Mycologia - Taylor & Francis Online

Mycologia, 101(4), 2009, pp. 449–472. DOI: 10.3852/08-163 # 2009 by The Mycological Society of America, Lawrence, KS 66044-8897

Discovery of the parnafungins, antifungal metabolites that inhibit mRNA polyadenylation, from the Fusarium larvarum complex and other Hypocrealean fungi Gerald F. Bills1 Gonzalo Platas David P. Overy Javier Collado Asuncioń Fillola Marıá Rosa Jimeńez Jesu´s Martıń Antonio Gonza´lez del Val Francisca Vicente J. Rubeń Tormo Fernando Pelaéz

of these data led to the conclusion that the diversity within the FLC exceeded the one-to-one correspondence between F. larvarum and its teleomorph Cosmospora aurantiicola. Based on multiple gene sequence analyses, strains of the FLC formed a monophyletic clade inclusive of the parnafunginproducing strains. The FLC, including newly discovered parnafungin-producing strains, could be resolved into at least six different lineages, possibly representing cryptic species, of which one was not fully resolved from F. larvarum var. rubrum. Fusarium larvarum var. rubrum represents a species distinct from var. larvarum. Finally we report that two other species from the Hypocreales, Trichonectria rectipila and Cladobotryum pinarense, are able to produce parnafungins and their open-ring forms. Key words: antibiotics, brefeldin A, chemotaxonomy, Cladobotryum pinarense, Cosmospora, entomopathogenic fungi, helvolic acid, lichenicolous fungi, secondary metabolites, Trichonectria rectipila

Centro de Investigacioń Ba´sica, Merck, Sharp & Dohme de Espan ˜ a S. A., Josefa Valca´rcel 38, Madrid, E-28027, Spain

Kathleen Calati Guy Harris Craig Parish Deming Xu Terry Roemer Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, New Jersey 07065

INTRODUCTION

Abstract: Evaluation of fungal fermentation extracts with whole cell Candida albicans activity resulted in the identification of a novel class of isoxazolidinonecontaining metabolites named parnafungins. Chemical-genetic profiling with the C. albicans fitness test identified the biochemical target as inhibition of polyadenosine polymerase, a component of the mRNA cleavage and polyadenylation complex. Parnafungins were discovered from fermentation extracts of fungi resembling F. larvarum isolated from plants, plant litter and lichens. Furthermore authentic strains of F. larvarum var. larvarum and F. larvarum var. rubrum could be induced to produce parnafungins and their degradation products in low titers. Relationships among strains of the F. larvarum complex (FLC), including parnafungin-producing strains, were examined by cladistic analyses of rDNA, mitochondrial rDNA, and two protein-coding genes, comparisons of antifungal activity and antifungal metabolite profiles, and morphological phenotypes. Integrated analyses

The Candida albicans fitness test (CaFT) is a targetbased antifungal screening platform that employs heterozygote strain sensitivities on a genome scale to predict the mechanism of action of compounds that are inhibitory against this clinically relevant fungal pathogen (Haselbeck et al 2002, Roemer et al 2003, Xu et al 2007, Jiang et al 2008). Applying this approach, MOA (mechanism of action) determination has been successfully validated with known bioactive compounds whose MOA is well characterized and extended to elucidating MOA of antifungal synthetic compounds and purified natural products (Roemer et al 2003, Rodriguez-Suarez et al 2007, Xu et al 2007, Ondeyka et al 2009). CaFT recently was applied to crude natural products extracts and incorporated into our antifungal discovery platform. Evaluation of natural products extracts with whole-cell C. albicans activity resulted in the identification of a family of interconverting natural products, named parnafungins (poly A RNA-fungin) based on their inhibition of poly-adenosine polymerase (PAP), one component of the mRNA cleavage and polyadenylation complex (Jiang et al 2008, Parish et al 2008).

Accepted for publication 23 January 2009. 1 Corresponding author. Present address: Fundacioń MEDINA, Armilla, Granada, 18100, Spain. E-mail: [email protected]

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FIG. 1. Structures of parnafungins: parnafungin A (1) and B (2) and their associated benzoquinoline analogs (3, 4), parnafungin C (5) and D (6) and their associated benzoquinoline analogs (7, 8).

Parnafungins (FIG. 1) are a novel class of isoxazolidinone-containing natural products. The fivemember isoxazolidinone ring with a nitrogenoxygen bond is unprecedented in an isolated natural product (Parish et al 2008). A mixture of the two major and two minor isomeric forms, parnafungins A and B (FIG. 1 [1 and 2]) demonstrated potent, broad spectrum antifungal activity. The isoxazolidinone ring of 1 and 2 is prone to opening, thus generating the benzoquinoline analogs 3 and 4 (FIG. 1). After ring opening of the isoxazolidinone antifungal activity of these natural products was no

longer observed. In addition to the isoxazolidinone ring parnafungins contain a xanthone ring system, which is related to the monomeric ergochrome unit of secalonic acids. Secalonic acids are polyketidederived natural products that are broad spectrum antimicrobial and cytotoxic agents (Buckingham 2007). We identified the parnafungins with Candida albicans-based chemical genetic profiling of crude fungal fermentation extracts with uncharacterized antifungal activity. The parnafungin containing extracts were selected for isolation because of their

BILLS ET AL: DISCOVERY OF THE PARNAFUNGINS distinct MOA profile, wherein multiple heterozygous mutants corresponding to subunits of the cleavage and polyadenylation complex displayed hypersensitivity to the bioactive entity. Isolation of the parnafungins was accomplished by tracking of anti-C. albicans activity, and the CaFT profile of the purified material matched that obtained from the original extract (Parish et al 2008). Mechanistically the parnafungin CaFT profile most closely matched that of cordycepin (39-deoxyadenosine), a natural product pro-drug which is converted to 39dATP within cells and inhibits mRNA polyadenylation (Muller et al 1977, Rose, Bell, Jacob 1977). The molecular target of the parnafungins was further dissected with a Saccharomyces cerevisiae-based cell-free cleavage and polyadenylation assay where 39 mRNA polyadenylation was preferentially impaired versus cleavage of the mRNA transcript. Indeed recombinant C. albicans PAP enzyme activity was inhibited by the natural product at picomolar levels, with IC50 levels fivefold lower than S. cerevisiae or human PAP. A purified mixture of parnafungins A and B displayed potent and broad spectrum activity across diverse clinically relevant fungal pathogens and significant in vivo efficacy in a murine model of disseminated candidiasis (Jiang et al 2008). The first two fermentation extracts used to isolate and characterize the antifungal parnafungins were from fungal strains resembling Fusarium larvarum Fuckel (Ascomycota, Hypocreales) isolated from lichens from the province of Madrid, Spain (Jiang et al 2008, Parish et al 2008). New fermentation extracts of strains resembling F. larvarum from plants, plant litter and lichens subsequently yielded CaFT profiles highly related to parnafungins. As predicted by CaFT profiling and rDNA sequencing of the strains, mass spectrometry (MS) data confirmed that the bioactivity within extracts of these F. larvarum-like fungi also were attributable to parnafungins. Features common to these fungi of the F. larvarum complex (FLC) were slow growth, dull orange, orange brown to yellow mycelia in agar culture, development of vinaceous pigments in the aerial mycelium of old cultures, erratic in vitro sporulation, and when cultures sporulated, sporulation was a phialidic anamorph conforming to F. larvarum, the conidial state of Cosmospora aurantiicola (Berk. & Broome) Rossman & Samuels (; Nectria aurantiicola Berk. & Broome). Sequences of the large subunit (LSU) D1D2 regions of rDNA of these fungi were highly similar to a D1D2 sequence of F. larvarum var. larvarum (Gerlach 1977) from a San Jose´ scale insect on a plum tree (Prunus domesticus) in Iran (CBS 738.79, NRRL 20473, GenBank U88107) and strains of an undescribed species of Fusarium pathogenic on lichens in the

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eastern United States (Lawrey, Torzilli, Chandhoke 1999, Torzilli et al 2002). Nectria aurantiicola has been included in genus Cosmospora because of its reddish, thin-walled, smooth perithecia with little stromatic development, and its light brown, verrucose, two-celled ascospores (Rossman et al 1999). However genus Cosmospora has been recognized as being polyphyletic. For example Cosmospora coccinea Rabenh., the type species, is characterized by its Verticillium olivaceum W. Gams anamorph. Therefore reclassification of Co. aurantiicola and other fungi with Fusarium anamorphs in sections Coccophilum and Episphaeria is needed. Cosmospora aurantiicola and its anamorph F. larvarum var. larvarum are associated with scale insects and aphids (Booth 1971, Gerlach 1977, Rossman et al 1999, Tyson et al 2005). Cosmospora aurantiicola also has been observed to be hyperparasitic on Septobasidium clelandii Couch, a parasite of Callococcus leptospermi (Homoptera, Asterolecaniidae) in Australia (Booth 1981) and has been observed on the lichens Teloschistes chrysophthalmus (L.) Th. Fr. and T. sieberianus (Laurer) Hillmann in New Zealand (B. Paulus and P. Johnston, Landcare Research, Auckland, pers comm). The objective of this study was to clarify the relationships of the parnafungin-producing strains of the FLC to the two varieties of F. larvarum. At the time of the discovery of the parnafungins rDNA sequence data from Co. aurantiicola or F. larvarum var. rubrum were unavailable for comparisons (Parish et al 2008). The fact that some of the parnafungin-producing strains were recovered from unidentified lichens and plant material, plus the observations of F. larvarum-like fungi on lichens in the USA and New Zealand, also brought into question whether F. larvarum is strictly associated with insects. Perhaps F. larvarum is a widespread species with cryptic phases of its life cycle that might colonize plant or lichen surfaces awaiting the establishment of an appropriate insect host. On the other hand different cryptic species might be embedded within the morphological species concept of F. larvarum, as has been the case in other fungal species complexes (Taylor et al 2000, Starkey et al 2007). These questions were addressed by cladistic analyses of rDNA, mitochondrial (mt) rDNA, and two protein-coding genes, comparison of antifungal activity and antibiotic metabolite profiles and morphological phenotypes from insect-, plant- and lichen-derived strains referable to the FLC. We also report that two other species from the Hypocreales, Trichonectria rectipila and Cladobotryum pinarense, are able to produce parnafungins and their open-ring forms.

NRRL 20475 NRRL 22170 NRRL 26803 NRRL 26790 F-265,963, CBS 123556 F-242,915, CBS 123347 F-253,264, CBS 123557 F-159,080, MF7022, ATCC PAT 7894 F-159,081, MF7023, ATCC PAT 7895 F-155,597, CBS 123558 F-257,517, CBS 123348 CBS 267.81

F. larvarum var. rubrum F. larvarum var. rubrum F. sp.

F. sp. F. sp.

F. larvarum complex

F. larvarum complex

F. larvarum complex

F. larvarum complex F. larvarum complex F. merismoides var. merismioides Corda

F. larvarum complex

CBS 638.76, ex-isotype

F. larvarum var. rubrum

Living stem of Fragula alnus Unidentified lichen Rhizoctonia solani, old sclerotia on old tubers

Unidentified lichen

Litter of Pinus nigra subsp. pallasiana Unidentified lichen

Living stems of Pinus kochiana

Puntelia rudecta Unidentified lichen

Scale insect on Salix 3 reichardtii Scale insect on Citrus maxima Aphid on Pyrus communis Quadraspidiotus perniciosus on Prunus vulgaris Quadraspidiotus perniciosus on Prunus vulgaris Derived from CBS 638.76 Derived from CBS 638.76 Lasallia paulosa

ICMP 11047 CBS 158.57 CBS 169.30 CBS 738.79, NRRL 20743

Co. aurantiicola F. larvarum var. larvarum F. larvarum var. larvarum F. larvarum var. larvarum

Substratum/origin Scale insect

Strain numbers ICMP 5444

Identification

Location

— — St. Mary’s Wilderness, Augusta Co., Virginia, USA Massachusetts, USA Green Mountain National Forest, Vermont, USA Djavakheti, Akhalkalaki, Republic of Georgia Gardabani, Kumisi Lake Area, Republic of Georgia Miraflores de la Sierra, Madrid, Spain Miraflores de la Sierra, Madrid, Spain Uncastillo, Zaragoza, Spain Palancares, Guadalajara, Spain The Netherlands

Near Rasht, Gilan, Iran

Tauranga, Bay of Plenty, New Zealand Gisborne, New Zealand Unknown Japan Iran

Strains, accession numbers, and origins of strains of Fusarium larvarum complex and related fungi

Cosmospora aurantiicola

TABLE I.

— — —

(Parish et al 2008)

(Parish et al 2008)

—

—

(Torzilli et al 2002) —

— — (Torzilli et al 2002)

— — (Booth 1971) (Gerlach 1977, Torzilli et al 2002) (Gerlach 1977)

—

References

452 MYCOLOGIA

Cuyuni-Mazaruni Region, Guyana Bombacaceae, decaying fruit Viridispora alata (Samuels) Samuels & Rossman

CBS 421.88

(Castan ˜ eda Ruiz 1986) (Rossman et al 1999) Province of Pinar de Rıó, Cuba CBS 400.86

On Auricularia sp.

F-202,821, CBS 123559 CBS 132.87

— (Samuels 1988) Living tissue of Fragaria vesca On Diatrype stigma

CBS 740.79

Isaba, Navarra, Spain Chester Co., Pennsylvania, USA

Wooden pole in seawater

F-223,908, CBS 123378

F. merismoides var. acetilereum Tubaki F. merismoides var. crassum Wollenw. F. ciliatum Sacc. Trichonectria rectipila Samuels, Rogerson & M.E. Barr Cladobotryum pinarense

—

Tree bark with decaying basidiomycete Soil F-167,589, CBS 123349 F. merismoides var. violaceum

—

—

Varirata National Park, Central Province, Papua New Guinea Salazie Valley, Saint-Denis, Reúnion Island Bangahani, Grand Comore, Union of Comores Germany

—

(Gerlach 1977) Iran

Quadraspidiotus perniciosus on Prunus vulgaris Soil CBS 634.76, ex-isotype

CBS 860.95

Identification

F. merismoides var. violaceum Gerlach F. merismoides var. violaceum

TABLE I.

Continued

Strain numbers

Substratum/origin

Location

References

BILLS ET AL: DISCOVERY OF THE PARNAFUNGINS

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MATERIALS AND METHODS

Fungal strains morphological evaluation.—Parnafungin-producing strains of the FLC were isolated in our laboratories (TABLE I). Additional strains of related Fusarium species and other hypocrealen fungi were obtained courtesy of the National Center for Agricultural Utilization Research (NRRL) and Landcare Research (ICMP) or purchased from the Centraalbureau voor Schimmelcultures (CBS) (TABLE I). Strains were maintained as frozen mycelium in 10% glycerol at 280 C. Duplicates of newly discovered strains are deposited at the American Type Culture Collection or CBS (TABLE I). Strains were grown on cornmeal agar (CMA, Sigma), 2% malt agar and oat cereal agar (40 g oat cereal, 20 g agar/l distilled H2O) for morphological studies. Agar cultures were incubated at 22 C with 12 h fluorescent light and with or without treatment with near-UV light. Sporulation was evaluated after 3–4 wk, but in many cases agar plates were incubated up to 2–3 mo because the onset of sporulation was often slow. Photomicrographs were taken from structures mounted in 5% KOH with either Nomarski or phase contrast microscopy. Capitalized color names in parentheses are from Ridgway (1912). Fermentation.—Strains producing parnafungins were discovered successively employing two fermentation systems. Strains F-159,080, F-159,081 and F-155,597 were discovered from 12 mL fermentation agitated in 40 mL vials as described by Parish et al (2008). Antifungal activities caused by parnafungins in Fusarium strains F-242,915, F-253,264, F-257,517, Trichonectria rectipila (CBS 132.87) and Cl. pinarense (CBS 400.86) were discovered by screening strains in nutritional arrays in a semi-automated microplate screening system modified for fungi (Bills et al 2008). The tools and protocols for cultivation of microorganisms in the microplate screening system (Duetz 2007) can be found at www.enzyscreen.com. Empirical comparisons among the strains on different fermentation media (data not shown) indicated antifungal activity was observed most consistently in a medium designated MMK2 (mannitol 40 g, yeast extract [Becton Dickinson] 5 g, Murashuge & Skoog salts [Sigma M5524] 4.3 g; 1000 mL distilled water). All subsequent strain comparisons employed medium MMK2 in both 40 mL EPA vials and 24-well fermentation plates. Procedures for growing liquid inoculum and inoculating fermentations have been described by Bills et al (2008) and Ondeyka et al (2009). EPA vial fermentations employed 12 mL medium per vial. Strains were inoculated individually in triplicate with 0.5 mL liquid inoculum. Vials were agitated at (220 rev/min, 5 cm throw) and incubated 14 d at 22 C. For 24-well plate fermentations each well of a 24-well inoculum plate was filled with approximately 2 mL hyphal suspension from each inoculum tube. This master plate was used to inoculate 24-well fermentation plates that contained 4 mL MMK2 medium per well. Plates were clamped to a shaker board, agitated at (250 rev/min, 5 cm throw) 14 d at 22 C. Master plates and fermentation plates were checked

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for contamination with procedures described by Bills et al (2008). Metabolite extraction, MS and HPLC analysis.—Mycelia were extracted by adding 3.2 mL acetone to each well. Plates were capped with a polypropylene cap mat and agitated (220 rev/min) 1 h and centrifuged. To retain metabolites in solution during the solvent evaporation 640 mL pure DMSO was added to each of the wells, and after 5 min additional shaking the mixture was placed in a Genevac HT-24 computer-controlled evaporator 1 h to remove the acetone. After evaporation and centrifugation plates remained opened in a chemical hood while automated liquid handling of the upper water/DMSO extract was processed with a Beckman Biomek FX robot. The final aqueous extract was approximately 13 whole broth equivalent (WBE) with 20% DMSO. Two 500 mL aliquots from each well were transferred to two 800 mL well AB-gene AB-0765 storage blocks. Extracts were stored at 4 C 2 d and were mixed briefly before assay. Extracts prepared from multiwell plate fermentations were profiled by HPLC-DAD-MS (Agilent 1100 series HPLC coupled with an Agilent MSD [Santa Clara, California]) 1100 single quadrupole mass spectrometer) by injecting 2 mL extract onto a Zorbax SB-C8 column (2.1 3 30 mm), maintained at 40 C with a flow rate of 300 mL/min. Solvent A consisted of 10% acetonitrile (ACN) and 90% water with 1.3 mM trifluoroacetic acid and ammonium formate, while solvent B was 90% ACN and 10% water with 1.3 mM trifluoroacetic acid and ammonium formate. The gradient started at 10% B and went to 100% B in 20 min, kept at 100% B for 2 min and returned to 10% B for 2 min to initialize the system. Full diode array UV scans 100–900 nm were collected in 4 nm steps at 0.25 s/scan while mass spectra were collected as full scans 150–1500 m/z, with one scan every 0.77 s, in both positive and negative modes (source temp 325 C, capillary voltage 3500 V, nebulizer pressure 40 psig, drying gas flow, 11 l/min). Vial fermentations were extracted with an equivalent volume (12 mL) of ethyl acetate (EtOAc). After addition of the solvent, mycelia were macerated with a spatula, and the vials were agitated 2 h (150 rev/min). The vials were left to stand 1 h to allow for adequate phase separation before the organic solvent layer was removed by pipette. A 1 mL aliquot of each extract was transferred to a glass HPLC vial and the solvent was evaporated under a stream of N2 for HPLC-MS-DAD analysis. The dried residue was resuspended into 100 mL ACN of which a 5 mL aliquot was profiled by HPLC-DAD-MS (Agilent 1100 series HPLC coupled with a Waters QuattroPro triple quadrupole MS) on a Luna C-18 Phenomenex column (50 mm 3 2 mm i.d., 3 mm particles, pore size 100 A˚) with a 33 min H2O : ACN gradient (both solvents containing 20 mM formic acid), eluting at a flow rate of 0.3 mL/min, beginning at 15% ACN, increasing to 100% ACN in 20 min, maintaining 100% ACN 5 min, returning to 15% ACN in 3 min and maintaining 15% ACN for an additional 5 min to re-equilibrate the column. UVVIS spectra were collected from 200–900 nm in 4 nm steps at 0.25 sec/scan with an Agilent DAD. Nominal mass spectral data were acquired across a range of 200–

1500 m/z with a scan-time of 0.5 s and interscan delay of 0.1 s. MS source parameters were capillary voltage 3 kV, cone voltage 30 V, extractor voltage 2 V, Rf lens voltage 0.2 V, source temperature 150 C, desolvation temperature 325 C and desolvation gas flow 600 l/h. Genomic DNA extraction, PCR amplification and sequencing.—Aerial mycelia of strains grown on malt-yeast extract agar were used for DNA extraction (Pelaéz et al 1996). A preliminary rDNA phylogeny based on the ITS and D1D2 regions was used to approximate the phylogeny of strains F-155,080, F-155,081 (Parish et al 2008) and F-155,597. Ambiguities in species level resolution prompted us to pursue a multigene phylogeny strategy including authentic F. larvarum strains to refine phylogenetic relationships among parnafungin-producing strains and their putative relatives. PCR reactions were performed with standard procedures (5 min at 93 C, followed by 40 cycles of 30 s at 93 C, 30 s at 53 C and 2 min at 72 C) with Taq DNA polymerase (QBiogene Inc.), according to the manufacturer’s recommendations. Primer sequences for multilocus phylogeny of other Fusaria were suggested by K. O’Donnell, except for H3F2 which was designed in our laboratory. PCR was used to amplify these genes with their correspondent primers: elongation factor 1a (EF-1a): EF-1.59-ATGGGTAAGGARGACAAGAC-39, EF-2.59-GGARGTACCAGTSATCATG-39. btubulin: T1.59-AACATGCGTGAGATTGTAAGT-39, T22.59TCTGGATGTTGTTGGGAATCC-39. ITS+D1D2 nuclear rDNA: ITS5.59-GGAAGTAAAAGTCGTAACAAGG-39, NL4. 59-GGTCCGTGTTTCAAGACGG-39. Intergenic spacer region (IGS rDNA): NL11.59-CTGAACGCCTCTAAGTCAG-39, CNS1.59-GAGACAAGCATATGACTAC-39. RNA polymerase 2: 5f2.59-GGGGWGAYCAGAAGAAGGC-39, 7cr.59CCCATRGCTTGYTTRCCCAT-39. RNA polymerase 2: 7cf. 59-ATGGGYAARCAAGCYATGGG-39, 11ar.59-GCRTGGATCTTRTCRTCSACC-39. Calmodulin: CL1.59-GARTWCAAGGAGGCCTTCTC-39, CL2A.59-TTTTTGCATCATGAGTTGGAC-39. Histone H3: H3F1.59-TGGCAAGGCCCCTCGCAAGC-39, H3R1.59-TTGGACTGGATRGTAACACGC-39, H3F2.59-CCACTGGTGGCAAGGCCCC-39. Mitochondrial ribosomal small subunit DNA (mtSSU rDNA): MS1.59-CAGCAGTCAAGAATATTAGTCAATG-39, MS2.59-GCGGATTATCGAATTAAATAAC-39. a-tubulin: a-tub-1.59-CATCTGCAACACTGCGTGARG-39, a-tub-2.59-CTCAGCCTCCAARTCRTCYTC-39. Putative reductase: RED1d.59-TCTCAGAAAGACGCATATATG-39, RED2.59-CGTAACTGCGTCATTCGGC-39. Phosphate permase: PHO1.59-ATCTTCTGGCGTGTTATCATG-39, PHO6.59-GATGTGGTTGTAAGCAAAGCCC-39. Ammonia ligase: URA1. 59-ATGAAGGTTGTTCTTGTGAGCGGCGG-39, URA10. 59-GCAATCTTTGTGATGGTAGCTTGATC-39. Ammonia ligase: URA11. 59-GAGTATGCCCGCAACGTCATG-39, URA16. 59-AATTATCTCATCGAGACATCC-39. Additional primers were used to sequence long DNA fragments: b-tubulin: (T2. 59TAGTGACCCTTGGCCCAGTTG-39, T11. 59-AATTGGTGCTGCTTTCTGGCA-39, T12. 59-AACAACTGGGCCAAGGGTCAC-3, T224. 59-GAGGGAACGACGGAGAA GGTGG-39 (O’Donnell and Cigelnik 1997). For ITS + D1D2 nuclear rDNA: NL1.59-GCATATCAATAAGCGGAG-

BILLS ET AL: DISCOVERY OF THE PARNAFUNGINS GAAAAG-39 and its reverse primer NL1R (O’Donnell, 1993). Amplification products were sequenced with the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) following the manufacturer’s recommendations. Each strand of the amplification products was sequenced with the same primers used for the initial amplification. Partial sequences obtained in sequencing reactions were assembled with Genestudio 2.1.1.5. (Genestudio Inc., Suwanee, Georgia). DNA sequences were aligned with Genestudio 2.1.1.5. All sequences were deposited in GenBank (TABLE I). Alignments and trees were deposited in TreeBASE (SN4042). Sequence data and phylogenetic analysis.—Bayesian inference and Markov chain Monte Carlo simulations (B/ MCMC) were used to estimate phylogenetic hypotheses from individual gene datasets and combined datasets and were implemented in the computer program MrBayes 3.01 (Ronquist and Huelsenbeck 2003). Four incrementally heated simultaneous MCMC were run over 2 000 000 generations to improve mixing of the chain. Hierarchical likelihood ratio tests to calculate the Akaike information criterion (AIC) values of the nucleotide substitution models were run with MrModeltest 2.2 (Nylander 2004). The AIC selected the HKY+I+G model for the alignment of the b-tubulin gene fragment, which allowed two classes of substitution types, a portion of invariant alignment positions and mean substitution rates varying across the remaining positions according to a gamma distribution. For the alignment of the histone H3 gene fragment the ITS+D1D2 nuclear rDNA gene fragment and mtSSU rDNA gene fragment as well as the consensus alignment obtained by the addition of all the sequences of the four genes, the AIC selected the GTR+I+G model. This model allowed six classes of substitution types, a portion of invariant alignment positions and mean substitution rates varying across the remaining positions according to a gamma distribution. Priors used for the MCMC process were a Dirichlet distribution for substitution rates and nucleotide frequencies and a uniform prior for the rate parameter of the gamma distribution. For all analyses the sampling frequency at which the trees were stored was 100, and the first 1000 trees were discarded; the support of nodes was tested with clade credibility values. Antifungal assays.—Antifungal susceptibility was tested with C. albicans strain MY1055 from the Merck Culture Collection. Thawed stock inoculum suspensions from cryovials were streaked on Sabouraud dextrose agar plates (Becton Dickinson, 65 g/l H2O) to grow new colonies. A few colonies were inoculated in 10 mL Sabouraud dextrose broth (Becton Dickinson, 30 g/l H2O) that was incubated overnight at 37 C. The C. albicans suspension was adjusted to an optical density of 0.4 at 660 nm. This suspension was added to yeast nitrogen base-dextrose agar (Becton Dickinson, 6.75 g/l, dextrose 10 g/l, agar 15 g/l H2O) in the proportion of 30 mL/l. Twenty milliliter aliquots of the seeded agar media were poured into Omnitray plates. Wells (4 mm diam) were formed in the agar with a removable pin

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lid. Aqueous acetone-derived extracts (10 mL) were applied in the wells of the seeded assay plates, which were incubated at 30 C approximately 20 h. Zones of inhibition (ZOI) were recorded by image analysis. The tests were performed twice with the two acetone extracts prepared for each sample. Antibiotics with positive and negative responses were used as internal plate controls (amphotericin B, 0.25 mg/mL and kanamycin 5 mg/mL). Screening fungal extracts with CaFT.—Our approach to antifungal lead discovery used CaFT to identify fermentation extracts with distinct bioactivities (thus likely new chemical entities). CaFT has been described by Xu et al (2007). The version used to discover parnafungin employed 2868 genetically engineered strains, each of which was heterozygous for a unique gene. These strains represent , 45% of the C. albicans genome. The relative abundance of each strain in a compound-treated culture was determined by DNA microarrays that identify the two DNA barcodes introduced at the deleted allele. The variation in growth (fitness) in response to an inhibitor at a sublethal concentration was expressed by the normalized z-score, hypersensitivity if greater than zero, hyposensitivity if less than zero. A compendium of known compounds from natural sources was selected. They were tested in the CaFT, and their profiles constructed. This compendium was augmented when additional compounds were identified. Crude fermentation extracts with desirable antifungal activity and spectrum were tested in the CaFT at multiple concentrations. Informative and reproducible profiles were compared with those in the compendium. When a distinct profile was noted the extract was selected for isolation guided by an antifungal activity assay and the CaFT if multiple active entities were separated during the fractionation step. The purified compound was tested in the CaFT for confirmation. When a profile matched a known compound its presence in the extract was determined by analytical chemical means, usually LC-MS database matching. If the result was negative the extract was fractionated further to identify the component responsible for the CaFT profile.

RESULTS

Discovery of parnafungin-producing fungi.—Antifungal activities in crude fermentation extracts of F-159,080, F-159,081 and F-155,597 were found by empirical screening of a large collection of extracts in a wild-type C. albicans ZOI assay. The extracts were evaluated in the CaFT, with each producing highly related profiles that overlapped with that of cordycepin, a known inhibitor of 39 mRNA processing (Muller et al 1977, Rose, Bell, Jacob 1977) (FIG. 2). However the profiles of these extracts indicated a clear mechanistic distinction that was inconsistent with an adenonsine analog. Bioassay-guided isolation of antifungal activity led to the identification of an interconverting mixture of isomeric natural products, which were identified as parnafungins A and B

456

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FIG. 2. CaFT profiles of parnafungins, cordycepin, brefeldin A and the fermentation extracts of strains of the F. larvarum complex, T. rectipila and Cl. pinarense. Two independent CaFT experiments, with comparable inhibitory concentrations (ICs), were selected for each active. The relative behavior of each heterozygous deletion strain elicited by antiproliferative agents in each experiment is expressed statistically by two normalized z-scores, corresponding to two DNA barcodes introduced at the deleted allele, with positive value indicating hypersensitivity and negative resistance. For each strain in a particular experiment, the z-score of higher absolute value was selected (Xu et al 2007). To compare CaFT profiles strains with absolute value of z-scores no less than 3.5 in at least two experiments were selected. They (51 in total from the experiments chosen) were grouped by hierarchical clustering with the centroid linkage method (Cluster 3.0), the result of which is displayed in TreeView with z-scores between +2 and 22 masked, with scale of heat shown in the top left corner and the hierarchical linkage on the right. The identities of antifungal actives and experimental conditions are shown on the left. The heterozygous deletion strains are indicated by the orf19 designations of the corresponding genes, with C. albicans genes and S. cerevisiae homologs (if any) indicated on the top of the heat map. The C. albicans genome annotation is adapted from the Candida Genome Database (www.candidagenome.org). Highlighted are hypersensitive (red boxes) and resistant (green boxes) heterozygotes whose corresponding genes are implicated in MOA of antifungal compounds: Group 1, subunits of RNA cleavage and polyadenylation complex (for parnafungin and cordycepin); Group 2, proteins involved in ribosomal biogenesis (parnafungin); Group 3, subunits of RNA polymerase II (parnafungin); Pop2p, mRNA deadenylase (parnafungin and cordycepin); Group 4, targets (Sec7p and Arf2p) and efflux pump of brefeldin A; and Group 5, transporter (Cnt1p) and activating enzyme (Ado1p) of cordycepin. For detailed description of parnafungin and cordycepin experiments see Jiang et al (2008), for brefeldin A see Xu et al (2007).

(FIG. 1) (Parish et al 2008) and reproduced the original CaFT profile (FIG. 2). Biochemical and genetic studies determined that the molecular target of parnafungins was the poly(A) polymerase (Jiang et al 2008). In addition to parnafungins A and B (FIG. 1 [1 and 2]), the inactive open ring forms (FIG. 1 [3 and 4]) also were isolated and characterized from strains F-159,080 and F-159,081. Because the opening of the isoxazolidinone ring involves the addition and subsequent elimination of one molecule of water, all four compounds (1–4) are isobaric. HPLC-DAD-MS analysis of an extract from strain F-155,597 and a comparison of these data with a standard of parnafungins A and B (FIG. 3) and the other producing

strains indicated the presence of two additional parnafungin analogs 5 and 6 (FIG. 1) having similar UV absorption spectra but differing both in retention time and m/z. Positive and negative mode ESI-MS were consistent with molecular weights of 465 ([M+H]+ 466; [M2H]2 464) and 479 ([M+H]+ 480; [M2H]2 478) for parnafungins C and D, respectively. Further, high resolution mass spectrometric analysis indicated that the molecular formula of parnafungins C was C24H19NO9 and that of parnafungin D was C24H17NO10. Structural elucidation of these components identified parnafungin C (5) as an analog of parnafungin A that is methylated on the phenolic hydroxyl group and parnafungin D (6) as an epoxide derivative of parnafungin C (Overy et al 2009).


457

FIG. 3. HPLC-DAD-MS profiles comparing parnafungin A and B standard and an extract of strain F-155,597 of the F. larvarum complex, demonstrating the occurrence of two additional parnafungins, C and D (* indicates [M+H]+ isotopic pattern for co-eluting parnafungin A benzoquinoline analog).

After the initial discovery of parnafungins A and B we continued to screen newly acquired fungal isolates with eight-medium nutritional arrays. Once a sufficiently potent antifungal activity was detected from a strain, the remaining extract was profiled by LC-MS to determine whether a known antifungal agent was present. If the extract was judged to be of interest it was fermented again to generate 100 mL extract and a subsample was profiled in the CaFT. DNA extracted from the fermentation inoculum of the corresponding strain was PCR amplified to generate a sequence including the D1D2 region of the rDNA LSU. This process identified three more strains (based on DNA sequence relatedness), F-242,915, F-253,264 and F-257,517 (TABLE I) with strong antifungal activity. As predicted a scaled-up fermentation extract of F-253,264 produced a CaFT profile indicative of parnafungins (FIG. 2). LC-MS analysis confirmed the

presence of parnafungins A and B (FIG. 3). Similarly they were confirmed in the extract of F-257,517 (not tested in CaFT). To our surprise the extract of strain F-242,915 yielded a CaFT profile indicative of preponderance of brefeldin A (FIG. 2), which was confirmed by high resolution LC-MS (data not shown). After fractionation of this extract and concentration of the antifungal fractions parnafungins A and B were identified in low titer along with the steroidal antibiotic helvolic acid (data not shown). The relatedness of chemotypes (i.e. presence of parnafungins) of these extracts, as initially predicted by D1D2 sequences, was further supported by examination of culture morphology that suggested these producing fungi were highly related to the three original parnafungin-producing strains (see below). Subsequent screening of authentic strains of fungi

458

MYCOLOGIA

FIG. 4. Upper panel. Strains of the F. larvarum complex and other related fungi (TABLE I) fermented 14 d in compartments of a 24-well plate. See methods for fermentation conditions. Lower panel. Zones of inhibition caused by extracts applied to assay plates seeded with Candida albicans MY1055. Amphotericin B applied at position bottom right corner and kanamycin at upper right corner. Ten microliter aliquots were applied to assay plates in numbered columns. 1A NRRL

BILLS ET AL: DISCOVERY OF THE PARNAFUNGINS of the Hypocreales obtained from the CBS identified nutritional array extracts of T. rectipila (CBS 132.87) and Cl. pinarense (CBS 400.86) with antifungal activities, which upon scale-up provided CaFT profiles with a strong 39 mRNA processing effect (FIG. 2). The presence of parnafungins A and B and their open-ring forms in these extracts were confirmed by high resolution LC-MS experiments (data not shown). Antifungal activity and mass spectroscopic analysis.—A set of fungi including authentic strains of F. larvarum and some close relatives was assembled (TABLE I) and fermented side-by-side under the same conditions to determine their antifungal activity and production of parnafungins, if any (FIG. 4, TABLE III). While the parnafungin-producing strains isolated in our lab afforded extracts with consistent antifungal activities, three strains of F. larvarum var. larvarum and var. rubrum produced extracts with variable antifungal activities depending on the length of fermentation. No antifungal activity was detected in the extracts of strains of Co. aurantiicola (ICMP 5444, ICMP 11047), F. larvarum var. larvarum (CBS 738.79) and three undescribed lichenicolous Fusarium species (NRRL 26803, NRRL 26790, F-265,963). Parnafungin production varied depending on fermentation methods and times. Extract analysis generated from the initial screens performed on fermentation extracts generated in 96-deepwell plates indicated that parnafungin production was limited to the FLC strains isolated in our lab (F-242,915, F-253,264, F-159,081, F-159,080, F-155,597 and F-257,517) as well as T. rectipila and Cl. pinarense; however analysis was performed only on extracts that caused ZOI in primary assay screening, and single ion monitoring was not used to confirm the possibility of low titer production in nonactive strains. To confirm parnafungin production among the FLC all strains were fermented multiple times (n 5 12) in 24-well plates (at 7 d, 10 d and 14 d, SUPPLEMENTAL DATA 1) as well as in vial fermentations (n 5 3, 14 d) and single ion monitoring was performed for the monoisotopic protonated and deprotonated molecular mass ions ([M+H]+ and [M2H]2). Patterns of parnafungin production among the FLC strains were summarized by mapping the compound names onto the ITS-D1D2 Bayesian tree (FIG. 5). Most FLC

459

strains, including F-242,915, were found to produce parnafungin A and B. In 7 d an accumulation of benzoquinoline parnafungin analogs (e.g. 3 and 4) was observed while parnafungin production remained constant. Production of the monomethylated or epoxide parnafungin analogs was not observed in any of the multiple fermentations from the first discovered strains F-159,080 and F-159,081; only low titer production was observed for strain F-242,915. Strain F-155,597 produced parnafungin C and D in the greatest titers; however for this strain parnafungin B production occurred at extremely low titers and was discernable only by using single ion monitoring of the [M+H]+ ion. Although they were not prominent peaks in the full scan MS profile, parnafungin A–D production clearly was observable after 7 d with single ion monitoring of the [M+H]+ and [M2H]2 ions from fermentation extracts of F. larvarum var. rubrum strains CBS 638.76 and NRRL 22170; only lower titer production of these natural products was observed for strain NRRL 20475 with single ion monitoring. Extremely low titer production of parnafungin A and B was inferred from F. larvarum var. larvarum strains (CBS 169.30 and CBS 158.57) by the observation of their benzoquinoline analogs with single ion monitoring from fermentations at 10 and 14 d; however the intact isoxazolidinone ring structure was not detected. Phylogenetic analysis of the F. larvarum complex.— From all the PCR amplifications using different primer pairs only the ITS+D1D2 nuclear rDNA, mtSSU rDNA, histone H3 and b-tubulin gene fragments rendered positive amplifications from the majority of the strains tested. No amplifications fragment were observed with RNA polymerase 2, calmodulin, a-tubulin, putative reductase or ammonia ligase (primers URA11 and URA16), and multiple amplification fragments were obtained with phosphate permease and ammonia ligase (primers URA1 and URA 10). The length of the amplification fragments of the different genes were 1321–1453 for the b-tubulin gene, 460–503 for the histone H3 gene, 497–593 for the mtRNA and 1117–1157 for the ITS+D1D2 nuclear rDNA. Only the ITS+D1D2 nuclear rDNA and mtSSU rDNA primers yielded clear amplification fragments for all strains. Alignment of these sequences provided

r 26803, 1B CBS 158.57, 1C F-159,080, 1D F-167,589, 2A NRRL 26790, 2B CBS 169.30, 2C F-253,264, 2D F-202,821, 3A NRRL 20475, 3B CBS 738.79, 3C F-252,517, 3D CBS 860.95, 4A NRRL 22170, 4B F-223,909, 4C F-159,081, 4D CBS 267.81, 5A CBS 638.76, 5B CBS 634.76, 5C F-155,597, 5D CBS 132.87, 6A ICMP 110471, 6B ICMP 5444, 6C F-242,915, 6D CBS 421.88. (Refer to TABLE I for strain data.)

FIG. 5. Relationships among strains of F. larvarum complex (FLC) and related fungi inferred from Bayesian analysis of combined ITS and LSU sequences. Numbers at the nodes indicate clade credibility values in Bayesian analysis. Viridispora alata was designated as outgroup. Identified metabolites are labeled to the right of terminal branches. (Refer to TABLE II for strain data.)

460 MYCOLOGIA


461

FIG. 6. Relationships among strains of F. larvarum complex (FLC) and related fungi inferred from Bayesian analysis of appended ITS, LSU, b-tubulin, histone and mtRNA sequences. Numbers at the nodes indicate clade credibility values in Bayesian analysis. Viridispora alata was designated as outgroup.

sets of 1190 and 579 bp respectively. Initially the histone H3 gene fragment was amplified for 16 strains; H3F2 a new primer derived from 59 end of the preliminary alignment of the amplified sequences was designed. This primer in combination with H3R1 achieved positive amplification of four more strains. Positive amplification for this gene failed for F. larvarum var. larvarum CBS 738.79, F-155,597, F-253,264 and F. merismoides var. acetilereum F-223,908. The final alignment of these sequences was 507 bp. The b-tubulin amplicon was obtained from 20 strains. Fusarium sp. F-267,963 and FLC F-253,264

failed in all attempts to amplify the whole gene or partial sequences of this gene (with primers T1 and T2). After the alignment of the contigs from the sequencer trace files, a b-tubulin gene sequence was obtained only for F. merismoides var. crassum F-241,346, whose 59 end was not obtained because reverse internal primers (T2, T21 and T41) were unable to hybridize to the amplified gene fragment. Strains of F. larvarum var. larvarum CBS 158.57, CBS 738.79 and CBS 169.30 exhibited a double sequence in the chromatogram of the partial sequence reactions from most primers, a fact suggesting that these organisms might have b-tubulin-paralogs that were

462

MYCOLOGIA

FIG. 7. Parnafungin-producing strains of the F. larvarum complex (FLC) grown on oat cereal agar in 10 cm Petri dishes 3 wk. FLC-1 7A and 7B. FLC-3 7C, 7D, 7E. FLC-2 7C. F. larvarum var. rubrum 7G, 7 H. (Refer to TABLES I and II for strain data.)


463

FIG. 8. Conidiomata, conidiogenesis and conidia of parnafungin-producing strains of FLC-1 from CMA. A. F-159,081 conidioma. Bar 5 50 mm. B. F-159,080 conidioma and conidiogenesis. Bar 5 10 mm. C. F-159,081 conidiogenesis. Bar 5 10 mm. D. F-159,081 conidiogenesis. Bar 5 10 mm. E. F-159,080 conidia. Bar 5 10 mm. F. F-159,080 conidia. Bar 5 10 mm.

464

MYCOLOGIA

TABLE II.

GenBank accession numbers for stains of the Fusarium larvarum complex and other hypocrealean fungi ITS-D1D2 region of LSU

b-tubulin

mtSSU

Histone H3

ICMP 5444 ICMP 11047 CBS 158.57 CBS 169.30 CBS 738.79, NRRL 20743 CBS 638.76, ex-isotype NRRL 20475 NRRL 22170 NRRL 26803 NRRL 26790 F-265,963 F-242,915 F-253,264 F-159,080, MF7022, ATCC PAT 7894 F-159,081, MF7023, ATCC PAT 7895 F-155,597 F-257,517 CBS 267.81

EU860061 EU860062 EU860063 EU860064 EU860065 EU860072 EU860073 EU860074 EU860075 EU860076 EU860078 EU860071 EU860070 EU860067

EU860022 EU860023 EU860024 EU860025 EU860026 EU860018 EU860019 EU860020 EU860028 EU860029 — EU860016 — EU860017

EU859995 EU859996 EU859994 EU859992 EU859993 EU860003 EU860004 EU860005 EU859991 EU859990 EU860006 EU859997 EU859999 EU860001

EU860042 EU860043 EU860048 EU860049 — EU860050 EU860051 EU860052 EU860053 EU860038 EU860054 EU860046 — EU860044

EU860066

EU860014

EU860002

EU860045

EU860068 EU860069 EU860057

EU860015 EU860021 EU860027

EU860000 EU859998 EU860007

— EU860047 EU860039


EU860059

EU860013

EU860008

EU860041

F-167,589

EU860060

EU860032

EU860009

EU860040

F-223,908

EU860058

EU860031

EU859988

—

CBS 740.79

EU860056

EU860033

EU859987

EU860036

F-202,821 CBS 421.88

EU860077 EU860055

EU860030 EU860010

EU859989 EU859986

EU860037 EU860035

Identification

Strain numbers

Co. aurantiicola Co. aurantiicola F. larvarum var. larvarum F. larvarum var. larvarum F. larvarum var. larvarum F. larvarum var. rubrum F. larvarum var. rubrum F. larvarum var. rubrum F. sp. F. sp. F. sp. F. larvarum complex F. larvarum complex F. larvarum complex F. larvarum complex F. larvarum complex F. larvarum complex F. merismoides var. merismioides Corda F. merismoides var. violaceum Gerlach F. merismoides var. violaceum F. merismoides var. acetilereum Tubaki F. merismoides var. crassum Wollenw. F. ciliatum Sacc. Viridispora alata (Samuels) Samuels & Rossman

amplified in the same PCR reaction. Only the sequences obtained with internal primers T2 and T224 rendered a clean and defined DNA sequence. Those sequences once aligned corresponded to the 59 end of the amplification fragment. The sequences were clearly orthologous to the remaining sequences included in the study. Three additional primers were designed to complete the missing data, but all failed to provide a clear and trustworthy sequence. Because all sequenced strains except one had a complete 59 end of the b-tubulin gene, only a 500 bp region from the 59 end of the b-tubulin gene was considered for phylogenetic analysis and F. merismoides var. crassum F-241,346 was excluded. The final sequence alignment consisted of 560 bp. Bayesian analysis based on the combined ITS and D1D2 regions of the LSU revealed a well supported monophyletic group consisting of all the FLC strains, Co. aurantiicola strains from New Zealand, plus the North American lichenicolous strains (FIG. 5). Within

this group F. larvarum var. larvarum and Co. aurantiicola from New Zealand formed a monophyletic subclade that diverged significantly from the parnafungin-producing strains and the ex-type strains of F. larvarum var. rubrum. The branch corresponding to the parnafungin-producing strains and the extype strains of F. larvarum var. rubrum received a clade credibility value of 90 (FIG. 5). Internal subbranches reflected the proximity of strains F-159,080 and F-159,081, which originated from the same sample (clade credibility 100). However other internal sub-branches received only moderate to little statistical support. Therefore, based on ITS and D1D2 sequences alone, the relationships among the parnafungin-producing strains were unresolved. Furthermore it was unclear whether any of the parnafunginproducing strains were conspecific with F. larvarum var. rubrum. Subtle differences in both the ITS and D1D2 sequences were evident among these strains, and these minor sequence substitutions were reflect-

BILLS ET AL: DISCOVERY OF THE PARNAFUNGINS ed in subtle differences in gross colony morphology (TABLE III, FIG. 7) and metabolite profiles among the parnafungin-producing strains. Sequence data for the histone H3, b-tubulin, mtSUU, ITS and D1D2 genes were combined for the 17 strains for which complete DNA data were available; sequences were concatenated in the same order to yield a composite alignment of 2797 bp. The Bayesian tree resulted in a monophyletic clade that encompassed 11 FLC strains including the undescribed North American lichenicolous Fusarium strains (FIG. 6). Within the FLC clade at least six subclades or distinct individual strains could be differentiated (FIG. 6, TABLE III) comprising (i) the lichenicolous North American Fusarium strains that potentially consist of two distinct species (Torzilli et al 2002); (ii) F. larvarum var. larvarum; (iii) New Zealand Co. aurantiicola strains; (iv) FLC-1 strains F-159,080 and F-159,081; (v) FLC-2 strain F-242,915; and (vi) ex-types of F. larvarum var. rubrum and FLC-3, which consisted of the unresolved the parnafungin-producing strain F-257,517. Complete sequence data were not obtained for F-155,597 and F-253,264. Data from the ITS and D1D2 (FIG. 5) and the b-tubulin gene were unable to resolve the group, therefore whether these two strains were conspecific with F-257,517 only or all three were conspecific with F. larvarum var. rubrum remained unclear. For the time being the three strains are referred to as FLC-3 (TABLE III). When individual phylograms obtained for each gene region were analyzed separately (SUPPLEMENTAL DATA 3–5), the clade credibility values for the main FLC branch varied from 56 for histone H3, 83 for b-tubulin, 100 for the ITS plus D1D2 nuclear rDNA to 99 for the mtSSU rDNA loci. All individual gene trees recovered the lichenicolous group (SUPPLEMENTAL DATA 3–5). Significant divergences between the var. larvarum strains and the New Zealand Co. aurantiicola strains were observed in the b-tubulin and histone H3 sequences, suggesting that the New Zealand populations were isolated geographically. All three single gene trees recovered the parnafungin-producing strains as a coherent subclade that included the ex-type strains of F. larvarum var. rubrum. In each single gene tree FLC-1 and FLC-2 were recovered as separate branches but the distinction among strains of FLC-3 and F. larvarum var. rubrum was evident only with b-tubulin sequence data (SUPPLEMENTAL DATA 3). Taxonomy and observations on culture morphology.— The morphological characteristics of F. larvarum var. larvarum and Co. aurantiicola have been summarized in the literature, while the morphology of F. larvarum

465

var. rubrum was depicted in Gerlach’s (1977) original observations. Colony pigmentation, development of aerial mycelia and ability of strains to sporulate in vitro varied significantly among isolates of the FLC (TABLE III). A description of the parnafungin-producing strains of FLC-1 is presented to expand the range of morphological features known from fungi of the FLC and because these strains provided the original discovery of the parnafungins (Jiang et al 2008, Parish et al 2008). Fusarium lavarum complex-1 FIGS. 7, 8 On 2% malt extract agar, attaining 30–31 mm diam, mycelium submerged to appressed, with scant velvety mycelium at the center; colony margins were even and appeared hyaline at the edge; colonies were pale yellow to golden yellow or yellowish brown (Antimony Yellow, Wax Yellow, Primuline Yellow, Yellow Ochre), with the reverse the same. On CMA, attaining 40–45 mm diam, mycelium submerged and hyaline, and colony margins were minutely fimbriate. On oat cereal agar (FIG. 7A, B), attaining 39–40 mm diam, the mycelium was appressed to velvety or hispid at the center; ranging from white to pale yellow to golden brown with white aerial mycelium, sometimes with tufts of golden brown mycelium, sometimes developing vinaceous pink to vinaceous red (Purplish Vinaceous, Brownish Vinaceous) areas in age, becoming subzonate, with some sectoring and sectors lacking aerial mycelium; colonies dark golden in reverse, in some strains developing vinaceous red to vinaceous brown as colonies mature (Ochre Red, Prussian Red). No growth at 37 C. Sporulation in agar culture erratic or absent. On occasions sporulation observed on cornmeal agar at low temperatures (16 C), after prolonged incubation (more than 6 wk) after wounding cultures by extraction of agar plugs or by exposure to near ultraviolet light. Scattered sporodochia forming at the colony edges of mature, fully extended mycelium, or at edges of wounded mycelium. Sporodochia hyaline to pale orange or pinkish orange and up to 500 mm diam, consisting of few to multiple complex penicillate conidiophores arising from the agar surface (FIG. 8A, B). Conidiophores (FIG. 8B) consisting of short parallel branches arising from surface hyphae that give rise to sparse or dense arrangements of penicillately branched conidiogenous cells, or conidiogenous cells scattered along branched hyphae, with primary branches septate or not, consisting of cylindrical to clavate cells, up to 7.5 mm diam, branching two to four times, and terminating in conidiogenous cells. Conidiogenous cells (FIG. 8C, D) phialidic, enteroblastic, hyaline, 7–15 mm long, 2–3 mm

F. larvarum var. larvarum





Undescribed lichenicolous species 1 Undescribed lichenicolous species 2 Undescribed lichenicolous species 2







F. sp.

F. sp.

F. sp.

Co. aurantiicola

Co. aurantiicola

Possible new species, sister species to F. lavarum var larvarum. Possible new species, sister species to F. lavarum var larvarum. F. larvarum var. larvarum

Revised designation

White appressed mycelium

Orange, slimy appressed

F-265,963

NRRL 26790

Pale orange, waxy

Pale orange, waxy

White to dull orange with violet sectors, appressed or scant aerial mycelium NRRL 20475, derived White to dull orange with from CBS 638.76 violet sectors, appressed or scant aerial mycelium NRRL 22170, derived White to dull orange with from CBS 638.76 violet sectors, appressed or scant aerial mycelium NRRL 26803 Pale orange, waxy


CBS 738.79

CBS 169.30

Orange, slimy appressed

White aerial mycelium

ICMP 11047

CBS 158.57

White aerial mycelium

Colony colors on OA

ICMP 5444

Strain numbers

F. larvarum-like conidia observed on first subculture, sterile afterwards

No conidia observed

No AF observed None observed No AF observed None observed No AF observed None observed

AF variable Low A–D

No conidia observed

No conidia observed

AF strong but variable Low A–D

No AF observed None observed AF weak and variable Low A–D

AF variable Low A and B

AF variable Low A and B

No AF observed None observed

No AF observed None observed

Antifungal activity (AF) parnafungins

No conidia observed

No conidia observed

Abundant conidia, conidiophores scattered on agar surface Abundant conidia, conidiophores scattered on agar surface No conidia observed

Large orange sporodcochia, perithecia formed on water agar Large orange sporodcochia

Observations on sporulation

Observations on sporulation colony pigmentation, and antifungal activity among strains of Fusarium larvarum complex

Original identification

TABLE III.

466 MYCOLOGIA

Continued

FLC 3, possible undescribed F-155,597 species


FLC-1, undescribed species

FLC-1, undescribed species

F. larvarum complex

F. larvarum complex

F. larvarum complex

F. larvarum complex

F-159,081, MF7023, ATCC PAT 7895

F-159,080, MF7022, ATCC PAT 7894


F. larvarum complex

Strain numbers


Revised designation

F. larvarum complex

Original identification

TABLE III.

Dingy white to dull yellow, some vinaceous tint in aerial mycelium Dingy white to dull yellow, some vinaceous tint in aerial mycelium

Strong vinaecous red in aerial mycelium and colony reverse Dingy white to dull yellow, strong sectors with vinaceous aerial mycelium Unidentified lichen

Dull orange to carmine

Colony colors on OA

Conidia produced erratically in small sporodochia Conidia produced erratically in small sporodochia Conidia produced erratically in small sporodochia

Conidia never observed

Conidia and conidiophores abundant, in yelloworange sporodochia or scattered on agar surface Conidia produced erratically

Observations on sporulation

AF consistent A and B

AF consistent A and B

AF consistent Low A–D

AF consistent C and D, low B

AF consistent Low A–D

AF consistent Low A–D?, brefeldin A, helvolic acid

Antifungal activity (AF) parnafungins

BILLS ET AL: DISCOVERY OF THE PARNAFUNGINS 467

468

MYCOLOGIA

wide, cylindrical and tapered at apex, often with an inconspicuous collerette at the conidiogenous locus. Conidia (FIG. 8E, F) were strongly curved to lunate, hyaline, overwhelmingly 3-septate, rarely 2-septate, occasionally with slight truncated or flattened basal cells, but lacking foot cell, 20–15 mm long, 4–6 mm wide. Distribution and habitat. Miraflores de La Sierra, Madrid, Spain. Isolated from lichen thalli. Specimens examined. F-159,080 (ATCC Pat 7894), F-159,081 (ATCC Pat 7895). DISCUSSION

Fusarium larvarum var. larvarum and var. rubrum are usually associated with scale insects and aphids, although associations of Co. aurantiicola and F. larvarum var. larvarum with Septobasidium clelandii (Booth 1981) and with lichens have been reported (B. Paulus and P. Johnston pers comm). To our knowledge F. larvarum var. rubrum was known only from a single strain (BBA 62460 5 CBS 638.76) from a San Jose´ scale insect from Iran (Gerlach 1977) and strains deposited in other collections (e.g. NRRL) are its derivatives. However we have isolated genetically distinct strains from lichen thalli on trees, from plant litter and from living plants in Spain and the Republic of Georgia that share morphological and biochemical characteristics with F. larvarum var. larvarum and var. rubrum (TABLES I, II; FIGS. 4, 7). Collection and diagnosis of the F. larvarum based on insect associations apparently has overlooked cryptic lineages of the FLC that may have alternative trophic phases. Observation of these cryptic lineages of the FLC was made possible with particle filtration techniques (Bills et al 2004) or extinction culturing (Collado et al 2007) that can reveal the presence of slow-growing species embedded in an organic matrix. Considerable effort was invested in sporulating these fungi because of the interest in their chemistry. However, if these cryptic fungi were isolated as endophytes or isolated from plant litter, their relationships might be overlooked because of poor sporulation and a sterile isolate, such as FLC-3, F-155,597, could be easily ignored altogether. We attempted to test the boundaries of the FLC by inclusion of as many genes as feasible in a multilocus study. Up to 14 primer pairs previously designed to amplify single-copy genes for analysis of species phylogeny in Fusarium species associated with Gibberella teleomorphs (O’Donnell et al 2000, 2004, Starkey et al 2007) were tested. Due to the lack of data from protein-coding genes their application to the FLC could not be assessed directly. As described in the methods, from 14 specific primer pairs only those for

rDNA regions rendered positive amplifications across all 22 strains and another three pairs amplified a significant fraction of the strains. Considering the phylogenetic distance between Gibberella and the FLC, that intron-rich protein-coding regions may evolve faster than ribosomal genes and that PCR conditions were not optimized for each single-copy gene these results might not be surprising. Attempted multilocus phylogenies often result in partial datasets due to the nonamplification of certain genes (Miadlikowska et al 2006, Sung et al 2007). The ratios of nucleotide identity of the ITS and D1D2 genes among the FLC strains tested were 90–100%. Although there was a high homology among rDNA genes that have been used extensively for phylogenetic studies, evidently conserved regions of other genes from Gibberella that were selected for the design of specific probes differed significantly. Characterization of the parnafungin-producing strains of the FLC, defined as Fusarium-like anamorphs related to Co. aurantiicola forming only short, lunate, 3-septate conidia, has led to the conclusion that such strains form a monophyletic group based on cladistic analysis of the rDNA, mtSUU, histone H3 and b-tubulin genes. Within this primary lineage at least six different lineages, possibly representing cryptic species, could be recognized. Because phenotypic and ecological distinctions among these lineages remain ambiguous and because taxon sampling remains limited we think modifying taxonomic ranks or describing new species is premature. One species designation includes strains identified as F. larvarum var. larvarum. Sequence divergence in some genes suggested that more extensive strain sampling could segregate the geographically isolated populations in New Zealand identified as Co. aurantiicola as yet another distinct species. The two strains of F. larvarum var. larvarum, CBS158.57 and CBS 169.30, produced trace amounts of parnafungins, while parnafungins were not detected in New Zealand strains. The ex-type strains of F. larvarum var. rubrum should be recognized as phylogenetically distinct species, and they could be induced to produce parnafungins. Three more strains from Spain and the Republic of Georgia, referred to as the FLC-3, might represent a sister species or be conspecific to F. larvarum var. rubrum. Strain F-242,915 from the Republic of Georgia might represent yet another species distinct from var. rubrum and var. larvarum. Phenotypically the strain was distinguishable by its consistent sporulation on different media, dull carmine basal mycelium and a metabolite profile differing by the production of helvolic acid and brefeldin A. Until more strains can be analyzed we refer to this strain as FLC-2. Finally the

BILLS ET AL: DISCOVERY OF THE PARNAFUNGINS first two FLC strains isolated from a Spanish lichen thallus, designated FLC-1, were consistently recovered as closely related but distinct lineage. Another group, NRRL 26803, NRRL 26790 and F-265,963, consisted of the lichen-derived strains from the eastern United States with F. larvarum-like conidia. Our phylogenetic analysis supports conclusions that these strains represented one or more undescribed lichenicolous Fusarium spp. and were a distinct lineage (Torzilli et al 2002). Bayesian analysis of individual protein-coding genes, mtSSU rDNA, ribosomal DNA or their combination, consistently recovered a single monophyletic lineage distinct from F. larvarum. Isolation of an additional strain (F-265,963) from an unidentified lichen extended the geographic range of these fungi into Vermont. This strain sporulated in its first subculture but ceased to sporulate on subsequent transfers. Conidia were 3septate, without a foot cell, somewhat more fusiform than the typical larvarum conidia and strongly resembled those illustrated from the lichen pathogen collected in the Adirondacks of New York (Glenn, Gomez-Bolea, Orsi 1997). We were unable to induce in vitro antifungal antibiosis against C. albicans in these strains, therefore it remains unclear whether toxic metabolites were involved in the necrosis of lichen thalli (Glenn, Gomez-Bolea, Orsi 1997, Lawrey, Torzilli, Chandhoke 1999). Our work demonstrates that the competence among strains of the FLC to produce parnafungins varied greatly, but a strong correlation exists between those members of the FLC exhibiting antifungal activity and parnafungins being identified as the predominant bioactive metabolites. Indeed only FLC-2, F-242,915 produced a prominent alternative antifungal bioactivity (brefeldin A), yet low level parnafungins also were present. Of note data from CaFT profiles (FIG. 2) demonstrated that parnafungins were produced in two Hypocrealean fungi beyond the FLC (i.e. T. rectipila and Cl. Pinarense). Both fungi are mycoparasites. The significance and extent of those results requires further optimization of fermentation conditions and analysis of additional strains. Moreover we observed that parnafungins were produced during early in vitro vegetative growth when the fungi were on adequate media formulations, but these compounds rapidly degraded to their respective inactive benzoquinoline analogs as the cultures matured to a stationary phase of development (SUPPLEMENTAL DATA 1). The spontaneous degradation of these products has been correlated with a change in pH, where ring opening was promoted in neutral or alkaline conditions while a stabilization of the molecule is favored in acidic conditions (Overy et al 2009, Parish et al 2008). Many fungi lower the pH

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of their growth medium through the secretion of organic acids during their initial phase of growth, and later as the fungus reaches stationary growth the medium’s pH progressively becomes more alkaline (Griffin 1994); in this case alkalization of the growth medium would promote the accumulation of the benzoquinoline analogs. Each strain of the FLC behaved as a recognizable individual with respect to its colony morphology, DNA sequence substitutions and patterns of parnafungin production. Long-term culture storage and subculture might have contributed to some of these differences. For example the three ex-isotype derived strains of F. larvarum var. rubrum should have behaved similarly. All three had lost the ability to sporulate in agar media, and each to some degree manifested different patterns of degenerated aerial mycelium (FIG. 7). Evaluation of parnafungin chemotypes could have had very different outcomes depending on which strain was evaluated, NRRL 20475 or CBS 638.76 with poor production or strain NRRL 22170 with somewhat stronger and thus consistent production. Continued degeneration of mycelial traits caused by long-term subculturing include loss of virulence and sporulation (Ryan et al 2002, Butt et al 2006), loss of metabolite production (MacDonald 1968) and the formation of mycotoxins and sclerotia (Horn and Dorner 2002). Although parnafungin production was confirmed from these strains, low titers were observed in most cases. In light of the degenerative condition of the cultures it is difficult to conclude whether the extreme low titer expression of the parnafungins was a result of the physiological condition of the organism or the cultivation parameters used in this study (e.g. insufficient repetitions of fermentations, effects of fermentation times and media composition). Perhaps parnafungin production could be further stimulated from these strains via manipulations of the growth medium composition. Stimulation in the production of low titer metabolites from pathogenic fungi and overall enhanced metabolite expression in cultured fungi has been achieved by employing media derived from host tissues (Overy et al 2006), empirical manipulation of growth parameters (Bode et al 2002) and most recently by activation of silent biosynthetic gene clusters with small-molecule epigenetic modifying agents (Williams et al 2008). The biosynthesis of parnafungins is likely related to that of other ergochrome-derived secondary metabolites. Anthraquinone natural products, such as emodin, are generated through polyketide condensations and cyclizations. Emodin is oxidatively cleaved, leading to the formation of the xanthone ring system

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and after dimerization to secalonic acids (Kurobane, Vining, McInnes 1979, Frank, Bringmann, Flohr 1980). The extended ring system in parnafungins, relative to the xanthone unit in secalonic acids, might be derived from an initially longer polyketide that can undergo further cyclization. The more interesting biosynthetic question is the process that adds the nitrogen atom and closes the isoxazolidinone ring. No xanthone-based natural product that includes nitrogen has been reported. Further because an isoxazolidinone has not been identified previously in an isolated natural product the biosynthetic mechanism of this ring closure is unclear and will require further investigation. Our study of the strains of the FLC has revealed parnafungins C and D as additional members of the parnafungin family of natural products. These analogs are likely produced directly from parnafungin A by methylation and subsequent oxidation (Overy et al 2009). Parnafungins inhibit the growth of C. albicans, S. cerevisiae and A. fumigatus, and both biochemical and genetic evidence demonstrated their mechanism of action as potent inhibitors of poly-(A) polymerase (PAP) enzyme (Jiang et al 2008). Because the PAP enzyme constitutes only one subunit of the cleavage and polyadenylation macromolecular complex, parnafungins also might indirectly affect assembly, stability and function of the complex, as suggested by weak effects on 39 mRNA cleavage also observed in the cell-free cleavage and polyadenylation assay (Jiang et al 2008). Although we can only speculate on their biological role and benefit to the producing organisms, parnafungins display broad growth inhibitory activity across taxonomically diverse ascomycetes and thus might act as potent antifungals providing a competitive advantage for growth of the producer in its natural habitat. Moreover because PAP is broadly conserved throughout eukaryotes the parnafungins also could be a virulence factor mediating interorganism interactions while colonizing hosts (e.g. lichenized and non-lichenized fungi, insects or plants). Moreover their spontaneous degradation might be a self defense mechanism preventing the producing organisms from accumulating toxic levels of parnafungins. ACKNOWLEDGMENTS

We are grateful for the help of Kerry O’Donnell who offered advice, access to strains at the NRRL and suggested sets of primer sequences. Plant materials for isolation of fungi from the Republic of Georgia were collected in collaboration with Manana Khutsishvili, The Institute of Botany of the Georgian Academy of Science, Tbilisi, Georgia.

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