Cultures of the Marine Bacterium Pseudovibrio ... - ACS Publications

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Cultures of the Marine Bacterium Pseudovibrio denitrif icans Ab134 Produce Bromotyrosine-Derived Alkaloids Previously Only Isolated from Marine Sponges Karen J. Nicacio,† Laura P. Ióca,† Adriana M. Fróes,‡ Luciana Leomil,‡ Luciana R. Appolinario,‡ Christiane C. Thompson,‡ Fabiano L. Thompson,‡ Antonio G. Ferreira,§ David E. Williams,⊥ Raymond J. Andersen,⊥ Alessandra S. Eustaquio,∥ and Roberto G. S. Berlinck*,† †

Instituto de Química de São Carlos, Universidade de São Paulo, CP 780, CEP 13560-970, São Carlos, SP, Brazil Instituto de Biologia, Centro de Ciência da Saúde, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Fo. 373, Bloco A, Anexo A3, Sl. 302, Cidade Universitária, CEP 21941-599, Rio de Janeiro, RJ, Brazil § Departamento de Química, Universidade Federal de São Carlos, CEP 13565-905, São Carlos, SP, Brazil ⊥ Departments of Chemistry and Earth, Ocean & Atmospheric Sciences, University of British Columbia, Vancouver, BC V6T 1Z1, Canada ∥ College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, Center for Biomolecular Sciences, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, Illinois 60607, United States ‡

S Supporting Information *

ABSTRACT: Herein we report the isolation and spectroscopic identification of fistularin-3 (1), 11-hydroxyaerothionin (2), and verongidoic acid (3), as well as the UPLC-HRMS detection of aerothionin (4), homopurpuroceratic acid B (5), purealidin L (6), and aplysinamisine II (7), from cultures of the marine bacterium Pseudovibrio denitrificans Ab134, isolated from tissues of the marine sponge Arenosclera brasiliensis. These results unambiguously demonstrate for the first time that bromotyrosine-derived alkaloids that were previously isolated only from Verongida sponges can be biosynthesized by a marine bacterium.

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secondary metabolite production, has been the challlenge of isolating and growing marine sponge-derived bacteria in artificial media. Despite this difficulty, a number of reports that cultures of microorganisms isolated from sponge tissues produce secondary metabolites previously isolated from the sponges have identified a microbial biosynthetic origin for the compounds. These include diketopiperazines from Tedania ignis and Micrococcus sp.,13 a polybrominated phenol from Dysidea f ragilis and Vibrio sp.,14 13-demethylisodysidenin from Dysidea sponges also detected in cells of the cyanobacterium Oscillatoria spongeliae obtained from Dysidea herbacea,15 the mixed PKS-NRPS-derived andrimid from both the sponge Hyatella sp. and cultures of Vibrio M22-1,16 and the alkaloid manzamine A first isolated from Haliclona sp. and more recently from cultures of a Micromonospora sp.17 Lithistid sponge metabolites, in particular, have been extensively investigated since these animals present a very diverse

arine sponges (Porifera) are the oldest metazoans on Earth, distributed over all the ocean biomes, from polar to tropical waters and from shallow to very deep substrates.1,2 During their evolution, Porifera have acquired long-term and stable associations with a wide diversity of bacteria, cyanobacteria, archaea, and other groups of microbes that constitute up to 60% of the biomass of these animals and are essential for their survival.3−5 Remarkably similar microbiomes have been reported in a variety of sponges belonging to different taxa.1,2,6−8 The significance and function of sponge− microbe relationships, including symbiosis, is starting to be deciphered, but is not yet fully understood.5,6,9 However, there is now strong genomic evidence that many secondary metabolites originally isolated from marine sponges are in fact biosynthetic products of associated microbes.2,10,11 These microorganisms offer the potential to overcome the issue of sustainable supply of bioactive marine-sponge-derived natural products for drug development and the opportunity to study the biosynthesis of sponge-derived metabolites with novel chemical scaffolds.12 A major barrier to the effective study of sponge/microbial associations, and in particular their role in © 2017 American Chemical Society and American Society of Pharmacognosy

Received: September 12, 2016 Published: February 13, 2017 235

DOI: 10.1021/acs.jnatprod.6b00838 J. Nat. Prod. 2017, 80, 235−240

Journal of Natural Products

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Chart 1

series of chromatographic separations of the MeOH/acetone wash of the resins, monitored by HPLC-UV-MS and UPLCQTOF analyses of the fractions, led to the isolation of fistularin-3 (1), 11-hydroxyaerothionin (2), and verongidoic acid (3). Structures of compounds 1−3 were unambiguously established by analysis of spectroscopic data, including 1H, 13C, COSY, HSQC, HMBC, and NOESY NMR spectra, as well as by HRESIMS analysis. Comparison with literature NMR data and our own NMR data obtained previously for the same compounds confirmed our structural assignments.23 The absolute configuration of fistularin-3 (1) was established as 1(R), 6(S), 1′(R), 6′(S), that of 11-hydroxyaerothionin (2) as 1(R), 6(S), 1′(R), 6′(S), and that of verongidoic acid (3) as 1(R), 6(S) by circular dichroism analysis (Supporting Information). Compounds 1−3 are identical to the same natural products isolated a number of times from marine sponges.23 We have also been able to detect aerothionin (4),24 homopurpuroceratic acid B (5),25 purealidin L (6),26 and aplysinamisine II (7)27 by UPLC-HRMS in fractions obtained from chromatographic separations of the XAD resins extract of P. denitrif icans Ab134 cultures (see the Supporting Information). Homopurpuroceratic acid B (5), tentatively identified by HRMS and HRMS-MS analysis, is a higher homologue of purpuroceratic acid B previously isolated from the sponge Aplysina f ulva.25 The identity of 6 could be further confirmed by analysis of 1H, 13C, COSY, HSQC, and HMBC NMR data in a yet impure fraction. Previous investigations on the chemistry of A. brasiliensis,28 from which the strain P. denitrif icans Ab134 has been isolated, did not reveal any bromotyrosine derivative. The successful culture of P. denitrif icans Ab134 was strictly dependent on growth conditions as has been observed for related Pseudovibrio strains.29 Growth and production of bromotyrosine-derived alkaloids by P. denitrif icans Ab134 was stable over three independent experiments, but considerably diminished over time. Good titers of 1−3 could be obtained

secondary metabolism, which now appears to be clearly of bacterial and cyanobacterial origin.10,11,18 Bromotyrosine-derived sponge metabolites are some of the very first natural products isolated from marine sponges.19 They are typically found in sponges belonging to the order Verongida, and they have been considered as reliable chemotaxonomic markers.20 However, bromotyrosine-derived metabolites have also been found in phylogenetically unrelated organisms, weakening the case for a taxonomic link.20a Speculation about the actual biological source(s) of spongederived bromotyrosine compounds has been in the literature for several years,20a,21 but to date there has been no hard evidence demonstrating that bromotyrosine-metabolites found in Verongida sponges are produced by sponge or microbial enzymes.20b As part of an ongoing program aimed at discovering novel bioactive secondary metabolites produced by cultured microorganisms isolated from marine habitats, we have isolated the bacterium Pseudovibrio denitrif icans Ab134 from tissues of the sponge Arenosclera brasiliensis (order Haplosclerida). This bacterium was selected for investigation because it displayed antibacterial activity in agar plate assays.22 Herein we report the isolation and spectroscopic identification of fistularin-3 (1), 11hydroxyaerothionin (2), and verongidoic acid (3), as well as UPLC-HRMS detection of aerothionin (4), homopurpuroceratic acid B (5), purealidin L (6), and aplysinamisine II (7), from the XAD resin extracts of axenic P. denitrif icans Ab134 cultures. This is the first report of bacterial production of bromotyrosine-derived natural products that had previously only been isolated from marine sponges in the order Verongida. P. denitrif icans Ab134 was isolated from specimens of a A. brasiliensis collected by hand using scuba at João Fernandinho Beach, Búzios, state of Rio de Janeiro. Axenic cultures of P. denitrif icans Ab134 were grown for 5 days at 30 °C in liquid marine broth (HiMedia 2216) containing a 1:1:1 mixture of XAD-2, XAD-4, and XAD-7 Amberlite resins. A 236



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Figure 1. UPLC-QTOF analysis of Sephadex fraction Ab134-H (see Experimental Section). (Top) Chromatogram by total ion current detection; (middle) selected ion current chromatogram for ion m/z 834.8 (tR 6.35 min.); (bottom) HRMS of 11-hydroxyaerothionin (2) detected at 6.35 min.

XAD extract after chromatography on Sephadex LH-20 (see Figure 1 for 11-hydroxyaerothionin, 2). We decided to use a mixture of XAD-2, XAD-4, and XAD-7 resins, which have a

only when the bacterium was grown in the presence of the mixture of the XAD resins (Figure S1). Bromotyrosines could be detected by UPLC-QTOFMS in fractions obtained from the 237



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can be addressed, with exceptional outcomes for the development of sponge-derived drug leads. Our results provide the first evidence for microbial production of the bromotyrosine-derived natural products 1− 7 previously only known from Verongida sponge extracts, evidencing a microbial origin for these compounds isolated from the sponge tissues. Ongoing work in our laboratory is aimed at identifying the bacterial gene cluster(s) responsible for the biosynthesis of 1−7.

range of polarities and distinct adsorption properties, aiming to maximize the recovery of secondary metabolites released in the growth medium by P. denitrif icans Ab134. Cultures of P. denitrif icans Ab134 form a dense and thick biofilm, which is difficult to disrupt using organic solvents. When the mixture of XAD resins was added to the bacterium growth medium at the start of the growth experiments, the formation of the biofilm was significantly reduced presumably by physical disruption. P. denitrif icans is an obligate marine bacterium,30 commonly found associated with sponge tissues.29b,e Bacteria in the genus Pseudovibrio are thought to have biochemical and physiological traits, including a tolerance to oligotrophy and being capable of living on diverse sources of nutrients and trace elements, that favor the establishment of symbiosis with marine holobionts.29 Only two secondary metabolites, tropodithietic acid29f and a prodigiosin,31 have been isolated from cultures of Pseudovibrio spp. prior to this work. Thus, while the isolation of P. denitrif icans Ab134 from tissues of the sponge A. brasiliensis is not unexpected, its production of bromotyrosine-derived alkaloids is completely without precedent. For nearly 50 years since the original isolation of the amide 8 from Verongia cauliformis by Sharma and Burkholder19 and aeroplysinin-1 (9) from Verongia aerophoba by Fattorusso, Minale, and Sodano,32 related bromotyrosine-derived alkaloids have been repeatedly isolated from many species of Verongida sponges.20b,33,34 The strong link between the sponge taxonomy and the occurrence of related bromotyrosine-derived alkaloids provided a compelling correlational argument for the biosynthesis of the compounds by sponge cells.35 Investigations of the localization of bromotyrosine alkaloids in Verongida sponge tissues, which showed that brominated metabolites accumulated in spherulous cells, provided further evidence that the sponges are the actual producers of these metabolites.21,35 Rinehart and collaborators performed a series of pioneering biosynthetic experiments feeding 14C-labeled precursors into tissues of the sponge Aplysina f istularis, resulting in incorporation of [U−14C]phenyalanine, [U−14C]tyrosine, [methyl-14C]methionine, and more advanced precursors into 8 and 9, consistent with either sponge-cell or microbial-cell biosynthesis.36

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a Polartronic H Schmidt+Haensch polarimeter. Ultraviolet spectra were recorded on a Shimadzu UV-3600 instrument. Circular dichroism spectra were recorded on a Jasco J-815 spectrometer. Infrared spectra were recorded on a Shimadzu, IRAffinity model. NMR spectra were recorded on a Bruker AvanceIII 9.4 T instrument, operating at 400.35 MHz for 1H and 100.10 MHz for 13C channels, respectively, or on a Bruker AvanceIII 14.1 T instrument with a 5 mm cryoprobe, operating at 600.23 MHz for 1H and 150.94 MHz for 13C. Water was purified using a Rios/Milli-Q Gradient A 10 double filtering system. MeCN, MeOH, and formic acid (HCO2H) were of HPLC or MS grade. CH2Cl2, EtOAc, and acetone were of analytical grade. Size-exclusion chromatography was performed with Sephadex LH-20 (25−100 μm). Column chromatography was performed with Waters cartridges (silica gel or cyanopropylbonded silica gel columns). HPLC-UV-MS analyses were performed using a Waters Alliance 2695 coupled online with a Waters 2996 photodiode array detector, followed by a Micromass ZQ2000 mass spectrometry detector with an electrospray interface. The photodiode array scanned the samples within a λmax range between 200 and 400 nm. The mass spectrometer detector was optimized to the following conditions: capillary voltage 3.00 kV, source block temperature 100 °C, desolvation temperature 350 °C, operating in electrospray positive mode, detection range 200−1200 Da with total ion count extracting acquisition. The cone and desolvation gas flow were 50 and 350 L/h, respectively. Data acquisition and processing were performed using Empower 2.0 software. HPLC-UV-MS analyses were performed on a C18 reversed-phase Waters X-terra column (250 × 4.6 mm, 5 μm particle size) with a mobile phase flow rate of 1 mL/min. The mobile phase consisted of (A) H2O containing 0.1% HCO2H and (B) 1:1 (v/ v) MeOH/MeCN containing 0.1% HCO2H. A linear gradient elution was applied, as follows: 0−1.0 min hold at 10% B, then a 1.0−22.0 min linear gradient to 100% B, followed by 22.0−30.0 min hold at 100% B, then 30.0 to 40.0 min hold at 10% B for column reequilibration. The total run time was 40 min. The injection volume was 20 μL of a 1.0 to 1.5 mg/mL sample. HRMS UPLC-qTOF analyses were performed on a Waters UPLC Xevo G2-XS Q-TOF, using a C18 reversed-phase Waters Acquity UPLC BEH column (2.1 mm × 100 mm, 1.7 μm). The elution was a gradient of MeCN (+0.1% HCO2H) in H2O (+0.1% HCO2H), starting at 10% to 100% MeCN (+0.1% HCO2H) in 4 min. Mass spectrometry detection were acquired with the following parameters: MSE continuum during 5 min, m/z 100−1200 Da range, in ESI(+) detection mode, with a scan time of 0.2 s and a ramp collision energy of 20−30 V. Pseudovibrio denitrif icans Ab134 Isolation and Growth. P. denitrif icans Ab134 was isolated from specimens of the marine sponge Arenosclera brasiliensis collected at approximately 10 m depth by scuba, at João Fernandinho Beach, Búzios, state of Rio de Janeiro (22°44′49″ S 41°52′54″ W) in January 2011.22 The strain P. denitrif icans Ab134 has been identified by 16S rRNA amplification and sequencing.22 The nearly complete 16S rRNA sequence of P. denitrif icans Ab134 is deposited in GenBank under the acession number KX990273. P. denitrif icans Ab134 was grown in 500 mL Schott flasks containing 100 mL of marine broth (HiMedia 2216) at a concentration of 37.4 g/ L with 15 g of a 1:1:1 mixture of XAD-2, -4, and -7 Amberlite resins in each flask. The total growth volume was 3.7 L. After 5 days of growth

Our finding that a P. denitrif icans isolate obtained from tissues of a marine sponge is able to biosynthesize compounds 1−7 illustrates that Pseudovibrio spp. have important unrecognized biosynthetic capabilities and they should be considered as potential microbial sources of putative sponge natural products. The importance of developing strategies for culturing marine bacteria, and in particular sponge symbionts, should be stressed,37 since the large majority of sponge-associated microbial strains are considered to be resistant to cultivation.38 This is particularly relevant to the knowledge of microbial physiology and ecology of sponge symbionts,6 and also for the investigation of unique biosynthetic pathways that are not found elsewhere in Nature but in sponges. The isolation of bromotyrosines from cultures of P. denitrif icans Ab134 illustrates that cultivation of marine sponge microorganisms 238



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at 30 °C and orbital agitation at 160 rpm, the culture medium was filtered through cotton wool under vacum in order to recover the resin mixture and cell suspension. The resin mixture was washed with H2O and extracted with 1:1 (v/v) MeOH/acetone (4 × 100 mL) and acetone (4 × 100 mL). The organic fractions were pooled and evaporated. The organic extract was solubilized in MeOH (500 mL) and partitioned with hexane (3 × 300 mL). The MeOH fraction was collected and filtered through a 0.2 μm membrane (Millipore). Evaporation of the MeOH fraction yielded 2.153 g of this material. Isolation of Brominated Metabolites. The MeOH fraction (2.153 g) was subjected to size-exclusion chromatography on Sephadex LH-20, eluted with MeOH to give 10 fractions. The third fraction (Ab134-C) (184.6 mg) was subjected to chromatography on a silica gel column (Waters Sep-Pack, 10 g), with a gradient of EtOAc in hexane from 2:8 to 100% EtOAc, then a gradient of MeOH in CH2Cl2, to give four fractions. The second (Ab134-C2) (26.9 mg) and third (Ab134-C3) (27.2 mg) fractions of this separation were pooled and purified by HPLC using a reversed-phase C8 InertSustain column (250 × 10 mm, 5 μm), with 36:32:32 H2O/MeOH/MeCN as eluent, detection at 254 nm, and a flow rate of 2 mL/min. Fistularin-3 (1, 3.1 mg) was obtained in 0.83 mg/L yield. Fractions Ab134-F to Ab134-I obtained from the separation by chromatography on Sephadex LH-20 were pooled (512.8 mg) and subjected to normal-phase chromatography on a cyanopropyl-bonded silica gel column (Waters, Sep-Pak 10 g), using a gradient of EtOAc in CH2Cl2, then a gradient of MeOH in EtOAc. Five fractions were obtained. Fractions Ab134-FI-1 to Ab134-FI-3 were pooled (153.2 mg) and separated by HPLC using a reversed-phase C8 InertSustain column (250 × 10 mm, 5 μm), with 4:3:3 H2O/MeOH/MeCN as eluent, detection at 254 nm, and a 2 mL/min flow rate to give nine fractions. The second fraction (Ab134-FI-13-B, 9.3 mg) was purified by HPLC using a reversed-phase C8-4 InertSil column (250 × 4.6 mm, 5 μm), with 70:19:11 H2O/MeOH/MeCN as eluent, detection at λmax 254 nm, and a 1 mL/min flow rate to yield verongidoic acid (3, 5.1 mg) in 1.4 mg/L yield. Fraction Ab-134-FI-13-E (4.8 mg) obtained above was purified by HPLC using a reversed-phase C8-4 InertSil column (250 × 4.6 mm, 5 μm), with 50:28:22 H2O/MeOH/MeCN, detection at λmax 254 nm, and a 1 mL/min flow rate, to yield 11hydroxyaerothionin (2, 2.8 mg) in 0.74 mg/L yield. Fistularin-3 (1): white, amorphous powder; [α]23D +120 (c 1, MeOH), lit.23a [α]D +100 (c 1, MeOH); UV (MeOH) λmax (log ε) 206 (4.4), 232 (4.0), 286 (3.7) nm; ECD (c 0.1, MeOH) λmax (Δε) 293 (5.4), 292 (5.7); IR νmax 3315, 2938, 2841, 1657, 1586, 1432 cm−1; 1H (600 MHz) and 13C (100 MHz) NMR data, Table S1; HRESIMS m/z 1108.7085 [M + H]+ (calcd for C31H30Br6N4O11, 1108.7084). 11-Hydroxyaerothionin (2): white, amorphous powder; [α]23D +200 (c 0.5, MeOH), lit.23d [α]D +190 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 229 (3.6), 285 (3.4) nm; ECD (c 0.1, MeOH) λmax (Δε) 250 (8.2), 291 (7.9); IR νmax 3336, 2939, 2835, 1657, 1587, 1546, 1427 cm−1; 1H (600 MHz) and 13C (100 MHz) NMR data, Table S1; HRESIMS m/z 830.8531 [M + H]+ (calcd for C24H27Br4N4O9, 830.8506). Verongidoic acid (3): white, amorphous powder; [α]23D +140 (c 1.1, MeOH); UV (MeOH) λmax (log ε) 229 (3.6), 286 (3.3) nm; ECD (c 0.1, MeOH) λmax (Δε) 249 (9.4), 292 (5.3); IR νmax 3318, 2939, 2842, 1691, 1594, 1430 cm−1; 1H (600 MHz) and 13C (100 MHz) NMR data, Table S1; HRESIMS m/z 380.9092 [M]+ (calcd for C10H10Br2NO5, 380.8847).

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AUTHOR INFORMATION

Corresponding Author

*Tel: +55-16-33739954. Fax: +55-16-33739952. E-mail: [email protected]. ORCID

Raymond J. Andersen: 0000-0002-7607-8213 Roberto G. S. Berlinck: 0000-0003-0118-2523 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank FAPESP for financial support to R.G.S.B. (BIOTA/BIOprospecTA program, 2013/50228-8), as well as a scholarship to L.P.I. (2016/05133-7). The authors also thank the Brazilian funding agencies CAPES and CNPq for scholarships to K.J.N., A.F., L.L., and L.R. Startup funds from the Department of Medicinal Chemistry and Pharmacognosy of the College of Pharmacy of UIC and a Hans W. Vahlteich Research Award provided financial support to A.S.E. The Canadian funding agency NSERC provided financial support to R.J.A.

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REFERENCES

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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.6b00838. HRESIMS, 1H and 13C NMR, and circular dichroism data for compounds 1−3 and HRESIMS-MS for compounds 4−7 (PDF) 239



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