on different cell types (18, 20). Host production of PGs upon infection is well documented (18); however, recent studies suggest that PG production by eukaryotic microbes could be contributing to the infection process (23, 42, 44). The potential link between pathogen eicosanoids and modulation of host immunity is intriguing and potentially a target for future pharmaceuticals (42).
Initial efforts to elucidate an oxylipin biosynthetic pathway in fungi stemmed from interest in deciphering potential oxylipin-driven cross-communication between plants and mycotoxigenic aspergilli (8, 64). Studies of the genetic model A. nidulans resulted in the characterization of three dioxygenase-encoding genes, ppoA, ppoB, and ppoC, required for biosynthesis of oleic and linoleic acid-derived oxylipins similar in structure to plant defense molecules and important in integrating asexual and sexual spore balance in A. nidulans (58-61). Because studies have established that oxylipin-generating enzymes exhibit activity toward more than one substrate, and all three of these putative dioxygenases showed high homology to mammalian cyclooxygenases (COX) (prostaglandin H synthases), we have here investigated the possibility that fungal Ppo proteins could be involved in PG biosynthesis. We show that both A. nidulans and A. fumigatus ppo genes contribute to PG production in these species. The A. fumigatus PG mutant showed increased virulence in a murine model of pulmonary aspergillosis and enhanced resistance to environmental stress. We suggest the possibility of oxylipins as cross talk bioactive lipids that induce host defense mechanisms important in retarding the development of pulmonary and invasive aspergillosis.
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 were grown at 37°C and stored as glycerol stocks. Appropriate supplements corresponding to the auxotrophic markers were added to the media as required. Sexual crosses and fungal transformation through polyethylene glycol-mediated fusion of protoplasts were conducted according to standard techniques for both Aspergillus species (4). RDIT64.2 (ppoA ppoB) is a recombinant strain resulting 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 and TTMK2.60. The A. nidulans ppoA ppoB ppoC triple mutant was created by a sexual cross between RDIT54.13 and TTMK2.60 (58). All strains were maintained 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 in 100 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, maintenance, and isolation of recombinant plasmids (51). Fungal chromosomal DNA was isolated and analyzed from lyophilized mycelia by using previously described techniques (65). Cultures for RNA extractions were grown by inoculating 30 ml of liquid GMM with 1 x 106 spores/ml of the appropriate strain before incubation 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. Approximately 20 μg of total RNA was used for Northern blot analysis using a 1.2% agarose-1.5% formaldehyde gel transferred to a Hybond-XL membrane (Amersham Pharmacia Biotech). Expression studies for the different genes were performed with appropriate probes. Probes for A. fumigatus were generated from genomic 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, and ppoC2f (5'-ATCCAAGCGCACGTTCGCCG-3') and ppoC2r (5'-TGAACTCCTTGCTGGCCTTTCC-3') for AfppoC. Nucleotide sequences were analyzed and compared 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 previously cloned, sequenced, and disrupted by replacement with marker genes: ppoA was replaced with metG, ppoB with pyroA, and ppoC with trpC (58, 59, 61). Briefly, disruption vectors were constructed by flanking the A. nidulans marker genes (metG, pyroA, and trpC) with 1-kb DNA fragments from upstream of the corresponding ppo gene start codon and downstream of the corresponding ppo stop codon. Double and triple ppo mutants were created by sexual crosses.
Aspergillus fumigatus AfppoA, AfppoB, and AfppoC genes were obtained from the TIGR database (http://www.tigr.org/tdb/e2k1/afu1/) based on a homologous search using A. nidulans ppoA, ppoB, and ppoC sequences. The polymerase Thermal ACE (Invitrogen Co.) was used for PCR amplifications. RNA interference (RNAi) technology was used to create an A. fumigatus vector that would silence expression of all three ppo genes (AfppoA, AfppoB, and AfppoC) simultaneously by sequentially arranging segments of each gene in both a forward and a 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 combinations with the indicated restriction enzyme sites introduced. The primers used were PpoAf (5'-CTTCGGCGCGCCATGGATCCCGATAGAGGGCCTTGCCCATC-3'), PpoAr (5'-CCCTCATATGATTGTGGAAGACGCGAAAGAGT-3'), PpoBf (5'-GGCTCATATGCGCGAAATATCCACCTGGTTT-3'), PpoBr (5'-TCAAGCATGCAAACCTGACGAACTGGGG-3'), PpoCf (5'-CCCAGCATGCACAAGACCTCTGGTTACTTGGA-3'), and PpoCr (5'-TAATCCATGGCGGCCGCAGGGTATCCAGCTGCGT-3') (f, forward; r, reverse). The ppoA PCR product was digested by NdeI, the ppoB PCR product by NdeI-SphI, and the ppoC PCR product by SphI. The three ppo fragments were ligated together, and the ligation mixture was used as a template to obtain a 1.5-kb PCR product (AfppoA-ppoB-ppoC) using the primer pair PpoAf-PpoCr. This 1.5-kb PCR product was initially digested with AscI and NcoI and ligated into the corresponding sites of pTMH44.2 (19) to generate plasmid pCEJ1. Digestion of pCEJ1 with BamHI and NotI yielded once more the 1.5-kb triple-ppo PCR product, which was further ligated into the corresponding sites of pTMH44.2 (19) in a forward orientation to yield the pCEJ2.7 vector. Next, the 1.5-kb AscI-NcoI fragment was released from pCEJ1, allowing it to be placed in the AscI-NcoI site of pCEJ2.7 in a reverse orientation, to create the pCEJ2.7.4 vector. An internal 280-bp spacer green fluorescent protein fragment separated the inverted repeats, and the A. nidulans gpdA promoter, which has been successfully used in different fungal systems for high levels of transcription (48), drove the transgene. The Aspergillus parasiticus pyrG gene (pBZ5) (53) was inserted into an EcoRI site of pCEJ2.7.4 to give pJW66.3 (Fig. 1). This final plasmid was used for transformation of AF293.1 to silence expression of AfppoA, AfppoB, and AfppoC in A. fumigatus.
Prostaglandin analysis. A. nidulans and A. fumigatus wild-type and ppo mutant strains were grown for 7 days at 37°C in RPMI medium (a defined medium devoid of fatty acids; Sigma Chemical Co.) with shaking at 300 rpm. After 7 days the cultures were incubated for an additional 2 h with 1 mM AA (Cayman Chemicals, Ann Arbor, Mich.). Culture supernatants from both AA-fed and non-AA-fed fungi were analyzed for prostaglandin production by using an enzyme-linked immunosorbent assay (ELISA) kit (catalog no. 514012; Cayman Chemicals) according to the manufacturer's instructions. The antiserum used in this assay exhibits high cross-reactivity for most PGs, allowing quantification of PGE1, PGE2, PGF1, PGF2 (100% specificity), PGF3, PGD2, PGE3 (<50% specificity), and thromboxane B2 (TXB2; 5% specificity). It does not detect PGA, PGB1, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2, or misopristol. The cultures without AA measure the endogenous production of PGs in the absence of exogenous fatty acid substrates. Student's t test and analysis of variance were used to analyze the significance of differences between the experimental groups using the Statistical Analysis System (SAS Institute, Cary, NC).
Physiological studies. Conidium production studies for wild-type A. fumigatus and Afppo IRT mutant strains were performed on plates containing 30 ml of solid 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 each plate. Cultures were incubated in continuous dark at 37°C. A core with a diameter of 12.5 mm was removed from each plate after 2, 4, and 6 days and was homogenized for 1 min in 3 ml of sterile water supplemented with 0.01% Tween 80 to release the spores. Spores were counted using a hemacytometer. The experiments were performed with four replicates. Radial growth tests were performed in triplicate with approximately 104 conidia centered on 30-ml GMM plates, and growth rates were recorded as colony diameter over time at five temperature 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 h at 300 rpm and 37°C. Samples were examined at 2-h intervals, and the germination rate was determined by counting 100 conidia. The mycelial weight of lyophilized tissue was assessed after 4 days of culture in liquid GMM. Physiological 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 study included arachidonic acid (20:4) and PGE2 obtained from Cayman Chemicals Co. Amounts of 0.1 mg and 1 mg were dissolved in 50 μl of methanol and dried on 12.5-mm-diameter paper filter disks. A paper filter disk treated with methanol was used as the solvent control. After drying, the fatty acid-containing disks and the methanol-containing disks were laid on the agar surfaces of plates containing 30 ml of solid 1.5% YGT medium (9) with a 5-ml top layer consisting of cool melted 0.7% agar-YGT containing 105 conidia of the wild-type A. nidulans (RDIT9.32) or A. fumigatus (AF293) strain. The cultures were incubated under light and dark conditions for 8 days.
Stress tolerance assays. Determination of tolerance levels against heat and oxidative stress was performed as previously described (10, 31). For the thermal tolerance assay, wild-type or Afppo IRT strain conidia were inoculated on solid GMM 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 for an additional 36 h at 37°C. Surviving colonies were counted. For the hydrogen peroxide 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 spore suspension was then diluted with sterile distilled water, and conidia were inoculated on solid GMM. After incubation at 37°C for 36 h, colony numbers were counted and calculated as a percentage of the control (10). For the assay of hyphal sensitivity to H2O2, plates containing 50 30-h-grown colonies were overlaid with 10 ml of 0, 50, 100, and 200 mM H2O2 solutions. After a 10-min incubation at room temperature, the H2O2 solution was removed, and the plates were washed twice with sterile distilled water and incubated further for 24 h at 37°C. The number of colonies that survived was calculated as a percentage of the control. All the experiments were performed in triplicate.
Animal model of Aspergillus infection. The virulence of isogenic wild-type and Afppo IRT strains was studied in a lung infection model, with the approval of the University of Wisconsin animal care committee. Conidia were harvested by flooding of fungal colonies with 0.85% NaCl with Tween 80, enumerated with a hemacytometer, 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 on GMM plates. Six-week-old outbred Swiss ICR mice (Harlan Sprague-Dawley) weighing 24 to 27 g were immunosuppressed by intraperitoneal injection of cyclophosphamide (100 mg/kg of body weight) on days –4, –1, and 3 and with a single dose of cortisone acetate (200 mg/kg). Mice were anesthetized via halothane inhalation in a bell jar at day 0. Sedated mice (10 mice/fungal strain) were infected by nasal instillation of 50 μl of the inoculum (day 1) and monitored three times daily for 7 days postinfection. All surviving mice were sacrificed at day 7. The tissue fungal burden of a whole-lung homogenate was quantified by serial dilution and enumeration of CFU (CFU/2 lungs). The duration of survival (in days after inoculation) was recorded for each animal. Moribund animals were sacrificed and cumulative survival recorded. Survival and clearance of residual fungal burden in tissue (CFU/2 lungs) were used as the outcome variables to assess the relative virulence of isogenic strains.
Mouse lung metabolite analysis. Chloroform extracts from mouse lungs were redissolved in 400 μl methanol (high-performance liquid chromatography grade) and loaded onto a Strata X (Phenomenex, Torrance, Ca) 60-mg SPE column already containing 3 ml water (Milli-Q). The SPE column had previously been sequentially activated with 2 ml methanol and 2 ml water. After the sample was loaded, the column was washed with 1 ml water, and the sample was eluted with 4 ml methanol-water (9:1) containing 0.5% formic acid (analytically pure). The samples were then evaporated in vacuo on a SpeedVac, redissolved twice in 50 μl methanol, and filtered through a 4-mm-diameter 0.45-μm-pore-size polytetrafluoroethylene (Teflon) syringe filter. Samples (3 μl) were then analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HR-MS) on an Agilent 1100 LC system equipped with a UV photo diode array detector and coupled to an LCT orthogonal time-of-flight mass spectrometer (Waters-Micromass, Manchester,United Kingdom) (40). Separation was performed on a Phenomenex (Torrance, CA) Luna II C18 (II) column (50 by 2 mm; inner diameter, 3 μm) using a water-acetonitrile system at a flow rate of 0.3 ml/min, starting at 15% acetonitrile, increasing the concentration linearly to 100% in 20 min, and holding at 100% for 5 min. The water was buffered with 10 mM ammonium formate and 20 mM formic acid (both analytical grade) and the acetonitrile with 20 mM formic acid. Gliotoxin and other metabolites were identified by comparison to reference standards (40) of known A. fumigatus metabolites (11, 12) and extracts from agar cultures, based on their retention time, UV spectra, and positive electrospray spectra (ESI+). For secondary metabolites reported from A. fumigatus (11, 12) but not available to us as standards, we used their known [M+H]+ ions as a basis for detecting them by selected ion chromatograms. The secondary 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; fumiquinazolines A to E; and tryptoquivalines A to H.
RESULTS
A. nidulans and A. fumigatus ppo genes encode cyclooxygenase-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 with the biochemically characterized oxylipin-producing linoleate diol synthase (lds) gene from the filamentous fungus Gaeumannomyces graminis (GenBank accession no. AF124979), horse COX-2 (GenBank accession no. O19183), and human COX-1 (GenBank accession no. P23219) revealed the presence of three genes named ppoA, ppoB, and ppoC in both aspergilli (A. fumigatus genes are distinguished by the "Af" prefix). Disruption of ppoA (AY502073) (61), ppoC (AY613780) (59), and ppoB (AY940146) (58) in A. nidulans led to strains defective in producing monohydroxy linoleic and oleic acid-derived oxylipins. Protein domain searches against the PFAM database (http://pfam.wustl.edu) indicated that both A. nidulans Ppo proteins and A. fumigatus Ppo proteins AfPpoA (58.m07572), AfPpoB (67.m03008), and AfPpoC (59.m09493) have domains similar to those of animal heme peroxidases and cytochrome P450 oxygenases. Comparative sequence analysis (ClustalW) between the predicted Ppo amino acid sequences of the two species led to the phylogenetic tree shown in Fig. 2A and the corresponding comparison values (Fig. 2B). AfPpoA showed 48% and 47% identities with AfPpoB and AfPpoC, respectively, whereas AfPpoB and AfPpoC showed 56% identity.
The amino acid sequences of A. nidulans and A. fumigatus Ppo proteins also revealed similarity with various mammalian COX, 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 prostaglandin PGH2 (54). PGH2 is converted to the biologically active end product PGD2, PGE2, PGF2, PGI2, or TxA2 (thromboxane A2) by other specific prostaglandin synthases (18, 54). Homology between A. nidulans and A. fumigatus Ppo and mammalian COX amino acid sequences over the conserved catalytic domains ranged from 25% to 29% identity and 40% to 45% similarity for COX-2 paralogs (E values, 10–24 to 10–18) and 25% to 26% identity and 38% to 40% similarity for COX-1 paralogs (E values, 2 x 10–18 to 5 x 10–18).
A more detailed sequence alignment of the Aspergillus Ppo sequences with horse COX-2 and human COX-1 was performed with the ClustalW program to identify conserved functional motifs. The sequence similarity appeared to be restricted to the catalytic domain of COX and was most striking along the -helices (predicted to be present in Ppo proteins by using the PredictProtein program, available at http://cubic.bioc.columbia.edu/predictprotein/) as shown in Fig. 3A. Structural homology shows that both the distal and proximal His heme ligands and the important Tyr residue, which are required for enzyme activity and are completely conserved within the COX family, aligned in context with identical amino acids of A. nidulans PpoC, A. nidulans PpoA, and A. fumigatus AfPpoA but not with the other Aspergillus proteins. The core helix H2 harbors the distal His heme ligand (consensus THXXFXT), and the core helix H8 contains the proximal heme ligand and the important Tyr residue (consensus EFNXXYXWH) of PGH synthases (54). In contrast to the structural conservation found within the catalytic domain, the regions of COX falling outside of this domain, that is, the epidermal growth factor-like domain and the membrane-binding domain, do not seem to have equivalent residues in the Ppo proteins. G. graminis Lds also has similar conserved regions in its sequence, as was previously reported (24); however, the biochemical involvement of Lds in PG 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 development and virulence, a gene-silencing approach was undertaken to inactivate the three genes simultaneously. The absence of a sexual cycle in A. fumigatus renders the incorporation of many deletions in the same individual a difficult task in this species. To evaluate the potential of RNAi technology as a means of silencing multiple genes in A. fumigatus, a vector was designed to simultaneously produce AfppoA, AfppoB, and AfppoC double-stranded RNA molecules in the fungal thallus (Fig. 1). Double-stranded RNA molecules are processed by RNAi machinery to produce small interfering RNA (siRNA) molecules that 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 two genes within each vector (19, 39).
The triple Afppo mutant (Afppo IRT) was created by transformation of strain 293.1 with pJW66. PCR and Southern blot analysis of 85 transformants revealed the introduction of the IRT Afppo construct in five transformants. Macroscopically, all five transformants were identical to each other and to the wild type. Transformants TJW62.2, TJW62.5, and TJW62.10 and the wild-type AF293 were selected for mouse virulence studies (see below); TJW62.2, TJW62.10, and AF293 were used for ELISA analysis (see below); and TJW62.2 was used for further physiological and molecular analyses. As predicted, the A. fumigatus transformants, which incorporated the Afppo IRT plasmid into their genome, displayed an Afppo silencing phenotype, as monitored by expression studies of the three ppo genes. Figure 4 shows that in contrast to the wild type, all three genes 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. Like the ppoA ppoB ppoC mutant of A. nidulans, the Afppo IRT mutant had no alterations in vegetative development or spore germination relative to the wild type in liquid GMM. However, in contrast to the A. nidulans ppoA ppoB ppoC strain, which demonstrated a significant reduction in asexual spore production and a significant increase in sexual spore production (58), spore counts of the A. fumigatus triple ppo mutant did not show any alterations in asexual spore production 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 growth experiments on solid GMM with glucose as the sole carbon source 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 there is no difference at 37°C.
Ppo mediation of prostaglandin production in A. nidulans and A. fumigatus. One primary goal of this study was to determine if any of the ppo genes could be involved in PG production. Recent reports indicate that several fungi can utilize exogenous sources of arachidonic acid to produce a number of different eicosanoids (44, 45); however, no candidate enzyme has been uncovered. By following procedures used by other labs in establishing that 90% of fungal PGs are secreted (36, 45), supernatants from Aspergillus strains grown in RPMI medium were examined for PG production. Dry weights of recovered mycelium were identical, but there was a decrease in production of PGs in A. nidulans strains carrying a ppoA allele (14%) and in all strains carrying the ppoC allele (36% to 37%) (Fig. 5A) (P < 0.01). These data suggest that PpoC and PpoA are involved in PG production by A. nidulans. Examination of two IRT Afppo mutants (TJW62.2 and TJW62.10) showed that both mutants had a 12% to 15% reduction in PG biosynthesis (Fig. 5B) (P < 0.01). The amount of PGs that was detected in non-AA-fed cultures of both aspergilli was below the assay's threshold of detection (40 pg/ml).
Increased virulence of Afppo IRT mutants in a murine model of pulmonary aspergillosis. The relative virulence of the wild type and three Afppo IRT mutants (TJW62.2, TJW62.5, and TJW62.10) was evaluated in a murine pulmonary infection model where mice were monitored for survival on a daily basis (Fig. 6). The Afppo IRT strains caused an increased fatal infection rate in the murine model (P, 0.02 to >0.001) relative to the wild type. By day 3 postinoculation, mice infected with the Afppo IRT strains began to die, with a 90 to 100% mortality rate reached by day 5 for TJW62.2 and TJW62.10 and a 50% mortality rate by day 5 for TJW62.5. In contrast, the wild type showed a 30% mortality rate at day 5. By day 6 none of the mice treated with TJW62.2 or TJW62.10 survived, whereas 50% of those infected with the wild type were still alive. The virulence experiment was repeated twice with similar results. From all mice culled, fungal colonies were recovered from the lungs, indicating that the pulmonary distress was due to aspergillosis. No statistical differences were observed in the number of CFU recovered from the lungs of wild-type- or Afppo IRT-infected mice sacrificed 3 days after inoculation (data not shown). The isolated fungal colonies from each group of mouse lungs were confirmed as wild type or Afppo IRT by diagnostic PCR and Southern blot analysis (data not shown).
Given the numerous studies implicating gliotoxin as a potent virulence factor in aspergillosis (41), five lungs from sacrificed mice were extracted and analyzed for gliotoxin content 3 and 4 days after infection with 107 wild-type (AF293) or Afppo IRT (TJW 62.2) conidia. LC-HR-MS (Fig. 7) showed no differences in the quantities of gliotoxin detected between the wild type and an Afppo IRT mutant. Gliotoxin was unambiguously detected at the same retention time in a reference standard, and both HR-MS and UV spectra were correct. Noninfected mouse lungs showed no detectable levels of gliotoxin (Fig. 7). No other gliotoxin analogs or secondary metabolites normally produced during in vitro culture of A. fumigatus (e.g., helvolic acid, fumigaclavines A to C, tryptoquivalines A to H, fumiquinazolines A to E, pseurotins A to F2, fumagillin, fumitremorgins A to C, verrucologen, or TR-2) could be detected in the wild-type- or Afppo IRT-infected lung tissue. Most of these compounds ionize significantly better than gliotoxin and would thus have much lower detection limits (40), showing that gliotoxin is the primary fungal metabolite produced in the lungs.
Increased stress sensitivity of the A. fumigatus triple ppo mutant. Although we could not detect any difference in spore pigmentation and gliotoxin production (both implicated as virulence factors) that could explain the increased virulence of the Afppo IRT mutant, we considered the possibility that mechanisms of resistance to host defenses might be altered in the Afppo IRT mutant. To test this hypothesis, the Afppo IRT mutant was subjected to oxidative and thermal stress. By following procedures established for A. fumigatus and A. nidulans (30, 47), both conidia and hyphae were exposed to increasing levels of H2O2, a treatment designed to mimic exposure to host reactive oxygen species (ROS), typically activated during pathogen ingress. Germination and survival rates of spores in varying concentrations of H2O2 were determined after 24 h at 37°C. As shown in Fig. 8, conidia of the Afppo IRT mutant showed a statistically significant 15-to-20% increase (P < 0.05) in survival at 20, 40, and 80 mM of H2O2 relative to the wild type, which showed a steady decrease in oxidative tolerance. No colonies of either strain survived the 250 mM H2O2 treatment. In contrast to the increased resistance of conidia to H2O2 treatment, Afppo IRT hyphae were not more resistant to H2O2 than wild-type hyphae.
The resistance of the Afppo IRT mutant to thermal stress was evaluated by treating germlings at 50°C for 0, 3, and 4 h and determining their ability to form viable colonies at 37°C. The wild type showed mean decreases of 10% and 93% in colony survival, whereas the Afppo IRT mutant showed 1% (P < 0.1) and 87% (P < 0.1) decreases, at 3 h and 4 h, respectively. Based on these data, Ppo proteins and/or their products, among several other possible roles, might mediate signaling cascades that regulate heat and oxidative stress responses at different developmental stages of the fungus.
Effects of arachidonic acid and PGE2 on Aspergillus physiology. Earlier studies in our lab showed that C18 unsaturated fatty acids and their derived oxylipins induced developmental changes in A. nidulans including changes in sexual-to-asexual spore ratios and secondary metabolite production (8, 9). Similar studies have shown that PGs can induce developmental changes in Candida albicans (28, 43). We therefore thought it possible that PGs and their progenitor, arachidonic acid, might affect Aspergillus physiological processes.
Wild-type A. fumigatus and A. nidulans cultures were examined for reactions to PGE2 and arachidonic acid. The addition of PGE2 to A. nidulans cultures inhibited the formation of conidia, in sharp contrast to arachidonic acid, which significantly induced conidiation (visualized as a green halo of conidia around the disk) (Fig. 9). These results were reminiscent of the differential reaction of A. nidulans to the plant oxylipin 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) and its precursor, linoleic acid, where the former induced conidial formation at 0.1 mg and the latter inhibited conidiation at the same concentration (9). Neither PGE2 nor arachidonic acid affected sporulation in A. fumigatus; however, they both inhibited the production of hyphal pigments (Fig. 9).
DISCUSSION
Oxylipin production is widespread among fungi, and progress has recently been made in identification, regulation, and cellular localization of the dioxygenases generating fungal oxylipins (17, 22, 33, 42, 58, 59, 61). In this study, we provide the first genetic evidence for fungi that Ppo proteins similar in sequence to mammalian COX are involved in the production of prostaglandins, a major group of oxylipins that regulate immune responses in mammals. Silencing of the three ppo genes in the human pathogen A. fumigatus yielded hypervirulent strains in the mouse pulmonary model system. These studies extend the involvement of oxylipins in aspergilli from control of developmental and metabolic functions, such as spore balance and fatty acid regulation (42, 58, 59, 61), to virulence in a host-pathogen interaction.
The types of oxylipins produced by fungi are numerous, and likely many remain to be characterized. Although many studies have focused on C18 oxylipins, several experiments have revealed that both pathogenic and nonpathogenic fungal species produce detectable amounts of both C20 cyclooxygenase and lipoxygenase products (36, 44, 45). The arachidonic acid metabolites PGF2 and PGF2-lactone have been detected in a number of environmental yeasts of the family Lipomycetaceae (Dipodascopsis, Lipomyces, Myxozyma, and Zygozyma) (22, 33, 34, 55) as well as in Saccharomyces cerevisiae (33). The pathogenic yeasts Cryptococcus neoformans and Candida albicans and the filamentous fungus A. fumigatus produce both PGs and leukotrienes, and their amounts are significantly increased after exogenous application of arachidonic acid (44, 45). Furthermore, COX inhibitors, including aspirin and other nonsteroidal anti-inflammatory drugs, inhibited hydroxyeicosa-tertraenoic acid, PGE2, and PGD2 production in several members of the family Lipomycetaceae, supporting the view that a COX-like enzyme is active in these fungi (5, 32, 36). However, no candidate protein has been identified in fungi prior to this report.
The structural similarity between Ppo proteins and COX led us to investigate the possibility that these enzymes could be involved in PG production. COX possesses two enzymatic activities, a cyclooxygenase that catalyzes the oxygenation of polyunsaturated substrates, such as arachidonic acid, to form prostaglandin G2 (PGG2) and a peroxidase that can use a variety of electron donors to reduce PGG2 to form prostaglandin H2 (PGH2) (54). The amino acid sequences of Ppo proteins are predicted to contain both an oxygenase and a peroxidase domain (58, 59, 61). Further detailed comparison of the amino acid sequences of all six Ppo proteins with human and horse COX revealed that three of the proteins, A. nidulans PpoC and PpoA and A. fumigatus PpoA, contained the conserved catalytic residues found in COX -helices, which included the proximal and distal heme ligands and the critical tyrosine residue of PGH synthases (24, 54). Considering the correlation of loss of PG activity in the respective A. nidulans ppoC and ppoA mutants (Fig. 5) and presence of catalytic residues, it is tempting to speculate that these amino acids are indicators of possible PG activity in fungi. Investigation of single A. fumigatus Ppo mutants may shed further light on the viability of this observation.
Through sexual genetics, we were able to analyze every possible combination of ppo mutant background in A. nidulans. The consistent decrease in PG production in all combinations of the ppoC allele suggests a major role for this enzyme in PG biosynthesis (Fig. 5A). The slight increase in production of PGs in ppoB and ppoA ppoB strains correlated with an upregulation in the expression level of ppoC (58, 59), further implicating PpoC in the synthesis of PGs. Previous biochemical data has shown that PpoC is also likely involved in the production of oleic acid-derived oxylipins (59). The ability of a fatty acid oxygenase to utilize different fatty acids as substrates is not uncommon. For instance, the G. graminis Lds can oxygenate 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 involves considerable effort and has not been reported to date. To address this issue, we attempted to silence all three Afppo genes by using one vector. As demonstrated by Northern blot (Fig. 4) and PG (Fig. 5B) analyses, this approach was successful. RNAi has emerged as an effective method for silencing gene expression in many eukaryotes and has recently been used successfully in genome-wide functional tests (2, 29); we believe this method will greatly help in further genomic studies of A. fumigatus. Caveats with this method are (i) the potential of off-target effects by the siRNAs that could jeopardize correct interpretation of gene function and (ii) the fact that some transcripts are still produced and not completely eliminated, unlike the situation in traditional gene replacement, as illustrated in Fig. 4. The fact that PG reduction in the Afppo IRT strain is not as pronounced as in the A. nidulans triple mutant could well be a reflection of this incomplete transcript suppression. Additionally, the presence of alternative enzymes or pathways for PG biosynthesis is expected, since both the A. nidulans triple mutant and the Afppo IRT strain still produced substantial amounts of PGs. It is also possible that levels of other eicosanoids are decreased in the ppo mutants, since our detection method was limited to specific PGs and did not cover the full array of known eicosanoids.
Despite a decrease of only 12% in PG production as measured by ELISA (Fig. 5B), the Afppo IRT strains displayed a significant increase in virulence in the murine pulmonary model (Fig. 6). To our knowledge this is the first report of a hypervirulent A. fumigatus mutant. Since a unique characteristic of mammalian PGs is their potency at very low (nanomolar) concentrations and their very short half-lives—they are produced de novo and act near the site of their synthesis—we speculate that the small decrease in the endogenous fungal PG levels can lead to a significant increase in the virulence of A. fumigatus (18). Two determinants implicated as virulence factors in A. fumigatus include spore pigmentation (6, 57) and gliotoxin production (41). Loss of pigmentation results in increased phagocytosis, whereas gliotoxin, a secondary metabolite produced in tissues of mice following development of invasive aspergillosis (15), is a potent immunomodulating agent and an inducer of apoptotic cell death in a number of cell types (62). Microscopic examination of Afppo IRT spores did not reveal any differences in pigmentation compared to the wild type. Chemical analysis indicated that gliotoxin production in mouse lungs by Afppo IRT strains was not altered from that by the wild type (Fig. 7). These factors, therefore, do not seem to play a role in the increased virulence of the Afppo IRT strains.
Physiological examination of one of the Afppo IRT strains did, however, indicate increased resistance to environmental stress as observed by H2O2 treatment (Fig. 8). H2O2 treatment of fungal propagules is an indirect method of measuring the putative resistance of the pathogen to host ROS, a major host antimicrobial effector system also active against Aspergillus conidia (63). ROS production in mammals occurs during the course of neutrophil and macrophage activation and is implicated in the defense against fungal pathogens (1, 49, 50). Increased resistance to a ROS-mounted defense could protect a pathogen and render it more virulent.
Although the hypothesis is not examined in this study, we also propose that part of the increased virulence of the Afppo IRT strains is due to changes in host physiology. A plausible explanation is that the Ppo-generated PGs enhance host defense mechanisms, perhaps through initiation of inflammation responses involved in recruiting phagocytic cells (16, 66). PGs and eicosanoids in general regulate both proinflammatory and anti-inflammatory responses of the immune system. A single PG molecule can have pleiotropic effects due to the existence of numerous receptors for each lipid species, and in turn, these receptors can elicit different responses on different cell types (18, 20). A decrease in PG signaling might lead to a decrease or slower response time in these host-mounted defenses. Finally, considering the detectable macroscopic reaction of Aspergillus spp. to exogenously applied arachidonic acid and PGE2, we propose that Aspergillus (and other eukaryotic pathogens) may share similar oxylipin signaling pathways with host cells. PGs are transported out of cells and interact with cell surface receptors linked to G proteins to initiate appropriate signaling pathways in mammalian cells (26). Our central hypothesis is that fungal oxylipins, similarly to the endogenous mammalian PGs, have the potential to mediate interkingdom signaling via a cross talk communication in which the pathogen triggers the host defense immune responses at the site of infection by binding to mammalian G protein receptors; this process may result in the retardation of pathogenesis (Fig. 10).
ACKNOWLEDGMENTS
This work was funded by NSF MCB-0196233 to N.P.K. and a Novartis (Syngenta) Crop Protection Graduate Fellowship to D.I.T.
We thank Courtney Jahn for experimental help and Thomas Hammond for providing the plasmid vector pTMH44.2. Genomic data for A. fumigatus were provided by The Institute for Genomic Research (www.tigr.org/tdb/e2k1/afu1) and The Wellcome Trust, Sanger Institute (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 data was enabled by an international collaboration involving more than 50 institutions from 10 countries and coordinated from Manchester, United Kingdom (www.cadre.man.ac.uk and www.aspergillus.man.ac.uk).
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, United Kingdom.
REFERENCES
1. Aratani, Y., F. Kura, H. Watanabe, H. Akagawa, Y. Takano, K. Suzuki, M. C. Dinauer, N. Maeda, and H. Koyama. 2002. Relative contributions of myeloperoxidase and NADPH-oxidase to the early host defense against pulmonary infections with Candida albicans and Aspergillus fumigatus. Med. Mycol. 40:557-563.
2. Ashrafi, K., F. Y. Chang, J. L. Watts, A. G. Fraser, R. S. Kamath, J. Ahringer, and G. Ruvkun. 2003. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421:268-272.
3. Bertout, S., C. Badoc, M. Mallie, J. Giaimis, and J. M. Bastide. 2002. Spore diffusate isolated from some strains of Aspergillus fumigatus inhibits phagocytosis by murine alveolar macrophages. FEMS Immunol. Med. Microbiol. 33:101-106.
4. Bok, J. W., and N. P. Keller. 2004. LaeA, a regulator of secondary metabolism in Aspergillus spp. Eukaryot. Cell 3:527-535.
5. Botha, A., J. L. Kock, and S. Nigam. 1997. The production of eicosanoid precursors by mucoralean fungi. Adv. Exp. Med. Biol. 433:227-229.
6. Brakhage, A. A., K. Langfelder, G. Wanner, A. Schmidt, and B. Jahn. 1999. Pigment biosynthesis and virulence. Contrib. Microbiol. 2:205-215.
7. Brookman, J. L., and D. W. Denning. 2000. Molecular genetics in Aspergillus fumigatus. Curr. Opin. Microbiol. 3:468-474.
8. Burow, G. B., T. C. Nesbitt, J. Dunlap, and N. P. Keller. 1997. Seed lipoxygenase products modulate Aspergillus mycotoxin biosynthesis. Mol. Plant-Microbe Interact. 10:380-387.
9. Calvo, A. M., L. L. Hinze, H. W. Gardner, and N. P. Keller. 1999. Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp. Appl. Environ. Microbiol. 65:3668-3673.
10. Chang, Y. C., B. H. Segal, S. M. Holland, G. F. Miller, and K. J. Kwon-Chung. 1998. Virulence of catalase-deficient Aspergillus nidulans in p47phox–/– mice. Implications for fungal pathogenicity and host defense in chronic granulomatous disease. J. Clin. Investig. 101:1843-1850.
11. Cole, R. J., B. B. Jarvis, and M. A. Schweikert. 2003. Handbook of secondary fungal metabolites, vol. III. Academic Press, Amsterdam, The Netherlands.
12. Cole, R. J., and M. A. Schweikert. 2003. Handbook of secondary fungal metabolites, vol. I and II. Academic Press, Amsterdam, The Netherlands.
13. Denli, A. M., and G. J. Hannon. 2003. RNAi: an ever-growing puzzle. Trends Biochem. Sci. 28:196-201.
14. Denning, D. W., M. J. Anderson, G. Turner, J. P. Latge, and J. W. Bennett. 2002. Sequencing the Aspergillus fumigatus genome. Lancet Infect. Dis. 2:251-253.
15. Eichner, R. D., P. Waring, A. M. Geue, A. W. Braithwaite, and A. Mullbacher. 1988. Gliotoxin causes oxidative damage to plasmid and cellular DNA. J. Biol. Chem. 263:3772-3777.
16. Filler, S. G., B. O. Ibe, A. S. Ibrahim, M. A. Ghannoum, J. U. Raj, and J. E. Edwards, Jr. 1994. Mechanisms by which Candida albicans induces endothelial cell prostaglandin synthesis. Infect. Immun. 62:1064-1069.
17. Fox, S. R., A. Akpinar, A. A. Prabhune, J. Friend, and C. Ratledge. 2000. The biosynthesis of oxylipins of linoleic and arachidonic acids by the sewage fungus Leptomitus lacteus, including the identification of 8R-hydroxy-9Z,12Z-octadecadienoic acid. Lipids 35:23-30.
18. Funk, C. D. 2001. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294:1871-1875.
19. Hammond, T. M., and N. P. Keller. 2005. RNA silencing in Aspergillus nidulans is independent of RNA dependent RNA polymerases. Genetics 169:607-617.
20. Hatae, N., Y. Sugimoto, and A. Ichikawa. 2002. Prostaglandin receptors: advances in the study of EP3 receptor signaling. J. Biochem. 131:781-784.
21. Herbrecht, R. 2002. Improving the outcome of invasive aspergillosis: new diagnostic tools and new therapeutic strategies. Ann. Hematol. 81(Suppl. 2):S52-S53.
22. Herman, R. P. 1998. Oxylipin production and action in fungi and related organisms, p. 115-130. In A. F. Rowley, H. Kuhn, and T. Schewe (ed.), Eicosanoids and related compounds in plants and animals. Princeton University Press, Princeton, N.J.
23. Herman, R. P., and C. A. Herman. 1985. Prostaglandins or prostaglandin like substances are implicated in normal growth and development in oomycetes. Prostaglandins 29:819-830.
24. Hornsten, L., C. Su, A. E. Osbourn, P. Garosi, U. Hellman, C. Wernstedt, and E. H. Oliw. 1999. Cloning of linoleate diol synthase reveals homology with prostaglandin H synthases. J. Biol. Chem. 274:28219-28224.
25. Jahn, B., F. Boukhallouk, J. Lotz, K. Langfelder, G. Wanner, and A. A. Brakhage. 2000. Interaction of human phagocytes with pigmentless Aspergillus conidia. Infect. Immun. 68:3736-3739.
26. Jump, D. B. 2002. Dietary polyunsaturated fatty acids and regulation of gene transcription. Curr. Opin. Lipidol. 13:155-164.
27. Kafer, E. 1977. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv. Genet. 19:33-131.
28. Kalo-Klein, A., and S. S. Witkin. 1990. Prostaglandin E2 enhances and gamma interferon inhibits germ tube formation in Candida albicans. Infect. Immun. 58:260-262.
29. Kamath, R. S., A. G. Fraser, Y. Dong, G. Poulin, R. Durbin, M. Gotta, A. Kanapin, N. Le Bot, S. Moreno, M. Sohrmann, D. P. Welchman, P. Zipperlen, and J. Ahringer. 2003. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421:231-237.
30. Kawasaki, L., and J. Aguirre. 2001. Multiple catalase genes are differentially regulated in Aspergillus nidulans. J. Bacteriol. 183:1434-1440.
31. Kawasaki, L., D. Wysong, R. Diamond, and J. Aguirre. 1997. Two divergent catalase genes are differentially regulated during Aspergillus nidulans development and oxidative stress. J. Bacteriol. 179:3284-3292.
32. Kock, J. L., D. J. Coetzee, M. S. van Dyk, M. Truscott, A. Botha, and O. P. Augustyn. 1992. Evidence for, and taxonomic value of, an arachidonic acid cascade in the Lipomycetaceae. Antonie Leeuwenhoek 62:251-259.
33. Kock, J. L., C. J. Strauss, C. H. Pohl, and S. Nigam. 2003. The distribution of 3-hydroxy oxylipins in fungi. Prostaglandins Other Lipid Mediat. 71:85-96.
34. Kock, J. L., P. Venter, D. Linke, T. Schewe, and S. Nigam. 1998. Biological dynamics and distribution of 3-hydroxy fatty acids in the yeast Dipodascopsis uninucleata as investigated by immunofluorescence microscopy. Evidence for a putative regulatory role in the sexual reproductive cycle. FEBS Lett. 427:345-348.
35. Kontoyiannis, D. P., and G. P. Bodey. 2002. Invasive aspergillosis in 2002: an update. Eur. J. Clin. Microbiol. Infect. Dis. 21:161-172.
36. Lamacka, M., and J. Sajbidor. 1998. The content of prostaglandins and their precursors in Mortierella and Cunninghamella species. Lett. Appl. Microbiol. 26:224-226.
37. Latge, J. P. 1999. Aspergillus fumigatus and aspergillosis. Clin. Microbiol. Rev. 12:310-350.
38. Marr, K. A., T. Patterson, and D. Denning. 2002. Aspergillosis. Pathogenesis, clinical manifestations, and therapy. Infect. Dis. Clin. N. Am. 16:875-894.
39. Mouyna, I., C. Henry, T. L. Doering, and J. P. Latge. 2004. Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol. Lett. 237:317-324.
40. Nielsen, K. F., and J. Smedsgaard. 2003. Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology. J. Chromatogr. A 1002:111-136.
41. Nieminen, S. M., J. Maki-Paakkanen, M. R. Hirvonen, M. Roponen, and A. von Wright. 2002. Genotoxicity of gliotoxin, a secondary metabolite of Aspergillus fumigatus, in a battery of short-term test systems. Mutat. Res. 520:161-170.
42. Noverr, M. C., J. R. Erb-Downward, and G. B. Huffnagle. 2003. Production of eicosanoids and other oxylipins by pathogenic eukaryotic microbes. Clin. Microbiol. Rev. 16:517-533.
43. Noverr, M. C., and G. B. Huffnagle. 2004. Regulation of Candida albicans morphogenesis by fatty acid metabolites. Infect. Immun. 72:6206-6210.
44. Noverr, M. C., S. M. Phare, G. B. Toews, M. J. Coffey, and G. B. Huffnagle. 2001. Pathogenic yeasts Cryptococcus neoformans and Candida albicans produce immunomodulatory prostaglandins. Infect. Immun. 69:2957-2963.
45. Noverr, M. C., G. B. Toews, and G. B. Huffnagle. 2002. Production of prostaglandins and leukotrienes by pathogenic fungi. Infect. Immun. 70:400-402.
46. Oren, I., and N. Goldstein. 2002. Invasive pulmonary aspergillosis. Curr. Opin. Pulm. Med. 8:195-200.
47. Paris, S., D. Wysong, J. P. Debeaupuis, K. Shibuya, B. Philippe, R. D. Diamond, and J. P. Latge. 2003. Catalases of Aspergillus fumigatus. Infect. Immun. 71:3551-3562.
48. Punt, P. J., N. D. Zegers, M. Busscher, P. H. Pouwels, and C. A. van den Hondel. 1991. Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene. J. Biotechnol. 17:19-33.
49. Roilides, E., C. A. Lyman, T. Sein, R. Petraitiene, and T. J. Walsh. 2003. Macrophage colony-stimulating factor enhances phagocytosis and oxidative burst of mononuclear phagocytes against Penicillium marneffei conidia. FEMS Immunol. Med. Microbiol. 36:19-26.
50. Roilides, E., K. Uhlig, D. Venzon, P. A. Pizzo, and T. J. Walsh. 1993. Enhancement of oxidative response and damage caused by human neutrophils to Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and gamma interferon. Infect. Immun. 61:1185-1193.
51. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
52. Segal, B. H., E. S. DeCarlo, K. J. Kwon-Chung, H. L. Malech, J. I. Gallin, and S. M. Holland. 1998. Aspergillus nidulans infection in chronic granulomatous disease. Medicine (Baltimore) 77:345-354.
53. Skory, C. D., P. K. Chang, J. Cary, and J. E. Linz. 1992. Isolation and characterization of a gene from Aspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis. Appl. Environ. Microbiol. 58:3527-3537.
54. Smith, W. L., D. L. DeWitt, and R. M. Garavito. 2000. Cyclooxygenases: structural, cellular, and molecular biology. Annu. Rev. Biochem. 69:145-182.
55. Strauss, T., A. Botha, J. L. Kock, I. Paul, D. P. Smith, D. Linke, T. Schewe, and S. Nigam. 2000. Mapping the distribution of 3-hydroxylipins in the Mucorales using immunofluorescence microscopy. Antonie Leeuwenhoek 78:39-42.
56. Su, C., and E. H. Oliw. 1996. Purification and characterization of linoleate 8-dioxygenase from the fungus Gaeumannomyces graminis as a novel hemoprotein. J. Biol. Chem. 271:14112-14118.
57. Tsai, H. F., Y. C. Chang, R. G. Washburn, M. H. Wheeler, and K. J. Kwon-Chung. 1998. The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence. J. Bacteriol. 180:3031-3038.
58. Tsitsigiannis, D. I., T. M. Kowieski, R. Zarnowski, and N. P. Keller. 2005. Three putative oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans. Microbiology 151:1809-1821.
59. Tsitsigiannis, D. I., T. M. Kowieski, R. Zarnowski, and N. P. Keller. 2004. Endogenous lipogenic regulators of spore balance in Aspergillus nidulans. Eukaryot. Cell 3:1398-1411.
60. Tsitsigiannis, D. I., R. A. Wilson, and N. P. Keller. 2002. Lipid mediated signaling in the Aspergillus/seed interaction, p. 186-191. In S. A. Leong, C. Allen, and E. W. Triplet (ed.), Biology of plant-microbe interactions, vol. 3. International Society for Plant-Microbe Interactions, St. Paul, Minn.
61. Tsitsigiannis, D. I., R. Zarnowski, and N. P. Keller. 2004. The lipid body protein, PpoA, coordinates sexual and asexual sporulation in Aspergillus nidulans. J. Biol. Chem. 279:11344-11353.
62. Waring, P., T. Khan, and A. Sjaarda. 1997. Apoptosis induced by gliotoxin is preceded by phosphorylation of histone H3 and enhanced sensitivity of chromatin to nuclease digestion. J. Biol. Chem. 272:17929-17936.
63. Washburn, R. G., J. I. Gallin, and J. E. Bennett. 1987. Oxidative killing of Aspergillus fumigatus proceeds by parallel myeloperoxidase-dependent and -independent pathways. Infect. Immun. 55:2088-2092.
64. Wilson, R. A., H. W. Gardner, and N. P. Keller. 2001. Cultivar-dependent expression of a maize lipoxygenase responsive to seed infesting fungi. Mol. Plant-Microbe Interact. 14:980-987.
65. Yelton, M. M., J. E. Hamer, and W. E. Timberlake. 1984. Transformation of Aspergillus nidulans by using a trpC plasmid. Proc. Natl. Acad. Sci. USA 81:1470-1474.
66. Yoshikai, Y. 2001. Roles of prostaglandins and leukotrienes in acute inflammation caused by bacterial infection. Curr. Opin. Infect. Dis. 14:257-263.
67. Zha, S., V. Yegnasubramanian, W. G. Nelson, W. B. Isaacs, and A. M. De Marzo. 2004. Cyclooxygenases in cancer: progress and perspective. Cancer Lett. 215:1-20.(Dimitrios I. Tsitsigianni)