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Division of Plant & Soil Sciences, West Virginia University, P.O. Box 6108, Morgantown, West Virginia 26506-6108
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ergot alkaloids are mycotoxins that affect the nervous and reproductive systems of exposed individuals through interactions with monoamine receptors. They have been studied more widely in ergot fungi and grass endophytes but also are found in Aspergillus fumigatus, an opportunistic human pathogen that reproduces and disseminates exclusively through conidia. The ergot alkaloids festuclavine and fumigaclavines A, B and C are present in or on conidia of A. fumigatus. Cultures of the fungus that are free of conidia are difficult to obtain, obscuring comparisons of conidia versus vegetative hyphae as sources of the ergot alkaloids. To create conidiation-deficient strains of A. fumigatus we manipulated the bristle A gene (brlA), which controls vesicle formation or budding growth necessary for conidiation in Aspergillus spp. Disruption of brlA in A. fumigatus, via homologous recombination, resulted in a nonconidiating mutant that produced bristle-like structures instead of conidiophores and conidia. Moreover the disrupted strain failed to produce ergot alkaloids as verified by HPLC analyses. Complementation with a wild-type allele restored conidiation and ergot alkaloid production. These results suggest that ergot alkaloids are not produced within the vegetative mycelium of the fungus and are associated directly with conidiation.
Key words: aspergillosis, bristle A gene, clavines, mycotoxins
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Gröger and Floss 1998, Panaccione 2005, Schardl et al 2006). The ecological roles of ergot alkaloids are poorly understood; however they have activities against mammals, insects, nematodes and bacteria (Schwarz and Eich 1983, Clay and Cheplick 1989, Ball et al 1997, Clay and Schardl 2002, Panaccione 2005, Panaccione et al 2006). Their effects on humans are thought to be mediated by their agonistic and/or antagonistic interactions with monoamine neurotransmitter receptors (Pertz 1996, Gröger and Floss 1998, Pertz and Eich 1999).
Aspergillus fumigatus Fres., a common saprophyte and opportunistic pathogen, produces several ergot alkaloids, including festuclavine and fumigaclavines A, B and C (Spilsbury and Wilkinson 1961, Cole et al 1977). We recently reported a high performance liquid chromatography (HPLC) procedure for identification and quantification of these alkaloids and demonstrated them to be present in or on conidia of A. fumigatus in quantities that collectively total approximately 1% of the dry mass of the conidium (Panaccione and Coyle 2005). Fumigaclavine C is the end product of the A. fumigatus pathway, with festuclavine, fumigaclavine B and fumigaclavine A (in that sequence) acting as the final three intermediates in its biosynthesis (Panaccione 2005, Schardl et al 2006, Unsöld and Li 2006). Differences in abundance and activity of the enzymes responsible for converting one intermediate to the next presumably account for the accumulation of festuclavine and fumigaclavine A to relatively high concentrations whereas fumigaclavine B is typically present in much lower concentrations (Panaccione and Coyle 2005, Panaccione 2005).
A. fumigatus in vitro sporulates prolifically, and cultures free of conidia are difficult to obtain. Our unpublished observations of ergot alkaloid yields from cultures of A. fumigatus on agar-based media (primarily conidia and limited vegetative hyphae) versus submerged, broth-based media (consisting mainly of hyphae and fewer conidia) suggested an apparent association between conidiation and ergot alkaloids.
Research on Aspergillus spp. has associated secondary metabolism and sporulation (Calvo et al 2002, Bok and Keller 2004). For example sporulation and sterigmatocystin production have been shown to be regulated by a FadA G
protein-dependent signaling pathway in Aspergillus nidulans Eidam (Winter) (Hicks et al 1997). More specifically sterigmatocystin production and conidiation both are regulated negatively by a cAMP-dependent protein kinase catalytic subunit (pkaA) (Shimizu and Keller 2001).
Conidiation has been studied extensively in A. nidulans. Conidiophore development is contingent on differentiation of the fungus from polarized apical extension to budding growth (Cole 1986). One gene in particular, brlA, has been determined to control conidiophore development and encodes a nucleic acid binding protein (BrlA) (Boylan et al 1987, Adams et al 1988, Adams et al 1998). BrlA is an early protein in a signaling cascade that controls the expression of many genes, including some that encode for other regulatory proteins by binding to brlA response elements (BRE) within the promoter regions of these genes (Chang and Timberlake 1992). In A. nidulans transcription of brlA may initiate at different sites resulting in two overlapping transcription units, brlA and brlAβ (Prade and Timberlake 1993). The products of these two forms are interchangeable functionally, translated in the same reading frame and differ only in that the product of brlAβ has an additional 23 amino acids at the amino terminus (Prade and Timberlake 1993). In this paper we report mutational analyses of the brlA gene of A. fumigatus to produce nonconidiating mutants for the purpose of examining whether ergot alkaloids are produced during vegetative growth or conidiation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and derivatives were cultured routinely on potato-dextrose agar (PDA) prepared from dehydrated potato flakes (Pillsbury, Minneapolis, Minnesota) (20 g/L), D-glucose (20 g/L) and agar (15 g/L). Cultures intended for preparation of protoplasts or DNA were grown overnight in 15 mL of malt-extract medium (50 g malt extract, 50 g lactose, 5 g asparagine and 0.4 g K2HPO4 per L) in a Petri dish (20 mm deep, 100 mm diam; Fisher Scientific, Pittsburgh, Pennsylvania) on an orbital shaker set at 37 C and 150 rpm. Nonconidiating mutants were cultured similarly except pieces of agar-based cultures containing hyphal fragments were used in place of conidia as inoculum.
Disruption and complementation of brlA.— –
The presence of a brlA orthologue in the A. fumigatus genome (Nierman et al 2005) was determined by tblastn with protein sequence deduced from A. nidulans brlA (Adams et al 1988) as a query sequence. A 640-bp fragment internal to the coding sequence of the putative brlA orthologue was amplified by PCR primed with oligonucleotides brlAF (5'-GCCACGCT-TACGACAGAGGT-3') and brlAR (5'-CGGTAGATAGGC-GACTGGTG-3'). The 50 µL PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% (v/v) Triton X-100, 1.5 mM MgCl2, 200 mM of each deoxyribonucleotide triphosphate, 1 µM of each primer and 2.5 units of Taq DNA polymerase (Promega, Madison, Wisconsin), which was added once the thermocycler reached 95 C in the initial denaturing period. The reaction began with an initial denaturing step of 2 min 30 s at 95 C, followed by 35 cycles of 1 min at 94 C, 1 min at 57 C and 1 min at 72 C, with a final extension step of 72 C for 7 min. A 4.6 kb disruption construct, pBRLA1, was generated by ligating the PCR product into the T/A overlap vector pCR2.1 (Invitrogen, Carlsbad, California). The construct was linearized at the unique NdeI site located within the brlA fragment before transformation of the fungus. Protoplasts were prepared and transformed according to the protocol described by Murray et al (1992) and with modifications noted by Coyle and Panaccione (2005). Protoplasts were cotransformed with the linearized disruption construct, pBRLA1, and a hygromycin resistance plasmid, pMOcosX (Orbach 1994), linearized at a unique NotI site (Coyle and Panaccione 2005). Nonconidiating transformants were identified by their altered colony morphology and color and were confirmed by light microscopy. Nonconidiating mutants were purified to nuclear homogeneity through repetitive subculturing from hyphal tips (because nuclear purification via single conidium isolation was not possible). Homologous recombination between pBRLA1 and the native brlA locus within the transformants was verified by three PCR assays similar to the PCR reaction described above but with different primers and annealing temperatures. The 5' border of the recombination event was confirmed by PCR (annealing temperature at 57 C) primed with oligonucleotides UF (5'-TGTAAAACGACGGCCAGT-GAAT-3', which anneals to vector sequences near the universal primer annealing site in pCR2.1) and brlAFscrn (5'-CTCCAACGAATGTCCGTCTATG-3', complementary to brlA sequences near the 5'-end of the brlA gene and flanking the intended site of integration) (FIG. 1a). The juxtaposition of sequences near the 3' border of the integration event was verified by PCR (annealing temp 55 C) primed from oligonucleotides UR (5'-AGCTATGAC-CATGATTACGCCA-3', complementary to vector sequences near the reverse primer annealing site of pCR2.1), and brlARscrn (5'-TAGTGACAAGCTCTGCTTGGA -3', which anneals to brlA sequences near the 3'-end of the gene and flanking the intended site of integration) (FIG. 1a). PCR across the entire brlA locus was conducted by priming with oligonucleotides brlAFscrn and brlARscrn (both described above) at an annealing temperature of 55 C.
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, except for substitution of a brlA-specific probe. The brlA probe was made by amplifying wild-type A. fumigatus genomic DNA via PCR from primers brlAF and brlAR, according to the PCR conditions described above, substituting 1x DIG DNA Labeling Mix (Roche, Mannheim, Germany) for unlabeled dNTP and increasing the annealing temperature to 57 C.
The brlA ko strain was complemented with a 2.9 kb fragment containing a full-length copy of the brlA coding sequences along with approximately 1 kb of 5'-flanking sequences and 0.6 kb of 3'-flanking sequences. The complementing fragment was amplified by PCR primed from WbrlAF (5'-CTCTCCAGGAACCTTGAAACAGGTTC-TC-3') and WbrlAR (5'-CCACATAGGACACTCGAGGATG-GAGACA-3') in a 50 µL PCR reaction containing 15 mM (NH4)2SO4, 50 mM Tris-HCl (pH 9.0), 0.1% (v/v) Triton X-100, 1.5 mM MgCl2, 200 mM of each dNTP, 1 µM of each primer and 1 unit of DyNAzyme DNA polymerase (New England Biolabs, Beverly, Massachusetts) added during the initial denaturing step. The thermocycler program was as described above; however the annealing temperature was 62 C and the extension step was 3 min 20 s. The PCR product was cotransformed into a brlA-disrupted mutant along with a phleomycin resistance plasmid, pBC-phleo (Fungal Genetics Stock Center, University of Missouri at Kansas City, Kansas City, Missouri). Transformants were selected on complete regeneration medium (Panaccione et al 2001) containing 100 µg/mL phleomycin and incubated at 37 C. Complemented transformants were cultured from individual germinated conidia to achieve a homokaryotic culture. To test for the presence of the original disruption construct PCR reactions were initiated from primers brlAFscrn and UF (for the 5' flank of the integration) and UR and brlARscrn (for the 3' flank). An additional PCR screen was performed with primers WbrlAF and WbrlaR (described above) to test for the presence of the introduced wild-type allele in the complemented transformants. PCR conditions are provided above with descriptions of the primers.
Analysis of ergot alkaloids and conidiation.— –
For routine analysis of ergot alkaloids small cubes of cultures grown on PDA (containing approximately 8 mm x 8 mm of colony surface area) were submerged in 0.5 mL of 80% methanol in a 1.9 mL microcentrifuge tube and pulverized with a plastic pestle. The samples were rotated end over end (44 rpm) 2 h and spin filtered through a 0.2 µM pore nylon filter. Ergot alkaloids were detected and quantified from 20 µL of the prepared sample by reverse-phase HPLC on a C18 column with fluorescence detection based on excitation and emission wavelengths of 272 nm and 372 nm respectively (Panaccione and Coyle 2005). The identities of peaks corresponding to festuclavine and fumigaclavines A, B and C were established previously by mass spectral analyses of native and de-esterified fractions (Panaccione and Coyle 2005) and confirmed by elimination of these peaks from extracts of strains in which the first gene in the ergot alkaloid pathway (dmaW) was inactivated by gene disruption (Coyle and Panaccione 2005).
To quantify ergot alkaloids and conidiation from the same samples cultures were gown on PDA 5 d at 37 C and an additional 9 d at room temperature. Samples containing 50.3 mm2 of colony surface area were extracted from cultures with the broader end of a disposable 1 mL pipette tip (Fisher Scientific, catalog No. 02–681–172). Samples were submerged in 1 mL 80% methanol, agitated by vortexing and rotated at 44 rpm 2 h. Separate aliquots were removed for HPLC analyses and for quantification of conidia. Concentrations of ergot alkaloids were based on peak area relative to an external standard of agroclavine (Sigma, St Louis, Missouri) as described by Panaccione and Coyle (2005). Conidia were enumerated in dilutions of the methanol extracts with a hemacytometer and light microscope. Six samples were analyzed for each treatment. Data from wild type and a complemented brlA disruptant were compared by Student’s t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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from A. nidulans (Mah and Yu 2006). Based on sequence analysis there is no evidence for capacity to encode the alternate BrlAβ product that has been observed in A. nidulans (Prade and Timberlake 1993). BrlAβ of A. nidulans is transcribed from the same locus as BrlA and is identical to BrlA except for containing an additional 23 amino acids at the amino terminus. In A. fumigatus only two of 19 amino acid residues preceding the initiator methionine of BrlA are identical to those in the analogous positions of BrlAβ of A. nidulans. A fragment of A. fumigatus DNA corresponding to nucleotides 191–830 of the BrlA coding sequence of A. nidulans was used to direct homologous recombination of the gene disruption construct pBRLA1 at the brlA locus of A. fumigatus (FIG. 1a
). Homologous recombination of pBRLA1 at brlA would interrupt coding sequences common to both brlA and brlAβ (if it were to occur) in A. fumigatus.
Cotransformation of pBRLA1 and NotI-linearized pMOcosX into A. fumigatus resulted in five nonsporulating colonies out of a total of 93 hygromycin-resistant transformants. Agar-based cultures of the putative brlA disruption strains were white and fluffy, unlike the dense, sooty, dark-green appearance of wild-type cultures (FIG. 2a). Observed by light microscopy the nonconidiating cultures had extensive, bristle-like, aerial hyphae that lacked the swollen apical vesicles, phialides and conidia of mature conidiophores (FIG. 2b). Putative disruptants that had been subcultured from hyphal tips six times in an effort to achieve a homokaryotic culture were analyzed by PCR and Southern blot hybridization. Amplification of transformant DNA annealed to primer sets UF and brlAFscrn resulted in an 899-bp product (FIG. 1). Similarly PCR from UR and brlARscrn produced an 863-bp product. Products of these sizes from transformants but not from the wild type indicated integration of the disruption construct into the native copy of brlA (FIG. 1). The lack of a PCR product from these transformants when amplification was primed from brlAFscrn and brlARscrn indicated the absence of a wild-type copy of brlA. Integration of one or more copies of pBRLA1 at brlA results in a large distance between priming sites, whereas in the wild type the primer annealing sites are separated by only 910 bp (FIG. 1). Moreover the absence of a product in the brlA disrupted strains (FIG. 1b) indicated nuclear homogeneity of these strains because any wild-type nuclei in the colony would provide template for amplification of the 910-bp product. Southern blot analyses confirmed the presence of multiple disruption constructs within brlA, which is evident by a large shift in the size of the AccI fragment containing the targeted gene region (FIG. 1c). The lack of multiple bands hybridizing to transformant brlA ko17 on the Southern blot verified that random integration of constructs elsewhere in the genome had not occurred (FIG. 1c).
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). Additional PCR analyses of the brlA locus in the complemented strains indicated that pBRLA1 was still present at the previously disrupted brlA locus because products of the predicted size were amplified from primer pairs flanking the site of integration of pBRLA1 (FIG. 3). Thus the complementing wild-type brlA fragment had integrated at a locus other than the native brlA and the recipient strain was derived from a brlA disruptant. Similar quantities of conidia were produced by a brlA-complemented disruptant and the wild type (TABLE I).
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, TABLE I). In contrast conidiating colonies of wild-type and the brlA-complemented strain accumulated festuclavine and fumigaclavines A, B and C in quantities that were similar by strain (FIG. 4, TABLE I). The relatively large peak eluting at 53 min in the brlA ko17 profile represents a metabolite associated with hyphae. It is present in low levels in wild-type and complemented extracts (FIG. 4), which come from colonies that contain abundant conidiophores and conidia but limited hyphae. Its presence in ergot alkaloid-deficient strains derived by knocking out the gene dmaW (Coyle and Panaccione 2005) indicates that it is unrelated to ergot alkaloids.
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, Schardl et al 2006) was examined for the BRE sequence, 5'-(C/A)(A/G)AGGG(G/A)-3' (Chang and Timberlake 1992), and its 26 occurrences were mapped (FIG. 5). Given the degenerate nature of the BRE sequence and its ability to function in both orientations it will occur by chance once in every 1024 bp of random sequence. BRE occurred once every 1404 bp in the coding sequences, whereas in the 5' noncoding regions these elements occurred once every 409 bp, making BRE about 3.4 times more common in the 5' noncoding regions. As assessed in a paired t-test BRE occurred at a higher frequency per bp in 5' noncoding sequences than in the corresponding coding sequences (P = 0.03). However there were no occurrences of the BRE sequences upstream of three of 11 genes including dmaW, the gene controlling the first step in the ergot alkaloid pathway (FIG. 5). Six other genes had less than three BRE within their upstream noncoding sequences. One 5' noncoding region contained four BRE sequences, and due to divergent orientation of genes this noncoding sequence was part of the promoter region of two ergot alkaloid cluster genes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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for brlA mutants of A. fumigatus constructed by a different knockout strategy. The absence of ergot alkaloids in our nonconidiating mutants indicates that ergot alkaloids are not produced during vegetative growth and are solely associated with conidiation in A. fumigatus.
The phenotype of the A. fumigatus brlA mutants indicates that brlA functions similarly in A. fumigatus as it does in A. nidulans. brlA first was discovered as the Mendelian locus defective in mutants that failed to conidiate but instead grew bristles reaching 20–30 times the normal conidiophore length (Clutterbuck 1969). The wild-type allele was cloned by complementation of the brlA mutant via transformation and recovery of the transforming DNA (Timberlake et al 1985). Adams et al (1988) demonstrated that expression of brlA under conditions that typically repress conidiation in A. nidulans (submerged broth culture) resulted in production of structures that produced conidium-like cells. Two developmentally regulated genes downstream of brlA, abaA and wetA were not expressed in the nonfunctional brlA mutant but were transcribed in the modified strains expressing brlA. Furthermore disruption of Cys2-His2-Zn(II) coordination sites resulted in the inability of BrlA to induce the conidiation pathway thus supporting the functionality of BrlA as a transcription factor that requires two zinc fingers to bind DNA (Adams et al 1990).
Links between sporulation and secondary metabolite production in Aspergillus spp. have been demonstrated by mutational analyses of other genes that regulate sporulation (Calvo et al 2002, Bok and Keller 2004). Relevant studies in A. fumigatus include work done on the regulatory genes stuA (Sheppard et al 2005) and laeA (Bok et al 2005). Strains mutated at stuA produced conidiophores that were stunted and appeared as vesicles that lacked phialides and arose directly from septate hyphae (Sheppard et al 2005). In microarray analysis stuA mutants showed changes in accumulation of transcripts from genes in two separate gene clusters that are hypothesized to control production of unidentified secondary metabolites. Numerous other genes were affected including those encoding genes and proteins involved in development and morphogenesis, such as the cell wall protein, PhiA, and a sexual development repressor protein, Pro1. Mutation of laeA impaired conidiophore (and conidium) development in broth cultures of A. fumigatus but laeA mutants conidiated normally on agar-based media. LaeA regulates expression of genes involved in conidiophore and rodlet formation as well as genes involved in production of secondary metabolites such as gliotoxin (Bok et al 2005).
Whether BrlA directly regulates the ergot cluster genes or whether those genes are controlled by a factor or factors whose expression depends upon brlA has not been established. Overall BRE were more abundant in the 5' noncoding sequences compared to their corresponding coding sequence. However the brlA response element was absent from the 5' noncoding sequences of three ergot alkaloid cluster genes (including dmaW) and was present in only one or two copies upstream of six other genes. Chang and Timberlake (1992) found that at least three BRE must be present in the upstream region to increase transcript accumulation from that gene. The occasional absence and relatively low copy number of the BRE associated with ergot cluster genes indicates that factors controlled by or in addition to BrlA regulate the ergot cluster genes. Previous work demonstrates that among the genes controlled by BrlA are genes encoding other regulatory factors such as AbaA and WetA (Boylan et al 1987, Adams et al 1988). A global analysis of gene expression in a brlA mutant compared to wild type, revealing the genes and secondary metabolite gene clusters controlled by BrlA, is already in progress (D.C. Sheppard, McGill University, pers comm).
Association of ergot alkaloids with conidiation might provide insight into the ecological significance and utility of these toxins to the fungus. When A. fumigatus grows pathogenically its hyphae colonize host tissue and conidia are not produced (Fraser 1993). Because ergot alkaloids are not produced in hyphae it is unlikely that they contribute to the invasive phase of aspergillosis. Conidia are produced during saprophytic growth and serve primarily for dissemination. Due to their exceptionally small size and low specific gravity (Panaccione and Coyle 2005) conidia can be inhaled deeply into the lungs of humans and animals. To initiate infection conidia must survive the initial host-defense response. The possibility that ergot alkaloids contribute to increasing the survival of inhaled conidia remains to be investigated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: Department of Plant Pathology, 406 Plant Sciences Hall, University of Nebraska at Lincoln, Lincoln, NE 68588–0722. 
2 Corresponding author. E-mail: danpan{at}wvu.edu
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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