Chemical and Physical Modulation of Antibiotic Activity in Emericella Species

Chemical and Physical Modulation of Antibiotic Activity in EmericellaSpecies

by Mercedes de la Cruza), Jesus Mart�na), Victor Gonzalez-Menendeza), Ignacio Perez-Victoriaa),Catalina Morenoa), Jose Ruben Tormoa), Noureddine El Aouada), Josep Guarrob),

Francisca Vicentea), Fernando Reyesa), and Gerald F. Bills*a)

a) Fundacion MEDINA, Centro de Excelencia en Investigacion de Medicamentos Innovadores enAndaluc�a, Avda. del Conocimiento 3, Parque Tecnologico de Ciencias de la Salud, ES-18100 Armilla,

Granada ([email protected])b) Unitat de Microbiolog�a, Facultat de Medicina i Ciencies de la Salut, Universitat Rovira i Virgili,

C/Sant LlorenÅ, 21, ES-43201 Reus, Tarragona

The addition of epigenetic modifying agents and ion-exchange resins to culture media and solid-statefermentations have been promoted as ways to stimulate expression of latent biosynthetic gene clustersand to modulate secondary metabolite biosynthesis. We asked how combination of these treatmentswould affect a population of screening isolates and their patterns of antibiosis relative to fermentationcontrols. A set of 43 Emericella strains, representing 25 species and varieties, were grown on a nutrient-rich medium comprising glucose, casein hydrolysate, urea, and mineral salts. Each strain was grown inuntreated agitated liquid medium, a medium treated with suberoylanilide hydroxamic acid, a histonedeacetylase inhibitor, 5-azacytidine, a DNA methylation inhibitor, an Amberlite non-ionic polyacrylateresin, and the same medium incorporated into an inert static vermiculite matrix. Species-inherentmetabolic differences more strongly influenced patterns of antibiosis than medium treatments. Theantibacterial siderophore, desferritriacetylfusigen (1), was detected in most species in liquid media, butnot in the vermiculite medium. The predominant antifungal component detected was echinocandin B.Some species produced this antifungal regardless of treatment, although higher quantities were oftenproduced in vermiculite. Several species are reported for the first time to produce echinocandin B (3). Anew echinocandin analog, echinocandin E (2), was identified from E. quadrilineata.

Introduction. – Culture medium composition can strongly affect the profile ofsecondary metabolites produced by a fungus and, therefore, its antibiotic phenotype[1– 5]. Other culture manipulations, including addition of epigenetic modifying agentsto modulate gene expression, addition of non-ionic polyacrylate resins to sequesterintermediate secondary metabolites, and changing the physical interactions of theorganisms with their medium via solid-state fermentations, have been promoted asways to modulate metabolite biosynthesis and stimulate expression of latentbiosynthetic gene clusters [6– 13]. Despite their potential utility in modulatingsecondary metabolism, the influences of these physical and chemical manipulationson secondary metabolite and antibiotic phenotypes across large populations of strainsremain poorly understood. The above cited references have provided useful examplesof where these techniques have stimulated or modulated a particular metabolite ormetabolite family; however, few studies offered examples of how combining different

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 1095

� 2012 Verlag Helvetica Chimica Acta AG, Z�rich

techniques can affect antimicrobial phenotypes during primary screening for antibioticactivity across microbial populations.

How will combining treatments designed to stimulate expression of latentbiosynthetic gene clusters affect a population of screening isolates? A test populationof representative strains of a metabolite-rich group of fungi, Aspergillus subgenusNidulantes, was selected from our culture collection. Aspergillus subgenus Nidulantescomprises a widespread and well-studied group of secondary-metabolite rich fungi [14]that are of concern in food and feed processing and storage, because many speciesproduce the mycotoxin sterigmatocystin [15]. When the sexual states of their life cycleare expressed, they are assigned to the genus Emericella [16]. A key species in thegroup is Emericella (Aspergillus) nidulans which is an important model organism forstudy of genetic regulation and biosynthesis of secondary metabolites [17] [18], and atleast 45 secondary metabolite gene clusters are predicted in its genome [19]. Emericellanidulans is the organism used to produce echinocandin B, the starting molecule for thesemi-synthetic antifungal lipopeptide, anidulafungin (ERAXISTM). Echinocandin B(3) has also been reported from E. rugulosa [3] [20] and A. nidulans var. echinulatus[21]. Therefore, because of their metabolic complexity and importance of theirmetabolites, we asume these fungi represent a good test case for understanding theeffects of culture-condition manipulations.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)1096

Will such treatments stimulate, repress, or reorder patterns of antibiosis relative tofermentation controls? A set of 43 authoritatively identified Emericella strainsrepresenting 25 species and varieties (Table 1) were grown on a nutrient-rich medium(NPF2) comprising glucose, casein hydrolysate, urea, and mineral salts, and buffered byCaCO3. Each strain was grown in control medium consisting of untreated agitatedliquid NPF2 medium. The medium was supplemented with suberoylanilide hydroxamicacid (SAHA), a potent inhibitor of histone deacetylases, 5-azacytidine (5AZ), a potentinhibitor of DNA methylation, or Amberlite XAD7, a non-ionic polyacrylate resin. Themedium was also formulated for solid-state fermentation (SSF) by incorporating it intoan inert vermiculite matrix (Fig. 1) [22]. To test the hypothesis that parallel physicaland chemical treatments could influence the pattern of antibiosis from individualspecies and from the set as whole, patterns of antibiosis against the human pathogenicbacterium, Staphylococcus aureus, and the human pathogenic yeast, Candida albicans,were compared between treatments and untreated controls. We also describe thestructure elucidation of a new echinocandin analog, echinocandin E (2), from E.quadilineata.

Results. – Distribution of Antibiotic Activities. A total of 39 of the 43 strainsproduced at least one visibly perceivable zone of inhibition (ZOI) against either C.albicans or S. aureus (Tables 2 – 4). Only E. desertorum, E. foveolata, one strain of E.striata (CBS 667.82), and one strain of E. variecolor (J. Guarro WO-036) expressed noantibiotic phenotype under these growth and assay conditions (Tables 3 and 4). At theother extreme, E. cleistominuta expressed an antibiotic phenotype in extracts in eight often possible assay�medium combinations.

From a total of 430 individual assays (43 strains�5 medium combinations�2 ZOIassays), 160 different ZOIs were observed (37% activity). The total number ofactivities observed against C. albicans among the different medium combinations (74ZOIs, Tables 2 and 4) did not differ significantly from a random distribution (c2¼1.0,p¼0.91, 4 df), but a disproportionate number of the ZOIs against S. aureus (86 ZOIs,Tables 2 and 3) was observed on XAD7-treated medium (c2¼12.5, p¼0.01, 4 df). Ingeneral, the most active extracts against S. aureus and C. albicans were exclusive to theXAD7-treated medium and SSF vermiculite medium, respectively (Tables 2 and 3).When individual treatments were compared to the untreated control (agitated NPF2),more ZOIs were observed on the XAD7-treated medium and SSF vermiculite medium(Table 2). Media treated with SAHA or 5AZ offered few ZOIs that were not observedin the control medium (Tables 2– 4). The most similar medium combinations for ZOIsagainst S. aureus were the NPF2 control and NPF2 treated with 5AZ, and thecombination of NPF2 treated with 5AZ and SAHA (both Jaccard�s index¼0.500); themost dissimilar media combination was NPF2 treated with 5AZ and the vermiculiteSSF (Jaccard�s index¼0.115). The most similar medium combinations for ZOIs againstC. albicans were the NPF2 control and NPF2 treated with 5AZ (Jaccard�s index 0.812);the most dissimilar media combination was NPF2 treated with XAD7 and thevermiculite SSF (Jaccard�s index 0.062).

Distribution of Echinocandin B (3) , Desferritriacetylfusigen (1) , and Sterigmato-cystin. Aliquots of extracts from each strain�medium combination were examined byLC/MS, and retention times (tR), and UV spectra, and ion signals were compared to



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those in a proprietary reference database of purified antibiotics and other secondarymetabolites. Database matching recognized that the lipopetide antibiotic echinocandinB (3) was produced by 16 strains (Table 1 and Fig. 2). Echinocandin B (3) was detectedin the known producers E. nidulans and E. rugulosa. Furthermore, to the best of ourknowledge, for the first time 3 was detected in E. cleistominuta, E. navahoensis, E.nidulans var. aurantiobrunnea, E. parvathecia, and E. quadilineata (Table 1, Fig. 2).


Fig. 1. Roller bottle with mycelium and vermiculite matrix formed by Emericella navahoensis (CBS351.81). See Exper. Part for medium preparation.

Most extracts where 3 was detected also caused moderate to large, transparent (zonequality A, B) ZOIs (Table 4). In some strains, e.g., E. cleistominuta, E. nidulans, E.rugulosa, and E. quadrilineata, echinocandin and strong antifungal activity wereproduced under nearly all medium conditions. The total ion counts were oftensignificantly stronger in vermiculite SSF (Fig. 2). Notably, in E. navahoensis and somestrains of E. quadilineata, echinocandin production and antifungal activity wererestricted to the vermiculite SSF (Fig. 2). In a few cases, e.g., E. heterothallica and E.variecolor var. astellata, strong ZOIs against C. albicans from liquid media extractswere produced, but LC/MS analysis did not displayed a known extract component.

LC/MS Signals also indicated the presence of a compound that was produced in allbut four strains. This component was isolated from one of the culture broths andidentified as desferritriacetylfusigen (1; iron-free form of triacetylfusigen; see below)by HR-MS and NMR spectroscopy. Its presence was highly correlated with detection ofsmall-to-large turbid ZOIs against S. aureus. Absorbance at 218 nm (UV max fordesferritriacetylfusigen) was often significantly stronger in the XAD7-treated medium,while totally absent in vermiculite medium (Fig. 3). This compound apparently wassynthesized by the organism in response to vigorous growth in a C-atom- and N-atom-rich, yet iron-limited medium. Trace iron sequestration by XAD7 resin may havefurther exacerbated its biosynthesis (Fig. 3). Because mineral vermiculite containsstructural iron, its biosynthesis was apparently suppressed in the SSF (Fig. 3).

Sterigmatocystin, a well-known mycotoxin from E. nidulans and other species ofsection nidulantes was only detected in E. nidulans var. echinulata, E. nidulans var. lata,and E. quadrilineata under these conditions (Table 1).

Isolation, Verification of Structure of Desferritriacetylfusigen (1) , and Determinationof Its Biological Activity. Desferritriacetylfusigen (1) was isolated from a 1-lfermentation of E. quadrilineata (J. Guarro WH-042) by acetone extraction, SP207ss


Table 2. Distribution of Zones of Inhibition (ZOIs) Caused by Emericella Extracts against Staph-ylococcus aureus and Candida albicans among Different Media and Culture Manipulations. All visible

ZOIs were considered regardless of zone quality.

Total screening set or subset considered Media and culture manipulationsa)

NPF 2Mediumb)

NPF 2mediumþSAHA

NPF 2mediumþ5AZ

NPF 2mediumþXAD7

NPF 2medium þvermiculite

Total ZOIs observed 32 23 24 44 37ZOIs against S. aureusc) 17 11 10 28 20S. aureus ZOIs exclusive to medium 0 0 0 6 5S. aureus ZOIs not present in control – 3 1 15 12ZOIs against C. albicans 15 12 14 16 17C. albicans ZOIs exclusive to medium 1 0 0 7 3C. albicans ZOIs not present in control – 0 1 7 4

a) See Exper. Part for medium formulations, SAHA, 100 mm suberoylanilide hydroxamic acid; 5AZ,100 mm 5-azacytidine; XAD7, 3% wet (w/v); vermiculite, fermentation in sterilized vermiculite. b) Un-treated agitated NPF 2 medium was considered the control. c) Significant deviation from homogeneousdistribution of ZOIs among media according to goodness-of-fit test (c2¼12.5, p¼0.01, 4 df).


Table 3. Antibiotic Activity of Different Strains of Aspergillus subsect. Nidulantes against Staphylococcusaureus. Activity is designated as ZOI (mm and zone quality). See Exper. Part for scoring of ZOIs.

Strain number Species Media and culture manipulationsa)b)

NPF 2 NPF 2þSAHA

NPF 2þ5AZC

NPF 2þXAD7

NPF 2þvermiculite

F-150,078 A. versicolor 0 0 0 0 7CCBS 425.77 E. bicolor 0 0 0 7C 0CBS 200.75 E. cleistominuta 5E 5E 0 10C 0CBS 655.73 E. desertorum 0 0 0 0 0CBS 279.81 E. foveolata 0 0 0 0 0CBS 489.65 E. heterothallica 14C 18C 16C 15C 0CBS 351.81 E. navahoensis 5E 0 6E 12C 0CBS 291.95 E. nidulans 0 0 0 0 5EF-109,962 E. nidulans 9E 0 0 0 0F-167,724 E. nidulans 5E 0 0 0 7DCBS 240.9 E. nidulans 5D 0 8C 15C 0CBS 542.83 E. nidulans 0 0 0 0 0J. Guarro WO-147 E. nidulans 9E 0 0 6E 7DCBS 467.88 E. nidulans var.

aurantiobrunnea5E 0 0 10C 0

CBS 114.63 E. nidulans var. dentata 10C 5D 0 15C 0CBS 564.8 E. nidulans var. echinulata 0 4D 0 8A 7DCBS 492.65 E. nidulans var. lata 0 4D 0 7A 0CBS 493.65 E. parvathecia 5E 0 0 15C 0J. Guarro WG-142 E. pluriseminata 0 0 0 7C 5ECBS 102705 E. pluriseminata 8E 8E 8B 5E 5EJ. Guarro WD-007 E. quadrilineata 0 0 0 8E 8BCBS 426.77 E. quadrilineata 7C 9C 5D 18C 0CBS 113684 E. quadrilineata 10C 0 5D 8C 0CBS 114511 E. quadrilineata 0 0 0 13C 0J. Guarro WE-155 E. quadrilineata 0 0 0 14C 5EJ. Guarro WD-005 E. quadrilineata 9D 0 0 16C 5EJ. Guarro WH-042 E. quadrilineata 0 0 0 16C 5EJ. Guarro WO-021 E. rugulosa 0 0 0 6D 5EJ. Guarro WO-148 E. rugulosa 0 0 0 0 5ECBS 171.71 E. rugulosa 0 0 0 9D 0CBS 852.96 E. rugulosa 0 0 0 11C 0J. Guarro WP-050 E. rugulosa 0 0 0 0 7EF-166,688 Emericella sp. 0 0 0 6E 7CJ. Guarro, WD-152 E. striata 0 0 0 7D 0CBS 667.82 E. striata 0 0 0 0 0CBS 261.88 E. undulata 0 0 0 0 12ACBS 595.65 E. unguis 0 8E 6E 7E 0CBS 691.93 E. unguis 0 0 0 0 12AJ. Guarro WE-010 E. variecolor 9A 8A 6E 8A 9AJ. Guarro WO-036 E. variecolor 0 0 0 0 0CBS 273.65 E. variecolor 0 0 0 0 9AF-202,719 E. variecolor 10E 5E 11C 0 10CCBS 470.88 E. variecolor var. astellata 13E 11E 12E 0 0J. Guarro, WD-004 E. violacea 0 0 0 5E 0

a) See Exper. Part for medium formulations; SAHA, 100 mm suberoylanilide hydroxamic acid; 5AZ,100 mm 5-azacytidine; XAD7, 3% wet (w/v); vermiculite, fermentation in sterilized vermiculite. b) Dataare ZOI diameters measured in mm, letter codes refer to zone quality ranging from A (absolutely clear)to E (very turbid).


Table 4. Antibiotic Activity of Different Strains of Aspergillus subsect. Nidulantes against Candidaalbicans. Activity is designated as ZOI (mm and zone quality).

Strain number Species Media and culture manipulationsa)b)

NPF 2 NPF 2þSAHA

NPF 2þ5AZC

NPF 2þXAD7

NPF 2þvermiculite

F-150,078 A. versicolor 0 0 0 0 0CBS 425.77 E. bicolor 0 0 0 6A 0CBS 200.75 E. cleistominuta 12A 12A 12A 6A 18ACBS 655.73 E. desertorum 0 0 0 0 0CBS 279.81 E. foveolata 0 0 0 0 0CBS 489.65 E. heterothallica 0 0 0 0 0CBS 351.81 E. navahoensis 0 0 0 0 10ACBS 291.95 E. nidulans 0 0 0 0 0F-109,962 E. nidulans 5C 9A 11A 0 11AF-167,724 E. nidulans 12A 11A 9A 0 12AJ. Guarro WO-147 E. nidulans 7C 0 13A 0 13ACBS 467.88 E. nidulans var.

aurantiobrunnea9A 10A 14A 6A 16A

CBS 114.63 E. nidulans var. dentata 0 0 0 0 0CBS 564.8 E. nidulans var. echinulata 0 0 0 8A 0CBS 492.65 E. nidulans var. lata 0 0 0 5A 0CBS 240.9 E. nidulans var. nidulans 0 0 0 0 0CBS 542.83 E. nidulans var. nidulans 0 0 0 10E 0CBS 493.65 E. parvathecia 3E 3E 15A 9A 16AJ. Guarro WG-142 E. pluriseminata 0 0 0 5B 0CBS 102705 E. pluriseminata 0 0 0 0 0J. Guarro WD-007 E. quadrilineata 9A 5D 11A 8A 18ACBS 426.77 E. quadrilineataCBS 113684 E. quadrilineata 0 0 0 0 0CBS 114511 E. quadrilineata 12A 12A 17A 14A 14AJ. Guarro WE-155 E. quadrilineata 0 0 5B 0 6BJ. Guarro WD-005 E. quadrilineata 0 0 0 0 5CJ. Guarro WH-042 E. quadrilineata 0 0 0 0 11AJ. Guarro WO-021 E. rugulosa 15A 9A 7A 0 19AJ. Guarro WO-148 E. rugulosa 12A 11A 9A 13A 19ACBS 171.71 E. rugulosa 9A 6A 0 5D 15ACBS 852.96 E. rugulosa 6A 5B 12A 6A 15AJ. Guarro WP-050 E. rugulosa 12A 5E 9A 0 21AF-166,688 Emericella sp. 0 0 0 5C 0J. Guarro, WD-152 E. striata 0 0 0 0 0CBS 667.82 E. striata 0 0 0 0 0CBS 261.88 E. undulata 6C 0 0 0 0CBS 595.65 E. unguis 0 0 0 0 0CBS 691.93 E. unguis 0 0 0 0 0J. Guarro WE-010 E. variecolor 0 0 0 16B 0J. Guarro WO-036 E. variecolor 0 0 0 0 0F-202,719 E. variecolor 0 0 0 0 0CBS 273.65 E. variecolor var. variecolor 0 0 0 0 0CBS 470.88 E. variecolor var. astellata 10A 11B 7AJ. Guarro, WD-004 E. violacea 0 0 0 0 0

a) See Exper. Part for medium formulations; SAHA, 100 mm suberoylanilide hydroxamic acid; 5AZ,100 mm 5-azacytidine; XAD7, 3% wet (w/v); vermiculite, fermentation in sterilized vermiculite. b) Dataare ZOI diameters measured in mm, letter codes refer to zone quality ranging from A (absolutely clear)to E (very turbid).

column chromatography, and preparative HPLC. The identity of the compound wasconfirmed by ESI-TOF MS (m/z 875.4029 ([MþH]þ ); calc. for C39H60N6NaOþ

15,875.4014) and comparison of its 1H-NMR spectrum with that reported in [23]. The (Z)-geometry of the C¼C bond present in each monomer of the molecule was secured by aNOESY correlation observed between the olefinic H-atom and the allylic Me group.

Purified desferritriacetylfusigen (1 mg ml�1 in DMSO) was active against S. aureusgrown in brain-heart infusion agar (ZOI 12 mm), but its activity was more pronounced,when S. aureus was grown on the low-iron medium, CAS (ZOI 15 mm) (Fig. 4).


Fig. 2. Relative abundance of echinocandin B (3) among 43 species and varieties of Aspergillus subgenusNidulantes grown on medium NPF2 with and without epigenetic modifying agents, Amberlite XAD7, andin vermiculite solid fermentations. Relative abundance measured by positive-ion intensity (POS) of m/z

1082. Strain order and culture medium and manipulations are the same as in Tables 1–3.

Isolation, Structure Elucidation of Echinocandin E (2) from E. quadrilineata, and ItsBiological Activity. A 1-l culture of E. quadrilineata (J. Guarro WD-007) grown inNPF2 vermiculite SSF was extracted by addition of acetone, centrifugation, andevaporation of the organic solvent under N2. The aqueous residue was subjected toSP207ss column chromatography followed by preparative HPLC to yield echinocan-dins E and B (2 and 3, resp.) [21]. The molecular formula of 2 was determined asC50H77N7O16 by HR-MS (ESI-TOF; m/z 1032.5517 ([MþH]þ ); calc. for C50H78N7Oþ

16

1032.5505). The 1H- and 13C-NMR spectra of 2 (Table 5) were nearly identical to those


Fig. 3. Relative abundance of desferritriacetylfusigen (1) among 43 species and varieties of Aspergillussubgenus Nidulantes grown on medium NPF2 with and without epigenetic modifying agents, AmberliteXAD7, and in vermiculite solid fermentations. Relative abundance measured by UV absorbance at218 nm (AU 218 NM). Strain order and culture medium and manipulations are the same as in Tables 1–3.

of echinocandin B (3), confirming that the new compound belonged to this family. Themajor differences were found in the intensity of the signal at d(H) 1.33 ppm(accounting for the CH2 H-atoms of the fatty acid residue) and the absence of twoC-atom signals in the spectrum of 2 compared with that of 3. These findings indicatedthe presence of a shorter fatty acid residue in the structure of 2 that was identified as(9Z,12Z)-hexadeca-9,12-dienoic acid on the basis of 2D-NMR correlations and 13Cchemical shifts. Thus, COSY and TOCSY correlations established the sequence fromthe olefinic H-atom H�C(13) to the terminal Me group of the fatty acid Me(16), andHMBCs from CH2(15) and Me(16) to the allylic C-atom C(14), from CH2(15) to theolefinic C-atom C(13), and from the allylic H-atoms CH2(14) to C(16) confirmed theposition of one of the C¼C bonds at C(12). On the other hand, the low-field d(H) valueof the doubly allylic CH2(11) (2.78 ppm) and its HMBCs with the olefinic C-atomsC(9), C(10), C(12), and C(13) confirmed the presence of the second C¼C bond atC(9). In addition, the high-field chemical shifts observed for the CH2 C-atoms C(8)(28.2 ppm) and C(11) (26.5 ppm) confirmed the (Z)-configuration for both C¼C bondsof the residue [24]. Finally, the configurations at all the stereogenic centers in themolecule were assumed to be the same as in echinocandin B (3) due to the closesimilarity in chemical shifts and multiplicities of the NMR signals of both compoundsand the optical-rotation value.

Purified echinocandin E (2 ; IC50 1.5 mm) was determined to be ca. 5.5 times lesspotent than echinocandin B (3 ; IC50 0.27 mm) in a liquid dilution series assay against C.albicans (Fig. 5).


Fig. 4. Zone of inhibition assay with Staphylococcus aureus (MB5393) on brain heart infusion agar(BHIA) and on low-iron casamino acids agar (CAS). Inhbition zones of crude extract from E.quadrilineata (WH-042) were compared to purified desferritriacetylfusigen (1; 10 ml of 1 mg ml–l inDMSO) on both media. Kanamycin and tunicamycin were positive controls. Amphotericin B was the

negative control. DMSO (20 and 100%) was inactive.

Discussion. – The high percentage of antibiotic phenotypes (37%) among extractsgenerated from the strain�media combinations and the high percentage of strainsexhibiting at least one antibiotic phenotype (88%) were consistent with the largenumber of biosynthetic pathways predicted to be present in Emericella species [19] andthe large number of metabolites that have been previously reported from these fungi[25]. If the experiment had included more media formulations for each strain, and ifmore different assays, e.g., other bacteria and mammalian cell cultures, had been tested,then an antibiotic phenotype could have been detected among all strains, species, andvarieties [3]. Although manipulation of secondary metabolism is a vital strategy inmetabolite discovery, the striking range of antibiotic phenotypes expressed among thedifferent species and varieties reinforces the idea that addition of new genotypes in thescreening process is also an essential discovery strategy [3].

Media manipulations leading to the greatest deviations in antibiotic phenotypesrelative to controls were the medium with XAD7 resin and the vermiculite SSF


Table 5. 1H- and 13C-NMR Data for Echinocandin E (2) in CD3OD; d in ppm, J in Hz. Assignmentsbased on COSY, TOCSY, HSQC, and HMBC experiments.

Position d(H) d(C) Position d(H) d(C)

3,4-Dihydroxyhomotirosine Thr-21 172.5 1 172.52 4.32 (m) 56.4 2 4.96 (dd, J¼8.8, 3.2) 58.73 4.22 (dd, J¼7.8, 1.7) 77.0 3 4.51 (m) 68.44 4.30 (d, J¼7.8) 75.7 4 1.21 (d, J¼6.3) 19.71’ 133.1 3-Hydroxyproline2’/6’ 7.14 (d, J¼8.6) 129.6 1 173.53’/5’ 6.75 (d, J¼8.6) 116.2 2 4.58 (dd, J¼11.4, 7.0) 62.54’ 158.5 3 2.43 (br. dd, J¼13.1, 7.9), 2.07 (m) 38.6Thr-1 4 4.56 (m) 71.31 170.0 5 3.97 (dd, J¼11.0, 3.2),

3.80 (br. d, J¼11.0)57.2

2 4.84 (dd, J¼9.1, 4.3) 56.9(9Z,12Z)-Hexadeca-9,12-dienoic acid3 4.15 (m) 69.71 176.24 1.21 (d, J¼6.3) 20.02 2.22 (t, J¼7.4) 36.83-Hydroxy-4-methylproline3 1.60 (m) 27.11 172.84 1.33 (m) 30.32 4.33 (d, J¼2.7) 69.75 1.33 (m) 30.33 4.19 (dd, J¼4.7, 2.7) 75.66 1.33 (m) 30.34 2.53 (m) 39.17 1.33 (m) 30.85 3.87 (dd, J¼9.2, 7.5),

3.38 (dd, J¼9.2, 9.2)52.9

8 2.05 (m) 28.24-Me 1.06 (d, J¼6.9) 11.3 9 5.37 (m) 130.74,5-Dihydroxyornithine 10 5.33 (m) 129.1*1 174.4 11 2.78 (t, J¼6.5) 26.52 4.39 (dd, J¼12.0, 5.2) 51.7 12 5.33 (m) 129.3*3 2.09 (m), 1.92 (m) 35.1 13 5.37 (m) 130.94 3.93 (m) 70.9 14 2.05 (m) 30.85 5.24 (dd, J¼8.8, 3.1) 74.7 15 1.39 (sext. , J¼7.4) 23.9

16 0.92 (t, J¼7.4) 14.1

(Tables 2– 4, and Figs. 2 and 3). The most dissimilar medium pairs were 5AZ andvermiculite (for S. aureus), and XAD7 and vermiculite (for C. albicans). Clearlyparallel treatments of strains with resin and vermiculite medium during antibiotic andsecondary metabolite screening deserve consideration.

XAD7 was chosen among other possible non-ionic exchange resins because of itssuccessful use in capturing metabolites in actinomycete and fungal fermentations[7] [10] [26], although other resins should be at least as effective. In the case of ZOIassays against S. aureus, a statistically significant increase in ZOIs was observed in theXAD7-treated medium, probably aggravated by iron depletion and further inducing ofbiosynthesis of the weakly antibacterial siderophore, desferritriacetylfusigen (1) [27].The iron-complexed form of 1, triacetylfusigen, has been observed in Aspergillusfumigatus, A. deflectus, A. nidulans, and Penicillium chrysogenum [28– 30]. In the liquidmedia, iron depletion probably occurred because NPF2 lacked an iron supplement.However, this unintentional effect was exacerbated in the presence of an ironadsorbing resin [31]. Growth on iron-free media, or addition of ion-exchange andchelating resins to media have previously been used to induce siderophore biosynthesisin Aspergilli and other fungi [32] [33]. This effect was ameliorated by growing the samestrains in a vermiculite matrix where presumably structural mineral iron is solubilizedfor mycelial uptake (Fig. 3). Therefore, although positive effects of resin additions tofermentation media, either via adsorption of unstable products or removal of pathwayfeedback repression, have been observed, some caution should be exercised to assurethat sufficient trace elements are added to the medium to prevent unwanted inductionof siderophore biosynthesis.

In the above example, treatment of fermentation media with either SAHA or 5AZcaused few changes in antibiotic profiles relative to the untreated control. Someevidence of changes in relative amounts of unidentified metabolites was observed in


Fig. 5. Dose responses of Candida albicans (MY 1055) growth in liquid RPMI-1640-modified mediumagainst echinocandin E (2) and echinocandin B (3). Statistical support for assay robustness and curve fit

were 0.82 Z’ factor and R2�0.992, respectively.

some strains (data not shown). Furthermore, a preliminary experiment treating strainE. rugulosa J. Guarro WO-148 with SAHA in other media showed changes inmetabolites profiles and spontaneous appearance of S. auerus activity relative to theuntreated control (unpublished data). Although epigenetically modifying compoundshave demonstrated potential for modulating expression of biosynthetic pathways, theprobability of provoking a change in an antibiotic metabolite against a specificpathogen is likely to be less, because not all of a given organism�s metabolites areantibiotic to a specific pathogen. In a recent example [9], twelve different fungi isolatedfrom marine sediments were fermented in a single medium treated with SAHA byusing a protocol similar to those described here. Extract profiling by LC-ELSD/MSrevealed only one significant new metabolite, although other changes in metaboliteprofiles were noted. The outcome of biological profiling of the same extract set was notmentioned, although one might speculate that more fungi may have needed to bescreened before observing a significant change in antibiosis.

Conclusions. – Within the test population of Emericella strains, the intrinsicmetabolic capacity of the species, rather than any particular medium treatment, morestrongly influenced patterns of antibiosis. Two media treatments, addition of AmberliteXAD7 and growth in vermiculite, caused the most divergent patterns of antibiosisrelative to controls. The antibacterial siderophore, desferritriacetylfusigen (1), wasdetected in most species in liquid media, but not in the vermiculite medium,presumably due to the low iron concentration in liquid medium relative to iron releasedfrom the vermiculite. This effect was exacerbated in medium with added resin. In caseof using ion-exchange resins in fermentation medium, possible immobilization of traceelements should be taken into account, and media should be supplemented with traceelements. The predominant antifungal metabolite detected was echinocandin B (3).Some species produced this antifungal metabolite regardless of treatment, althoughhigher quantities were often produced in vermiculite SSF. Several species are reportedfor the first time to produce 3. Echinocandin E (2), a new echinocandin analog, wasproduced by E. quadrilineata.

Experimental Part

General. Optical rotations: Perkin-Elmer polarimeter 341. NMR Spectra: Bruker Avance IIIinstrument (1H: 500 and 13C: 125 MHz). ESI-TOF-MS: Bruker maXis ; in m/z.

Fungal Strains. Authoritatively identified strains were purchased from the Centraalbureau voorSchimmelcultures (http://www.cbs.knaw.nl/) or received from the Universitat Rovira i Virgili, Reus,Tarragona, Spain. Additional strains were isolated and identified in our laboratory (strains designated,e.g., F-166,668). Their origins, strain numbers, and identifications are listed in Table 1. Strains werepreserved as frozen mycelial discs in 10% glycerol at �808 and maintained in the collection of FundacionMEDINA, Granada, Spain, unless designated otherwise. Frozen strains were revived by plating frozenagar discs onto YM agar (malt extract, 10 g; yeast extract, 2 g; agar, 20 g; 1000 ml of dist. H2O) at 228.

Cultivation of Strains. Mycelia discs from YM agar cultures were used to inoculate 250-ml flaskscontaining 50 ml of SMYA medium (Difco neopeptone, 10 g; maltose, 40 g; Difco yeast extract, 10 g;agar, 4 g; 1000 ml of dist. H2O). Flasks were incubated in an orbital shaker at 220 rpm for 4 d at 228. Thisseed inoculum was used to inoculate fermentations in 40-ml EPAvials containing 10 ml of liquid media orSSF in a vermiculite matrix incubated in 1-l roller bottles. The base medium for all fermentations was


NPF2 (150 g of glucose, NZ amine type A (Sigma�Aldrich), 4 g; urea, 4 g; K2HPO4, 0.5 g; MgSO4 ·7 H2O, 0.25 g; KCl, 0.25 g; ZnSO4 ·7 H2O, 0.9 g; CaCO3, 16.5 g; 1000 ml of dist. H2O) [34] [35]. Thecontrol medium was NPF2 dispensed at 10 ml in 40-ml EPA vials, inoculated with 1 ml of myceliainoculum, and was agitated at 220 rpm. The SAHA-treated medium was NPF2 prepared, inoculated, andagitated the same way. After 48 h, SAHA dissolved in DMSO was aseptically added to each vial to attaina concentration of 100 mm. The 5-azacytidine (5AZ) treated medium was prepared, inoculated, andagitated in the same ways as the control, and 5AZ dissolved in DMSO was aseptically added to each vialafter 48 h to attain a concentration of 100 mm. The resin-treated medium was prepared by washingAmberlite XAD7 (Sigma�Aldrich) twice with an equal volume of MeOH, followed by five washes withdist. H2O. Resin was stored in dist. H2O at 48 until used. Wet activated resin was added to each EPA vialto attain 3% weight of the fermentation. Medium was added, and the resin and medium were autoclavedtogether. The resin-treated medium was inoculated and agitated in the same way as the control. Toprepare SSFs (Fig. 1), 330 ml of coarse vermiculite were added to a 1-l medium bottle and doubleautoclaved. For each bottle of vermiculite, 115 ml of liquid NPF2 medium were autoclaved separately.Eight ml of inoculum were added to the liquid medium, mixed thoroughly, and the mixture wasaseptically added to the bottle of vermiculite. The inoculated vermiculite was incubated on tissue cultureroller machine (Bellco, Vineland, NJ, USA) at 4 rpm to homogeneously mix the fungal cells and mediuminto the vermiculite matrix. When fungal mycelia had grown enough to completely penetrate andimmobilize the matrix (4 d), the roller machine was turned off, and mycelia were further incubatedstatically until extracted. All fermentations were incubated for 14 d at 228.

To purify and determine the structures of echinocandins B and E (3 and 2 , resp.), strain E.quadrilineata J. Guarro WD-007 was grown in five 2-l roller bottles with 225 ml of liquid NPF2 in 675 mlof vermiculite as described above. To purify and confirm the structure of desferritriacetylfusigen (1),strain E.quadilineata J. Guarro WH-042 was grown in 20 250-ml Erlenmeyer flasks each containing 50 mlof NPF2 and 4% XAD7 as described above.

Extraction and Preparation of Screening Samples. Strains grown in EPA vials were extracted bymixing the 10-ml fermentation broths with 10 ml of ethyl methyl ketone (EtCOMe). After 1 h of orbitalshaking, the EtCOMe/aq. mixtures were centrifuged at low speed, and the org. phases were transferred toglass tubes. Strains grown in SSFs with vermiculite were extracted by adding 180 ml of EtCOMe to themycelium�vermiculite aggregation. The mycelial�vermiculite mass was macerated and then shaken for1 h. After filtering, 10 ml of the EtCOMe phases were transferred manually into glass tubes.

To each tube, H2O (4 ml) and DMSO (1 ml) were sequentially added to increase the solubility ofmetabolites during evaporation. The samples were concentrated during orbital shaking under a warm N2

stream to 5 ml. The final samples were 2� whole broth equivalents (2� WBE), including 20% DMSOrel. to original culture volume. Extracts were aliquoted in 96-well screening plates (AB gene AB-0765,800 ml wells) for biological and chemical evaluation.

Extraction and Isolation of Desferritriacetylfusigen (1). A culture of E. quadrilineata WH-042 (1 l)was extracted by addition of an equal volume of acetone, shaking for 1 h, centrifugation, filtration, andevaporation of the org. solvent under N2. The aq. residue was loaded on to a SP207ss column (65 g, 32�220 mm) that was eluted with a gradient of acetone in H2O (0 –100 % acetone in 15 minþ100% acetonefor 15 min, 8 ml/min, 18 ml/fraction). Half of Fr. 6 was subjected to prep. HPLC (Zorbax SB-C8, 21.2�250 mm, gradient H2Oþ0.1% CF3COOH (TFA)/MeCNþ0.1% TFA from 5 to 100 % of MeCN/TFA in80 min, 20 ml/min, UV detection). The peak eluting at 31 min was repurified on the same column with agradient from 35–45% MeCN/TFA in 80 min to afford 4.1 mg of 1.

Extraction and Isolation of Echinocandins E and B (2 and 3, resp.). A 1-l culture of E. quadrilineatawas extracted with acetone and SP207ss column chromatography under the same conditions used for theisolation of 1. Prep. HPLC of half of Fr. 8 of the latter, HPLC (Zorbax SB-C8, 21.2�250 mm, gradientH2Oþ0.1% TFA/MeCNþ0.1% TFA from 5 to 100% of MeCN/TFA in 80 min, 20 ml/min, UVdetection) yielded a group of fractions eluting between 42 and 50 min that were subjected to a new prep.HPLC separation on the same column with a gradient from 40 to 80 % MeCN/TFA in 80 min to give1.0 mg of 2 and 2.0 mg of 3.

Data of Echinocandin E (¼ (9Z,12Z)-N-{(2R,6S,9S,11R,12R,14aS,15S,16S,20S,23S,25aS)-23-[(1S,2S)-1,2-Dihydroxy-2-(4-hydroxyphenyl)ethyl]-tetracosahydro-2,11,12,15-tetrahydroxy-6,20-bis[(1R)-


1-hydroxyethyl]-16-methyl-5,8,14,19,22,25-hexaoxo-1H-dipyrrolo[2,1-c :2’,1’-l] [1,4,7,10,13,16]hexaazacy-clohenicosin-9-yl}hexadeca-9,12-dienamide ; 2). Amorphous solid. [a]25

D ¼ �30.0 (c¼0.06, MeOH). 1H-and 13C-NMR: see Table 5. HR-ESI-TOF-MS (pos.): 1032.5517 ([MþH]þ , C50H78N7Oþ

16 ; calc.1032.5505, D 1.6 ppm), 1054.5320 ([MþNa]þ , C50H77N7NaOþ

16 ; calc. 1054.5324, D 0.09 ppm).Antimicrobial Susceptibility. Antibacterial susceptibility was tested with the methicillin-resistant S.

aureus (MRSA), MB5393, while antifungal susceptibility was tested with the strain C. albicans MY1055,a clinical isolate from MEDINA�s Culture Collection.

For C. albicans, thawed stock inoculum suspensions from cryovials were streaked on Sabourauddextrose agar plates (SDA; 65 g l�1) to grow new colonies. A few of the colonies were inoculated into10 ml of Sabouraud dextrose broth (30 g l�1 H2O) that was incubated overnight at 308. The C. albicanssuspension was adjusted to an optical density of 0.4 at 660 nm. This suspension was added to yeastnitrogen base-dextrose agar (yeast nitrogen base, 6.75 g l�1; dextrose, 1 g l�1; agar, 17.5 g l�1 H2O) in theproportion of 30 ml l�1. One hundred-ml aliquots of the seeded agar media were poured onto 25 cm2

Nunc assay plates. Extracts and controls (10 ml) were applied onto the surface of the seeded assay plates,and plates were incubated at 308 for ca. 20 h. Amphotericin B (160 mg ml�1) and kanamycin (0.5 mg ml�1)were used as positive and negative internal plate controls, resp. Zones of inhibition (ZOIs) were handmeasured and photographed.

For S. aureus, thawed stock inoculum suspensions from cryovials were streaked onto Brain HeartInfusion Agar (BHIA; Difco) plates, which were incubated at 378 overnight to obtain isolated colonies.Single colonies were selected to inoculate an overnight culture to be used as assay plate inoculum. Thesingle colonies were inoculated into 10 ml of Brain Heart Infusion Broth (BHIB; Difco) in 250-mlErlenmeyer flasks. The flasks were incubated and agitated at 220 rpm overnight at 378. To prepare theassay plates, the BHIA medium was inoculated to 3% final with the overnight culture, which previouslywas adjusted to an optical density of 0.2 at 660 nm. Assay plates were prepared as described above for C.albicans, and 10-ml samples were applied directly to the agar surface, then plates were incubated for 18 to20 h at 378. Amphotericin B (200 mg ml�1) and tunicamycin (150 mg ml�1), and kanamycin (0.5 mg ml�1)were used as negative and positive controls, resp. Zones of inhibition (ZOIs) were hand measured andphotographed. The tests were duplicated with two EtCOMe extracts prepared for each fermentationsample. Measured values were usually very similar (differences <3 mm of ZOI), and the two valueswere averaged. Any extract producing a visibly discernable ZOI, regardless of zone quality, wasconsidered to be positive. ZOI Quality was subjectively evaluated by the same person from absolutelytransparent (A) to very turbid (E).

Purified 1 (10 mg ml�1 in DMSO) was assayed against S. aureus on BHIA an on a low iron medium,CAS, consisting of casamino acids, 10 g (Becton Dickinson); (NH4)2SO4, 1 g; MgSO4 · 7 H2O, 0.1 g;glucose, 2 g; KH2PO4, 3 g; K2HPO4, 7 g; Noble agar, 15 g, was autoclaved. After cooling, 2 ml of filter-sterilized vitamin soln. 1 (1000 ml of H2O, 40 mg of 4-aminobenzoate, 10 mg of l-biotin, 100 mg ofnicotinic acid, 50 mg of hemicalcium d-l-pantothenate, 150 mg of pyridoxamine hydrochloride, 100 mg ofthiamine chloride hydrochloride, and 50 mg of cyanocobalamin) and 6 ml of filter sterilized vitamin soln.2 (1000 ml of H2O, 10 mg of dl-6,8-thioctic acid, 10 mg of riboflavin, and 4 mg of folic acid) were added.

Candida albicans Liquid Growth Assay and MIC Determination of Echinocandins. Frozen stocks ofC. albicans MY1055 were used to inoculate Sabouraud Dextrose Agar (SDA) plates for confluentgrowth. Plates were incubated for 24 h at 308. The grown colonies were harvested from the SDA platesand suspended in RPMI-1640 modified medium. Medium RPMI-1640 modified was prepared as follows:20.8 g of RPMI powder (Sigma�Aldrich) were poured into a 2-l flask, together with 13.4 g of YNB, 1.8 lof milliQ H2O, 80 ml of HEPES (1m), and 72 ml of 50% glucose. The volume was adjusted at 2 l andfiltered. The OD660 was adjusted to 0.25 (ca. 108 CFUml�1) using RPMI-1640 modified as diluent andblank. The inoculum was diluted 1 : 10 and kept on ice until it was used to inoculate 96-well assay plates.

For the assay, 90 ml of the 1 : 10 diluted inoculum were mixed with 10 ml of sample. Amphotericin Band penicillin G were used as positive and negative controls at each plate, resp. After dispensing theinoculum, the assay plates were read in a Tecan Ultraevolution spectrophotometer at 612 nm for T0 (zerotime). Then, the plates were statically incubated at 308 for 18 –24 h. After incubation, the plates wereshaken in a DPC Micromix-5 and read again for Tf (final time). Percentage of growth inhibition was


calculated using the following normalization: % Inhibition¼ [1� [(TfSample�T0Sample)� (TfBlank�T0blank)/(TfGrowth�T0Growth)� (TfBlank�T0blank)]�100.

The screen data were summarized using the Genedata Screener program (Genedata AG, Switzer-land). Both antifungal dose�response curves for echinocandin B (3) and echinocandin E (2), started at16 mg/ml of compound per well in 1 : 2 serial dilutions, and the assay was carried out in triplicate asexplained above. Assay robustness (Z factor) [36] and the goodness-of-fit of the curves were calculated.The MIC value was defined as the lowest concentration of an antimicrobial that inhibited the visiblegrowth of a microorganism after overnight incubation.

Effect of Medium Manipulation on Antibiosis. Any extract producing a discernable ZOI, regardlessof zone quality, was considered to be positive and assumed to be indicative of production of ametabolite(s) perturbing cell growth, i.e., bioactivity. For scoring antibiotic phenotypes, eachrecognizable and measurable ZOI, regardless of turbidity and quality, was counted as an indicationthat some extract component disturbed pathogen growth.

Strains by medium matrix with the 215 individual assays were compiled (43 strains�5 media) foreach set antibiotic activity against S. aueus and C. albicans, and any observable ZOI was scored as 1 andabsence as 0 (Tables 3 and 4). In addition to observations of qualitative trends in the data, the hypothesisthat the total number ZOIs observed for each medium was homogenously distributed among all mediumconditions was tested with a c2 goodness-of-fit test. The degree of the intersection between patterns ofantibiosis of different media pairs was estimated with Jaccard�s coefficient of similarity.

Conditions for HPLC and ESI-LC/MS. In a first-line identification, 2 ml of the active extractsselected in the antibiotic assays were examined by liquid chromatography/mass spectrometry (LC/MS)and compared to a proprietary database, which, in many cases, yielded a positive match with a knownantibiotic or metabolite. Extracts were analyzed with an Agilent (Santa Clara, CA) 1100 singlequadrupole LC/MS, using a Zorbax SB-C8 column (2.1�30 mm), maintained at 408 and with a flow rateof 300 ml min�1. Solvent A consisted 10% MeCN and 90% H2O with 1.3 mm TFA and HCOONH4, andsolvent B contained 90% MeCN and 10% H2O with 1.3 mm TFA and HCOONH4. The gradient startedat 10% B and went to 100% B in 6 min, kept at 100% B for 2 min, and returned to 10% B for 2 min toinitialize the system. Full diode-array UV scans from 100 to 900 nm were collected in 4-nm steps at 0.25 s/scan. The eluting solvent was ionized using the standard Agilent 1100 ESI source adjusted to a drying gasflow of 11 l min�1 at 3258 and a nebulizer pressure of 40 psig. The cap. voltage was set to 3500 V. Massspectra were collected as full scans from m/z 150 to 1500, with one scan every 0.77 s, in both positive- andnegative-ion modes.

Database Search and Metabolite Identification. The DAD (diode array detection) spectra, retentiontime, and positive- and negative-ion mass spectra of the active samples were searched with an in-housedeveloped application and compared to the UV-LC/MS data of known metabolites stored in aproprietary database [37] [38], where metabolite standard data had been obtained under the same LC/MS conditions as the samples under analysis. Presence and relative abundance of different metaboliteswere verified in fermentation extracts by HPLC/high resolution (HR)-ESI mass spectra recorded on aBruker maXisTM QTOF-MS instrument (Bruker Daltronics GmbH, D-Bremen) coupled to the sameHPLC system as described above.

We gratefully acknowledge the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands,for providing strains. Marc Stadler made helpful suggestions on the text.

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Received November 3, 2011


Chemical and Physical Modulation of Antibiotic Activity in Emericella Species

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