Expanding the Chemical Repertoire of the Endophyte Streptomyces

Article pubs.acs.org/jnp

Expanding the Chemical Repertoire of the Endophyte Streptomyces albospinus RLe7 Reveals Amphotericin B as an Inducer of a Fungal Phenotype Fernanda Oliveira Chagas,†,# Andrés Mauricio Caraballo-Rodríguez,†,# Pieter C. Dorrestein,‡ and Mônica Tallarico Pupo*,† †

Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n, Ribeirão Preto, SP 14040-903, Brazil ‡ Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0751, United States S Supporting Information *

ABSTRACT: During an investigation of the chemistry of the endophytic actinobacterium Streptomyces albospinus RLe7, which was isolated from the roots of the Brazilian medicinal plant Lychnophora ericoides, three new natural products, (2R*,4S*)-2-((1′S*)-hydroxy-4′methylpentyl)-4-(hydroxymethyl)butanolide (1), (3R*,4S*,5R*,6S*)tetrahydro-4-hydroxy-3,5,6-trimethyl-2-pyranone (2), and 1-O(phenylacetyl)glycerol (3), together with known secondary metabolites (S)-4-benzyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (4), (S)-4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (5), and the diketopiperazines cyclo(LTyr-L-Pro) (6) and cyclo(L-Val-L-Pro) (7), were isolated. The role of isolated natural products in the interaction between S. albospinus RLe7 and the fungus Coniochaeta sp. FLe4, an endophyte from the same plant, was investigated. None of these isolated actinobacterial compounds were able to inhibit the fungus or induce the fungal red pigmentation observed when both endophytes interact. Further investigation using mass spectrometry approaches enabled identifying the well-known antifungal compound amphotericin B (9) as a microbial metabolite of S. albospinus RLe7. Finally, compound 9 was demonstrated as at least one of the agents responsible for both the antifungal activity and induction of redpigmented fungal phenotype.

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not necessarily reflect their true roles in nature.9 Characterizing chemical entities and understanding their biological function will give insights into how those molecules are used in nature.10 Small molecules are widely produced by microorganisms, and the explanation of their natural role is still in its infancy. Signal molecules and quorum sensing in bacteria appear to be very common and are not restricted to pathogens involved in infectious processes.10−12 Related compounds have been closely associated with specific functions such as acylhomoserine lactones13 and alkyl quinolones14,15 in cell signaling and quorum sensing, siderophores in growth development,16 and low-molecular-weight thiols having antioxidant function.17 However, in many cases, little is known about the functions that small molecules may play in nature,18 implying that the correlation between chemical structures and biological roles is yet to be established. A previous chemical investigation of the actinobacterium Streptomyces albospinus RLe7, an endophyte from the Brazilian

ndophytic actinobacteria associated with medicinal plants are interesting as a source of bioactive compounds.1 As in any microbial consortium, they are in continuous competition for nutrients, leading to the production of specialized molecules that enable survival in specific environments.2 Recent research suggests that some metabolites initially described as plantspecific are either microbial products or the result of a host− symbiont interaction, such as in endophyte−plant symbiosis.3−5 Laboratory conditions for microbial culturing do not necessarily reflect conditions in the natural environment. Simulating endophytic−host interactions in these conditions is challenging, although some examples have been published.6 Despite the long-standing efforts to discover new metabolites from microbial sources, the potential of microorganisms is much larger than previously expected.7 Genomic approaches have revealed a huge reservoir of cryptic metabolites from unexpressed genes, supporting the continuous development of tools to explore this hidden microbial arsenal.8 Specific biological activities have driven natural product discovery in recent decades, leading to innumerable characterized molecules from microbial sources. This bioactivity does © 2017 American Chemical Society and American Society of Pharmacognosy

Received: September 23, 2016 Published: April 4, 2017 1302

DOI: 10.1021/acs.jnatprod.6b00870 J. Nat. Prod. 2017, 80, 1302−1309

Journal of Natural Products

Article

Figure 1. Compounds produced by the endophyte S. albospinus RLe7.

Table 1. NMR Data (500 MHz, MeOH-d4) for the New γ-Lactone 1 Isolated from S. albospinus RLe7

a

position

δC,a type

1 2 3

180.0, C 47.5, CH 24.2, CH2

4 5

80.8, CH 64.6, CH2

1′ 2′

70.1, CH 34.2, CH2

3′

35.9, CH2

4′ 5′ 5″

28.8, CH 22.6, CH3 22.6, CH3

δH (J in Hz) 2.84, 2.18, 2.02, 4.48, 3.72, 3.60, 3.98, 1.48, 1.46, 1.34, 1.19, 1.54, 0.89, 0.90,

ddd (11.1, 9.6, 2.9) ddd (12.4, 9.6, 6.7) ddd (12.4, 11.1, 10.0) dddd (10.0, 6.7, 6.1, 3.4) dd (12.3, 3.4) dd (12.3, 6.1) ddd (10.3, 5.2, 2.9) m m m m m d (6.6) d (6.6)

gCOSY (H→H)

gHMBC (H→C)

3a, 3b, 1′ 2, 3b, 4 2.18; 2.84; 4.48 2, 3a, 4 3a, 3b, 5a, 5b 4, 5b 4, 5a 2, 2′a, 2′b 1′, 3′a, 3′b 1′, 3′a, 3′b 2′a, 2′b, 3′b 2′a, 2′b, 3′a 5′, 5″ 4′ 4′

1, 3, 1′ 1, 2, 5 C-2; C-4; C-5; C-1′ 2, 4, 5, 1′ n.o. 3 3, 4 n.o. 2, 1′, 3′, 4′ C-2; C-1′; C-3′ ; C-4′ 2, 1′, 3′, 4′ 2′, 4′, 5′ C-1′; C-2′; C-4′; C-5′ 1′, 2′, 4′, 5′ 2′, 3′, 5′ 3′, 4′, 5′ 3′, 4′, 5′

Assignments made indirectly based on gHMBC and gHSQC data; n.o.: not observed.

connected to heteroatoms (δC4 80.8 and δH4 4.48; δC1′ 70.1 and δH1′ 3.98; and δC5 64.6 and δH5 3.60 and 3.72). Carbon C-4 was more deshielded than expected for a carbon only connected to oxygen, suggesting the presence of an additional structural feature contributing to the chemical shift. Three additional methylene groups were observed (δC3′ 35.9, δH3′ 1.19 and 1.34; δC2′ 34.2, δH2′ 1.46 and 1.48; and δC3 24.2, δH3 2.02 and 2.18), as well as two additional methines (δC2 47.5 and δH2 2.84; δC4′ 28.8 and δH4′ 1.54) and two methyls (δC5′ 22.6 and δH5′ 0.89; δC5″ 22.6 and δH5″ 0.90) (Table 1). Since the multiplicities of the signals in the 1H NMR spectrum were intricate, the gCOSY correlations helped to determine the vicinity of some of the carbons. The H-4 showed correlations to H-3, and H-3 to H-2. Additionally, H-4 was also correlated to H-5, and H-2 to H-1′. According to the gHMBC, correlations of H-3 and H-2 to the ester carbonyl C-1 (δC 180.0) were observed. The chemical shifts of C-4 and C-2 suggested that C-2 was connected to the carbonyl C-1 and C-4 to the oxygen of the ester, forming a five-membered lactone. In addition, the gCOSY and gHMBC correlations showed that this γ-lactone was 2,4-disubstituted. A hydroxymethyl group

medicinal plant Lychnophora ericoides (falsa-arnica), led to the isolation of two siderophores, namely, nocardamine and dehydroxynocardamine, together with the pyrroloindole alkaloid physostigmine.19 However, the role they play in nature is still unknown. The aim of this study was to increase our knowledge of the chemical repertoire of this actinobacterium and investigate the potential role for its secondary metabolites during microbial antagonistic interactions with an endophytic fungus from the same plant host.

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RESULTS AND DISCUSSION The endophytic strain S. albospinus RLe7 was cultured in both PDB (potato dextrose broth) and rice supplemented with liquid ISP-2 (International Streptomyces Project−medium 2). Compounds 1, 2, and 3 were obtained from solid medium, while compounds 4, 5, 6, and 7 were isolated from liquid medium (Figure 1). Compound 1 was obtained in the S-2-14 fraction as a pale yellow wax. The carbon shifts of compound 1 were assigned from the gHSQC and gHMBC spectra. 1H NMR and gHSQC showed the presence of methine and methylene carbons 1303



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Streptomyces coelicolor, which have been manipulated to express part of the erythromycin biosynthetic gene cluster.27,28 As for other small molecules,10 the δ-lactone 2 may be involved in cellular and/or interspecific signaling. However, as for compound 1, its biological role with respect to its producer organism remains unclear. Compound 3 corresponds to 1-O-(phenylacetyl)glycerol and was obtained in fraction S-2-11. In addition to the aliphatic and aromatic monoacylglycerols that are widely distributed in nature,29,30 this is the first report of the isolation of compound 3 as a natural product. Synthetically, the aromatic monoacylglycerol 3 has been produced by esterification of glycerol with aromatic anhydrides employing biocatalysis.29,31 Our data (Supporting Information) are in agreement with reported data for 3.32 A specific optical rotation value for this compound was provided. The isolated alkaloids (S)-4-benzyl-3-oxo-3,4-dihydro-1Hpyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (4) and (S)-4-isobutyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (5) are biosynthetic congeners. These compounds have been isolated as natural products from sugar cane33 and produced artificially under very high temperatures by reaction of D-glucose with an α-amino acid.34,35 In smoked tobacco, the presence of those compounds and other similar molecules improves its flavor.34,36 In sugar cane, they are correlated with a sweet smell.33 Both compounds have also been isolated from the plant’s fruits.37,38 More recently, this class of compounds was isolated for the first time from a mangrove-derived actinobacterium, and the absolute configuration of their single stereogenic center was determined to be S,39 indicating that they are derived from L-amino acids. Their biological roles in producer organisms and potential biological activities have never been reported. These flavored compounds may be involved in insect signaling in nature. The diketopiperazines cyclo(L-Tyr-L-Pro) (6) and cyclo(LVal-L-Pro) (7) were also isolated from S. albospinus RLe7. The cyclic diketopiperazines derived from simple amino acids are compounds whose roles have not been defined.18 These compounds have been found in Gram-positive and Gramnegative bacteria, fungi, plants, and animals.40 Many biological activities have been correlated with cyclic diketopiperazines, including antibacterial, antifungal, antitumor, antiviral, and cell signaling. 41 However, some researchers have presented controversial results regarding biological activities.18,41 More recently, the discovery of a novel mechanism of biosynthesis of cyclic diketopiperazines has increased interest in this class of compounds.40 Nevertheless, their natural roles have not yet been identified. Additionally, there are no similarities between the secondary metabolites isolated from S. albospinus RLe7 and those isolated from the host plant L. ericoides, which include sesquiterpene lactones, flavonoids, chlorogenic acids, lignans, and triterpenes.42,43 Given the lack of knowledge about the ecological role of the compounds described above isolated from the endophytic actinobacterium S. albospinus RLe7 and the previously isolated alkaloid physostigmine (8),19 which possesses anticholinesterase properties, we decided to test them against another endophytic microorganism from the same plant. This approach is supported by our previous mixed culturing experiments with endophytic microorganisms that showed the role of an induced fungal secondary metabolite in the endophyte−endophyte and endophyte−host plant interactions.44

was attached to C-4. On the basis of the analysis of the remaining carbons and hydrogens, the group attached to C-2 was determined (see below). In the gCOSY data, the correlations of hydrogens H-5′ and H-5″ of both methyl groups to H-4′ suggested the presence of an isopropyl moiety in the structure, and the gHMBC correlations of those hydrogens to C-3′ indicated that C-4′ is linked to C-3′. In the same way, the correlations of H-4′ to C-2′ indicated that C-3′ is linked to C-2′. In addition to the previous observations, the gHMBC correlations of H-2′ to C-4′, C-3′, C1′, and C-2 and the gCOSY correlations of this hydrogen to H3′ and H-1′ suggested the presence of a hydroxylated branched aliphatic side chain attached to the C-2 of the lactone ring. The HRESIMS analysis confirmed the molecular formula of 1 as C11H20O4. The relative configurations of the stereogenic centers C-2, C4, and C-1′ were determined using NOEDiff experiments. First, by irradiation of hydrogens H-2 and H-4, the geometric relationship between H-2 and H-4 was determined as cis. Conformational analysis of similar compounds, based on molecular mechanics, predicted a hydrogen bond between the hydroxy at C-1′ and the ester carbonyl C-1.20 Indeed, the infrared carbonyl absorption of 1 appears at 1755 cm−1 (Figure S9, Supporting Information), an unusually low value for a γlactone, which is in agreement with the presence of a hydrogen bond. Compound 1 presents a semirigid structure restricting the spatial mobility of the side chain, and the conformational relationship between H-2 and H-1′ was determined to be synperiplanar based on irradiation of hydrogen H-2. The coupling constant between H-2 and H-1′ (J = 2.9 Hz) agrees with the value observed in an analogous compound displaying an H-2− H-1′ syn-periplanar conformation.20 The relative configuration of compound 1 was assigned as 2R*, 4S*, and 1′S* and is different from a closely related analogue, which was 2R*, 4R*, and 1′R*.21 Therefore, compound 1 corresponds to (2R*,4S*)2-((1′S*)-hydroxy-4′-methylpentyl)-4-(hydroxymethyl)butanolide. Compound 1 belongs to the butanolide class. Some butanolides, known as γ-butyrolactones (GBLs), have been isolated from Streptomyces strains and reported as regulators of cytodifferentiation and secondary metabolite production.22−24 However, compound 1 possesses a substitution pattern that is different from those of γ-butyrolactones, which are 2,3disubstituted. There are few reports of 2,4-disubstituted γlactones,20,21 and all analogues present stereochemistry that is different from compound 1. There are no reports on the ability of 2,4-disubstituted γlactone analogues to interact with GBL receptors and display regulatory activities similarly to GBLs. No other biological activity for this class of compounds has yet been described. Compound 2 was obtained in fraction S-2-8 and has thus been isolated here as a natural product for the first time. A δlactone presenting the same stereochemistry as compound 2 (3R*, 4S*, 5R*, and 6S*) has only been produced by synthetic methods. Our data (Supporting Information) are in agreement with data for the synthetic compound,25 corresponding to (3R*,4S*,5R*,6S*)-tetrahydro-4-hydroxy-3,5,6-trimethyl-2-pyranone. In addition, we included a specific optical rotation not yet reported in the literature. An epimer of 2, presenting the stereochemistry 3R*, 4S*, 5R*, and 6R*, has been isolated as a natural product from a Streptomyces strain26 and also from the genetically engineered strains Saccharospora erythraea and 1304



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Figure 2. Identification of amphotericin B in extracts of S. albospinus RLe7: (a) cluster from molecular networking representing amphotericin-related compounds; (b) amphotericin B node highlighted in yellow; (c) representative MS/MS spectrum of amphotericin B.

Figure 3. MALDI-TOF IMS of a coculture of S. albospinus RLe7 with Coniochaeta sp. FLe4. Coculture employing two colonies of S. albospinus RLe7 (at right in all pictures) and two colonies of Coniochaeta sp. FLe4 (at left in all pictures). Spatial distribution of amphotericin B (9), detected as the ion of m/z 924 [M + H]+, is represented by a heat map. Analysis occurred after 24 and 96 h.

In a preliminary experiment of microbial antagonism between S. albospinus RLe7 and the endophytic fungus Coniochaeta sp. FLe4, inhibition of fungal growth was visualized. Apart from the antifungal activity, a particular fungal response was observed: red pigmentation was induced at the fungal rim near the S. albospinus RLe7 colony. To determine whether the isolated compounds were responsible for the antifungal activity and/or induced fungal pigmentation, an agar diffusion assay of these compounds against Coniochaeta sp. FLe4 was performed. However, none of

the isolated compounds displayed antifungal activity or induced any fungal response (Figure S45, Supporting Information). To investigate other compounds that may be involved in this specific endophytic microbial interaction, we applied mass spectrometry approaches to detect additional secondary metabolites produced by the actinobacterial strain.45,46 Using molecular networking to analyze LC-MS/MS data from extracts of the microbial cocultures, amphotericin-related compounds produced by S. albospinus RLe7 were detected due to the characteristic fragmentation pattern of macrocyclic polyenes47 1305



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Each sample was purified at 10 mg mL−1 with injections of 200 μL each. The ESIMS system used to analyze fractions consisted of a quadrupole time-of-flight instrument (UltrOTOF-Q, Bruker Daltonics, Billerica, MA, USA) or a triple-quadrupole (XEVO TQ-S, Waters Corporation, Milford, MA, USA), both equipped with an ESI ion source. High-resolution mass spectrometry (HRESIMS) of purified fractions containing the compounds was performed using the following parameter settings: capillary voltage 3900 V; dry gas flow 4 L h−1; and nitrogen nebulizer gas. Sample analysis by MALDI-TOF IMS was performed on a Bruker Microflex (Bruker Daltonics) equipped with an N2 laser. Spectra were acquired in reflectron positive mode at a raster width of 500 × 500 μm and a range of 100−2200 or 100−3000 m/z. Acquired data were analyzed using FlexImaging 2.0 software. Detailed parameters for acquisition of IMS were as described previously.50 The LC-MS/MS profiles of crude extracts for molecular networking were obtained on an Agilent 1290 UHPLC using a Kinetex 2.1 mm, 50 mm, 1.7 μm C18 RP column coupled to a MicrOTOF-QII mass spectrometer (Bruker Daltonics) equipped with the standard Bruker ESI source. A mobile phase gradient was employed for chromatographic separation (5% solvent B for 1 min, linear gradient from 5% B to 95% B in 8 min, 95% B for 2 min, 5% B in 1 min, and 5% B for 1 min, for a total run time of 13 min at a flow rate of 0.5 mL min−1 throughout the run). Solvent B: ACN 0.1% formic acid; solvent A: water 0.1% formic acid; both solvents were of LC-MS grade. MS spectra were acquired in positive ion mode from 50 to 2000 m/z. External calibration was performed prior to data collection using ESI-L Low Concentration Tuning Mix (Agilent Technologies). Hexakis(2,2difluoroethoxy)phosphazene, used for internal calibration, was added to a calibrant reservoir and placed inside the ion source. Other instrument settings were as follows: capillary voltage 4000 V, nebulizer gas pressure (N2) 2.0 bar, ion source temperature 200 °C, dry gas flow 9 L min−1, and spectral rate 3 Hz for MS1 and 10 Hz for MS2. For MS/MS fragmentation, the 10 most intense ions per MS1 were selected for subsequent collision-induced dissociation. Isolation, Preservation, and Identification of the Endophytic Actinobacterium. The S. albospinus RLe7 strain was isolated from the roots of the endemic Brazilian plant L. ericoides, preserved and identified as previously described.19 The strain Coniochaeta sp. FLe4 was isolated from the leaves of the same plant and identified by sequencing the ITS region by Genotyping Biotecnologia LTDA (www. genotyping.com.br). Permission to access and research endophytic microorganisms from L. ericoides was provided by the Brazilian government under process number CNPq 010858/2014-8. Liquid and Solid Fermentation and Culture Extraction. The stored strain was reactivated in 4 mL of ISP-2 liquid medium (4 g of yeast extract (Sigma-Aldrich), 10 g of malt extract (Sigma-Aldrich), and 4 g of dextrose (Sigma-Aldrich) per 1 L of deionized water) at 120 rpm and 30 °C for 48 h. This culture was plated on ISP-2 agar and incubated at 30 °C for 7 d. Two 5-mm-diameter plugs of actinobacterium agar culture were transferred to 4 mL of ISP-2 liquid medium in Falcon tubes and incubated on a rotary shaker at 30 °C and 120 rpm for 72 h (preculture). To perform liquid cultures, a preculture was transferred to a 500 mL Erlenmeyer flask containing 200 mL of PDB medium. The fermentative liquid culture was incubated at 30 °C and 120 rpm for 11 d. After the fermentation, a liquid−liquid partition of the culture medium was performed. The culture medium and the actinobacterium mycelium were crushed in 200 mL of methanol (MeOH), sonicated for 10 min, and vacuum filtered. The hydroalcoholic fraction was partitioned with 700 mL of ethyl acetate, and the acetate portion was concentrated by rotary evaporation. The extract from the liquid culture yielded compounds 4, 5, 6, and 7. To prepare the solid culture, a preculture grown as described before was transferred to a 500 mL Erlenmeyer flask containing 45 g of autoclaved rice and 50 mL of ISP-2 liquid medium. The fermentative solid culture was incubated in a BOD (Biochemical Oxygen Demand) incubator at 30 °C for 28 d. The culture was extracted with 200 mL of MeOH for 24 h, sonicated for 5 min, and filtered. This process was repeated twice, and the methanol extract was concentrated by rotary evaporation. Compounds 1, 2, and 3 were isolated from the extract of the solid culture.

(Figure 2). In addition, MALDI-TOF IMS revealed the spatial distribution of amphotericin B (9) in coculture of S. albospinus RLe7 with Coniochaeta sp. FLe4 (Figure 3), showing direct association with the fungal phenotype. After MS detection of antifungal macrolides produced by S. albospinus RLe7, a standard amphotericin B was tested against Coniochaeta sp. FLe4. Surprisingly, beyond the predicted antifungal activity of this compound, the induced red fungal pigmentation was also observed in the presence of pure amphotericin B (Figure S46, Supporting Information). Agar diffusion of amphotericin B visualized by MALDI-TOF IMS over 4 days (Figure 3) was consistent with the red pigmentation induced in Coniochaeta sp. FLe4 during interaction with S. albospinus RLe7. In addition to amphotericin B, other chemicals may be involved in the microbial interaction, and fungal inhibition may be reinforced by the presence of diverse macrocyclic polyene analogues, as shown by molecular networking. These can act together to synergize their individual effects. Indeed, complex metabolic exchange may occur in nature where microbial communities interact.48 To verify whether an amphotericin B analogue was able to elicit a similar fungal response, a nystatin standard was tested at the same experimental conditions. Nystatin also induced red pigmentation in Coniochaeta sp. FLe4, similarly to amphotericin B (Figure S47, Supporting Information). Curiously, the induced fungal response was observed only with high concentrations of antifungal compounds, but lower concentrations were able to partially inhibit fungal growth without inducing any color change (Figures S46 and S47, Supporting Information). Thus, our results indicated that macrocyclic polyenes such as amphotericin B and nystatin are responsible for, in this case, inducing red fungal pigmentation. Similarly to this specific interaction, amphotericin B and other antimicrobial compounds may be responsible for the induction of unknown and diversified microbial responses in other environments, including human microbial communities treated with antimicrobial agents. More detailed chemical studies of the microbial interactions of endophytes from L. ericoides, including the fungal response induced by amphotericin B, are currently in progress and will be published soon. Our results are consistent with the suggested role of microbial natural products in interspecies interactions,44,48,49 where they may act as inducers of several specific microbial responses that are progressively being revealed by numerous research groups.

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EXPERIMENTAL SECTION

General Experimental Procedures. One- and two-dimensional NMR spectra were recorded with a 500 MHz spectrometer (DRX-500, Bruker UK, Coventry, UK) using deuterated chloroform or methanol. IR analysis was performed using an IRTracer-100 Fourier transform infrared spectrophotometer (Shimadzu, Kyoto, Japan). Specific optical rotations were recorded in a P-2000 digital polarimeter (Jasco, Tokyo, Japan).The instrumentation for the HPLC analysis consisted of an HPLC system (Shimadzu) consisting of an LC-6AD solvent pump, an SCL 10AVB system controller, a CTO-10ASVP column oven, a Rheodyne model 7725 injector, an SPD-M10AVP diode array detector, and Class VP software for data acquisition. The HPLC purifications were carried out at 3.0 mL min−1 with a reversed-phase C18 semipreparative column (Macherey-Nagel, VP 250/10 NUCLEOSIL 120-5 C18, 10.0 mm, 250 mm, 5 μm) attached to a guard column (Agilent, 9.4 mm, 15 mm, 7 μm) using aqueous acetonitrile (ACN). 1306



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ing workflow using the GNPS platform.47 In this study, only the amphotericin cluster was mentioned; the complete data set is part of an ongoing study of interkingdom interactions of endophytes from L. ericoides (paper in progress) and is currently available through the online repository MassIVE (gnps.ucsd.edu) at ftp://massive.ucsd.edu/ MSV000079048. The complete molecular networking output and parameters can be accessed via http://gnps.ucsd.edu/ProteoSAFe/ status.jsp?task=5dd7b0a5aeca4354945bf5bd1bddac27. Biological Assays of Isolated Compounds against Coniochaeta sp. FLe4. Pure compounds 1, 2, 3, 6, 7, and 8 and a 1:1 mixture of 4 and 5 were dissolved in MeOH at concentrations of 0.01, 0.1, 1, and 10 μg μL−1. All concentrations of compounds were tested in duplicate. To perform the experiment, 1 μL of a standardized liquid culture of Coniochaeta sp. FLe4 was inoculated on ISP-2 agar plates (total of eight spots), and 1 μL of tested compound was placed 0.5 cm apart. Plates were incubated at 30 °C for 96 h. Biological Assays of Amphotericin B against Coniochaeta sp. FLe4. Amphotericin B (Sigma-Aldrich) was first dissolved in 1:1 dimethyl sulfoxide (DMSO)/MeOH. One experiment was performed by adding 1 μL containing 0.01, 0.1, 1, 10, and 20 μg of the antifungal agent directly onto the center of the ISP-2 agar inoculated with fungus spread over the surface. As a positive control, 1 μL of a standardized liquid culture of S. albospinus RLe7 was placed in the center of a Coniochaeta sp. FLe4 plate. Plates were incubated at 30 °C for 96 h. Another experiment was performed by solubilizing the antifungal agent to a final concentration of 2 mg L−1 (∼2 μM) into previously sterilized ISP-2 agar in Petri dishes with Coniochaeta sp. FLe4 and incubating at 30 °C for 6 days. Biological Assays of Nystatin against Coniochaeta sp. FLe4. These experiments were similar to those performed with pure compounds and amphotericin B. In one experiment, nystatin (Sigma-Aldrich) was dissolved in 1:1 DMSO/MeOH, and 1 μL of solution at a concentration of 10 μg μL−1 was directly placed in the center of ISP-2 agar with fungus spread over the surface. Plates were incubated at 30 °C for 96 h. In another experiment, nystatin was dissolved in 1:1 DMSO/MeOH at concentrations of 0.01, 0.1, 1, and 10 μg μL−1. All compound concentrations were tested in duplicate. To perform the experiment, 1 μL of a standardized liquid culture of Coniochaeta sp. FLe4 was inoculated on ISP-2 agar plates (total of eight spots), and 1 μL of nystatin at different concentrations was placed 0.5 cm apart. Plates were incubated at 30 °C for 96 h.

Isolation of Secondary Metabolites and Structural Determination. The extract from a liquid culture (L = 1.05 g) of S. albospinus RLe7 was first fractionated using column chromatography with silica gel (230−80 mesh) and a gradient elution of hexane, ethyl acetate, and MeOH to give 12 fractions. Fractions L-5 and L-12 were subjected to further fractionation. Fraction L-5 (16.2 mg) was fractionated using a semipreparative HPLC C18 column and aqueous ACN gradient from 30% to 100% ACN for 30 min and ending in isocratic elution with 100% ACN for 10 min to afford 23 fractions. The fifth fraction (L-5-5 = 1.3 mg), collected at 21.1 min, was a mixture of compounds 4 and 5. Fraction L-12 (511.5 mg) was fractionated in silica gel with a gradient of hexane, ethyl acetate, and MeOH to give eight fractions, of which fraction L-12-7 (479.3 mg) was also fractionated in silica gel using gradient elution. The sixth fraction (L-12-7-6 = 14.8 mg) was further purified by semipreparative HPLC C18 column and aqueous ACN in isocratic elution with 5% ACN for 5 min, followed by gradient elution of 5% to 35% ACN for 30 min to obtain fraction L-12-7-6-2 at 19.4 min, which contains 6.7 mg of a mixture of compounds 6 and 7. This fraction was purified by semipreparative HPLC in a C18 column using aqueous ACN in isocratic elution with 15% ACN for 20 min to obtain fractions L-12-7-6-2-1 (2.5 mg) at 13.8 min and L-12-7-6-2-2 (2.4 mg) at 15.9 min, which contain compounds 7 and 6, respectively. The extract from a solid culture (S = 9.50 g) of S. albospinus RLe7 was fractionated with SPE C18 using gradient elution from pure water to pure MeOH and acetone to yield eight fractions. Fraction S-2 (43.2 mg) was fractionated by semipreparative HPLC in a C18 column using aqueous ACN in isocratic elution with 5% ACN for 10 min, followed by gradient elution of 5% to 100% ACN for 18 min, and ending in isocratic elution with 100% ACN for 5 min. The peaks at 19.3 min (fraction S-2-8 = 4.2 mg), 20.7 min (fraction S-2-11 = 1.8 mg), and 22.3 min (fraction S-2-14 = 3.4 mg) afforded compounds 2, 3, and 1, respectively. The isolated compounds were analyzed by one- and twodimensional NMR and HRESIMS, and the spectroscopic and spectrometric data of known compounds (Supporting Information) are in agreement with previously reported data.25,38,39,51,52 Coculture between Endophytic Microorganisms for MS Analysis. Endophytic microorganisms were grown in SFM (soy flour mannitol) agar (2 g of soy flour, 2 g of mannitol, 2 g of agar per 100 mL of deionized water) for 7−10 days at 30 °C. Two 5-mmdiameter plugs of SFM medium-containing mycelia were transferred to 4 mL of ISP-2 liquid and incubated for 72 h in a rotatory shaker at 200 rpm and 27−30 °C. Next, 1 μL of each cultured strain was spotted onto Petri dishes (90 mm diameter) containing 10 mL of ISP-2 medium (15 g of agar per 1 L of ISP-2) and incubated at 30 °C until required for MALDI-TOF IMS or extraction procedures for posterior LC-MS/MS analysis. Sample Preparation for MALDI-TOF IMS. After colonies were grown individually or in coculture, the region of interest was excised from the plate and placed on a Bruker MSP 96 stainless steel target plate and prepared as previously published.53 Briefly, the surface of the excised agar containing the microbial colonies was covered with universal MALDI matrix (1:1 mixture of 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid; Sigma-Aldrich) using a 53 μm sieve (Hogentogler & Co., Inc.). The target plate was kept at 37 °C until agar completely dried and adhered to the plate. Pictures were taken before and after matrix recovery. Sample Preparation for LC-MS/MS. After colonies were grown individually or in coculture, the region of interest was excised from the plate as described for MALDI-TOF IMS sample preparation and extracted with solvent mixtures of 1:1 ACN/MeOH; 1:1 ACN/water; 1:1 ACN/MeOH 0.1% formic acid, and 1:1 ACN/water 0.1% formic acid. Each solvent mixture was used to extract each of the quadruplicated cocultures and controls. Sonication was performed for 10 min, and the supernatant was transferred to a clean vial, centrifuged at 16 873 rcf (14000 rpm radius of rotor ∼76.865 mm Eppendorf centrifuge 5418) for 15 min, and diluted when necessary for LC-MS/MS. Amphotericin B Annotation in GNPS. Annotation of the detected amphotericin B was performed through molecular network-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00870. Tabulated NMR data and spectra, specific optical rotations, and bioassay results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (M. T. Pupo): +55 16 3315 4710. Fax: +55 16 3315 4879. E-mail: [email protected]. ORCID

Mônica Tallarico Pupo: 0000-0003-2705-0123 Author Contributions

# F. O. Chagas and A. M. Caraballo-Rodriguez contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS F.O.C., A.M.C.R., and M.T.P. would like to thank FAPESP, CNPq, and CAPES for financial support. A.M.C.R. would like to thank the Dorrestein lab members for their support 1307



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regarding the MS approaches. P.C.D. further acknowledges Bruker and NIH for support of the shared instrumentation infrastructure at UCSD. This work was supported by the São Paulo Research Foundation (FAPESP) [grant numbers 2008/ 09540-0, 2013/07600-3 (Center for Innovation in Biodiversity and Drug Discovery-CEPID-CIBFar)], and the National Institutes of Health (NIH) [grant numbers 5P41GM103484, GMS10RR029121]. We also acknowledge FAPESP fellowships to F.O.C. [2009/17695-6] and A.M.R.C. [2012/21803-1, 2014/01651-8] and the National Council for Scientific and Technological Development (CNPq) fellowship to F.O.C. [150572/2015-8].

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