Genomics-Guided Discovery of Endophenazines from Kitasatospora

Mar 11, 2014 - Biosynthetic Potential of Bioactive Streptomycetes Isolated From Arid Region of the Thar Desert, Rajasthan (India). Meeta Masand ...
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Genomics-Guided Discovery of Endophenazines from Kitasatospora sp. HKI 714 Daniel Heine, Karin Martin, and Christian Hertweck* Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstraße 11a, 07745 Jena, Germany, and Friedrich Schiller University, Jena, Germany S Supporting Information *

ABSTRACT: In this study we report on the genomics-guided exploration of the metabolic potential of the newly discovered strain Kitasatospora sp. HKI 714. The bioinformatics analysis of the whole genome sequence revealed the presence of a biosynthetic gene cluster presumably responsible for the biosynthesis of formerly unknown endophenazine derivatives. A 200 L cultivation combined with bioactivity-guided isolation techniques revealed four new natural products belonging to the endophenazines and the 5,10-dihydrophenazines. Detailed descriptions of their biological effects, mainly focused on antimicrobial properties against several mycobacteria, are given.

T

During preliminary screening programs including approximately 100 strains of actinobacteria we discovered broad antimicrobial activity in the culture supernatant of Kitasatospora sp. HKI 714. Sequencing of the complete genome revealed the potential for secondary metabolite biosynthesis that exceeds the complexity of the actual metabolic profile. We discovered a large gene cluster that is presumably responsible for the biosynthesis of phenazine antibiotics (Figure 1).19 Although we detected large amounts of phenazine-1carboxylic acid, we postulated the existence of additional derivatives due to the presence of several genes for tailoring enzymes. Specifically, the phenazine biosynthesis gene cluster codes for enzymes involved in the mevalonate pathway.20 Further, we noted a gene putatively encoding a prenyltransferase that shows high similarity to epzP and ppzP. Both genes are linked to the prenyl transfer in endophenazine biosynthesis in Streptomyces cinnamonensis DSM 1042 and in Streptomyces anulatus 9663, respectively.21−23 The whole gene cluster resembles the biosynthetic gene cluster of endophenazine A in S. cinnamonensis DSM 1042 with the exception that the former lacks a homologue of epzM, which is most likely responsible for the formation of an N-methyltransferase (Figure 1). After these preliminary genome analyses, the strain was cultivated on a 200 L scale in order to obtain sufficient amounts of metabolites for structure elucidation. After 4 days of fermentation, an intensive purple crude extract from the culture broth showed strong antimicrobial activity against Mycobacterium vaccae, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus faecalis (VRE).

he ongoing search for unique secondary metabolites as promising new drug lead candidates often guides scientists to remote areas in order to seek unexplored species.1−3 Notably, the involvement of genomic data and related innovative approaches have resulted in progress in the discovery of new, potentially clinically interesting chemical entities over the past few years.4−9 During our continuing program to isolate new members of rare actinobacteria, we discovered Kitasatospora sp. HKI 714, which displayed strong inhibition of the growth of several bacterial pathogens. Analysis of the genome indicated the potential of the strain to biosynthesize numerous secondary metabolites, including phenazine derivatives. Phenazines represent a structurally diverse class with numerous options for chemical derivatizations and are seen as potential drug candidates.10−12 One prominent representative is the clinically used therapeutic clofazimine.13 Clofazimine has been successfully applied in the treatment of leprosy and is effective against multiple-drugresistant Mycobacteria tuberculosis strains. However, there is a strong need for analogues and chemical derivatives with fewer side effects and higher activity.14,15 Although phenazines represent a potential source for anti-infectives, their synthetic access is still limited. Thus, genome mining and isolation from natural sources are promising tools for the expansion of existing compound libraries.10 In particular, terpenoid-substituted phenazines have aroused special interest in recent years due to the cumulated discovery of numerous new derivatives.16−18 Herein we report on a genome mining approach that led to the discovery of a previously unknown set of endophenazines and dihydrophenazines. We discuss the results of a broad bioactivity investigation and give details of the impact of the newly discovered secondary metabolites on several mycobacteria. © 2014 American Chemical Society and American Society of Pharmacognosy

Received: November 15, 2013 Published: March 11, 2014 1083

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Figure 1. Graphical comparison of the putative biosynthetic gene clusters of prenylated phenazine derivatives: (1) tentative biosynthesis gene cluster of endophenazine A1 (3) in Kitasatospora sp. HKI 714 containing genes epaA-epaU; (2) locus B and (3) locus A of biosynthesis gene clusters of endophenazine A in Streptomyces cinnamonensis DSM 1042. The dashed box between L and M represents a gap. The whole sequence has been deposited at GenBank under the accession number KJ207079.

shift of its signals in the 13C NMR spectrum at 144.2 and 110.7 ppm, respectively. The quaternary carbon atoms of the second aromatic ring feature signals at 138.3 and 127.0 ppm. We concluded that both parts are connected by two nitrogen atoms to form a dihydrophenazine core structure. The remaining acetyl group could be assigned due to the NOE coupling of proton 2′ to protons 4 and 6 and therefore turned out to be attached at the nitrogen atom 5. There was a high similarity of compound 2 with respect to its UV spectrum and the chemical shift of the majority of all 1H and 13C NMR signals. The detected pseudomolecular ion at m/z 285.0869 [M + H]+ indicates a molecular formula of C15H11O4N2 and 11 doublebond equivalents. We assumed a further hydroxylation, which could finally be assigned to position 2′ based on the chemical shift of carbon atom 2′ in the 13C NMR spectrum. Compound 3 features a UV spectrum slightly different from that of 1 and 2 and was identified as a phenazine derivative. 1H NMR and 13C NMR data point toward an additional partial unsaturated aliphatic side chain. COSY and HMBC coupling signals indicate a hydroxylated prenyl chain. Its location, which could be at either position 6 or position 9, could be assigned by comparison of the chemical shifts of the carbon atoms with those of endophenazine A26,27 and by selective NOESY experiments after methylation with trimethylsilyldiazomethane.28 NOESY experiments of compound 3 additionally revealed the trans-position of the hydroxymethyl group due to coupling signals for 2′ and 5′ in combination with lacking signals for 2′ and 4′. Endophenazine F (4) has been isolated as a strongly purple-colored compound. Structure elucidation revealed the presence of two prenyl side chains located at nitrogen atom 5 and carbon atom 9 due to 2D NMR studies. The UV signals resemble those of pyocyanin-like compounds. The substitution pattern could subsequently be assigned according to COSY and HMBC couplings. Compound 4 resembles endophenazine B but bears a prenyl chain instead of a methyl group,26 and there is a similarity between compound 4 and chromophenazine C, which represents a related amide lacking the prenyl chain at position 9.17 Another isolated compound, endophenazine G (5), appeared to be identical with a known natural product, with regard to the chemical shifts of the signals in the 1H and 13C NMR spectra.29 However, the position of the prenyl side chain seemed puzzling, opposed to the substitution pattern of 3. Methylation of 5 with trimethylsilyldiazomethane allowed NOESY experiments to

Bioactivity-guided isolation including separation by open column chromatography on silica gel and Sephadex LH-20 followed by preparative RP-HPLC led to the isolation of several active compounds. Besides the well-known phenazine-1carboxylic acid, bafilomycin C1,24 and ebracteatoside B,25 we succeeded in isolating 1 (0.9 mg), 2 (16.5 mg), endophenazine A1 (3) (6.2 mg), endophenazine F (4) (0.5 mg), and endophenazine G (5) (3.6 mg) as pure compounds.

Compound 1 is a yellow-colored substance with absorbance maxima in the UV spectrum at 241, 289, and 356 nm. According to HRMS (m/z 269.0925 [M + H]+) and 13C NMR data, the compound has a molecular formula of C15H12O3N2 and 11 double-bond equivalents. 2D NMR data suggested the presence of two aromatic parts, a 1,2,3-substituted phenyl ring and a 1,2-substituted phenyl ring. Hydrogen atom 1 is orthosubstituted next to a carboxylic acid group. The two remaining quaternary carbon atoms of the first ring feature a chemical 1084

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Table 1. 1H NMR Spectroscopic Data for Compounds 1−5 δH (J in Hz) 1b,c

position 2 3 4 6 7 8 9 NH OH 1′ 2′ 4′ 5′ 1″ 2″ 4″ 5″ a

7.85, 7.00, 7.60, 7.29, 7.01, 7.14, 6.90, 9.5

2a,d

dd (8.0, 1.3) dd (8.0, 7.8) d (7.8) d (7.8) m m dd (7.9, 1.0)

3a,c

7.76, d (7.8) 7.14, dd (7.8, 7.8) 7.51, d (7.8) 7.38, d (7.8) 6.97, m 6.96, m 7.07, d (7.8) 10.24, s

2.21, s

8.96, 8.03, 8.52, 8.20, 7.89, 7.83,

dd dd dd dd dd dd

(7.0, (8.7, (8.7, (8.8, (8.8, (6.9,

4a,c 1.5) 7.0) 1.5) 1.0) 6.9) 1.0)

8.37, 7.76, 7.58, 6.07,

dd (7.7, 1.1) dd (8.6, 7.7) dd (8.6, 1.1) d (2.0)

6.96, m

15.50, bs 4.09, d (7.5) 5.75, m 1.87, s 4.10, s

4.15, s

5b,d

14.68, bs 4.79, d (5.5) 5.08, m 1.92, s 1.78, m 3.60, d (7.0) 5.31, m 1.69, s 1.78, m

8.06−8.18, me 7.91−7.99, me 8.25, d (8.6) 8.06−8.18, me 7.91−7.99,me 8.06−8.18, me

8.36, 7.64, 7.05, 1.38, 1.38,

s d (16.3) d (16.3) se se

Recorded at 500.3 MHz. bRecorded at 600.3 MHz. cChloroform-d1. dDimethylsulfoxide-d6. eSignals partly obscured.

Gram-negative bacteria. Table S1 shows the dimensions of the inhibition zones for all compounds against several test strains. Compounds 3 and 4 especially show good activity against MRSA, while others, such as 1 and 2, seem to be quite effective against Escherichia coli. All tested phenazines appeared to be weakly active against the Mycobacterium vaccae. Minimal inhibition concentrations (MICs) against MRSA, Mycobacterium fortuitum, Mycobacterium aurum, Mycobacterium vaccae, and Mycobacterium smegmatis were determined (Table 3).

locate the position of the side chain. Selective 1D NOESY indicated that the isoprenyl side chain is positioned at position 9. Our findings raise the question whether the assignment in the original publication29 might be incorrect. In order to gain deeper insights into the antimicrobial properties of the new compounds, we performed an agar diffusion test of all new substances against 10 of our standard test strains, including several fungi as well as Gram-positive and Table 2. 13C NMR Spectroscopic Data for Compounds 1−5

Table 3. Minimal Inhibitory Concentration (μg/mL) of a Given Compound toward MRSA and Several Mycobacteriaa

δC position

1b,c

2a,d

3b,c

4b,c

5b,d

1 2 3 4 4a 5a 6 7 8 9 9a 10a COOH 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″

127.3 129.2 119.2 131.6 110.7 127.0 125.2 121.8 127.0 115.4 138.3 144.2 169.9 170.4 22.5

126.0 129.0 119.1 128.9 115.6f 125.5 124.9 121.1 115.6f 127.1 138.8 142.7 169.2 171.8 60.0

124.9 137.3 130.2 135.0 143.1 144.6 128.5 131.7 131.9 138.5 139.1 139.2 165.9 29.8 121.6 138.2 14.1 68.3

126.2 128.3 132.5 118.3 132.3 138.1 100.6 184.0 135.7 142.9 148.0 132.0 165.5 46.7 115.3 139.8 18.7 25.7 29.1 118.2 136.9 18.0 25.6

−e 130.0f 130.8 130.0f 142.3 142.9 127.8 131.3 126.8 135.6 139.6 139.1 168.4 119.8 143.7 69.7 29.9 29.9

compound

MRSA SG 511

M. fortuitum IMET 10667

M. Aurum SB 66

M. vaccae IMET 10670

M. smegmatis SG 987

2 3 5 PCA ciprofloxacin

100 100 100 100 12.5

50 25 25 50 0.1

50 25 25 50 0.1

25 25 25 25 0.1

50 50 50 50 0.78

a

MRSA: methicillin-resistant Staphylococcus aureus SG 511; M. fortuitum: Mycobacterium fortuitum IMET 10667; M. aurum: Mycobacterium aurum SB 66; M. vaccae: Mycobacterium vaccae IMET 10670; M. smegmatis: Mycobacterium smegmatis SG 987, PCA: phenazine-1-carboxylic acid (used as reference), clofazimine shows MIC values of 0.12−1.00 μg/mL against an isolated Mycobacterium avium complex30 and of 0.12−2.00 μg/mL against several strains of Mycobacterium tuberculosis.31

The endophenazine-related compounds 3 and 5 especially exhibit an increased activity in comparison with phenazine-1carboxylic acid against Mycobacterium fortuitum and Mycobacterium aurum.

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EXPERIMENTAL SECTION

General Experimental Procedures. All NMR measurements were performed on a Bruker AVANCE III 500 or on a 600 MHz spectrometer, equipped with a BrukerCryoplatform. The chemical shifts are reported in parts per million (ppm) relative to the solvent residual peak of chloroform-d1 (1H: 7.24 ppm, singlet; 13C: 77.00 ppm,

a

Recorded at 125.9 MHz. bRecorded at 150.9 MH. cChloroform-d1. Dimethylsulfoxide-d6. eSignals not detectable. fSignals partly obscured. d

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triplet), methanol-d4 (1H: 3.30 ppm, quintet; 13C: 49.00 ppm, septet), or DMSO-d6 (1H: 2.50 ppm, quintet; 13C: 39.52 ppm, septet). HRESIMS was carried out on an Accela UPLC system (Thermo Scientific) combined with an Exactive mass spectrometer (Thermo Scientific) equipped with an electrospray ion source. Solid phase extraction was carried out using Chromabond C18ec cartridges filled with 2000 mg of octadecyl-modified silica gel (Macherey-Nagel). HPLC-MS measurements were performed on an HPLC 1100 system connected to a 1100 Series LC/MSD trap using a Zorbax Eclipse XDB-C8, 4.6 × 150 mm column, particle size 5 μm (Agilent Technologies). For thin-layer chromatography TLC aluminum sheets with silica gel 60 F254 (E. Merck) were used. Open column chromatography was performed on silica gel 60, particle size 0.015− 0.04 mm (Macherey-Nagel), and on Sephadex LH-20 (SigmaAldrich). Semipreparative HPLC was performed on a 1260 Infinity System (Agilent Technologies) using the following columns: Zorbax Eclipse XDB-C8, 9.5 × 250 mm, particle size 5 μm (Agilent Technologies), Zorbax Eclipse XDB-C18, 9.5 × 250 mm, particle size 5 μm (Agilent Technologies), and EC 250/4 Nucleodur Sphinx RP, particle size 5 μm (Macherey-Nagel). All solvents for analytical and preparative HPLC measurements were obtained commercially at least in gradient grade and were filtered prior to use. To avoid microbial growth, 0.1% formic acid was added to the water used for analytical and preparative HPLC. UV spectra were obtained using a UV-1800 spectrometer (Shimadzu). IR spectra were recorded on an FT/IR-4100 ATR spectrometer (Jasco). Strain Isolation and Characterization. Stock cultures of the isolate (HKI 714T) in liquid organic medium 7932 supplemented with 5% DMSO were maintained either in the vapor phase of liquid nitrogen or at −80 °C by adding a glycerol−medium (1:1) that consisted of K2HPO4, 1.26%; KH2PO4, 0.36%; MgSO4, 0.01%; Nacitrate, 0.09%; (NH4)2SO4, 0.18%; and glycerol, 8.8%. The strain represents a new species of the genus Kitasatospora and was originally isolated from a soil sample. The 16S rDNA sequence has been deposited in GenBank under the accession number KJ173610. Fermentation. A preculture of 3.5 L of strain HKI 714, grown for 24 h in soy medium, consisting of soy flour (20 g/L), glucose (20 g/ L), NaCl (5 g/L), and CaCO3 (3 g/L), was used to inoculate 200 L of the same medium without CaCO3 in a 300 L vessel. Cultivation was accomplished at 28 °C at a pH range of 7.0−8.3 for 4 days, maintaining an aeration of 50 L/min. Stirring was performed at 200 rpm throughout the whole fermentation process. The pH was 7.2 after inoculation and has not been regulated during the fermentation process. Isolation of the Active Compounds. The whole fermentation broth was filtered through 8 depth filter sheets KS 50 (PALL) having a removal rate of 0.5−0.85 μm. The filtrate (140 L) was loaded onto an Amberchrome 161 M resin LC column (200 cm × 20 cm, 6 L). A linear gradient (eluent: methanol−water (v/v) 0% to 100% in 27 min, followed by 100% methanol for 10 min; flow rate 1 L/min) was used for elution of fractions F1 to F7. Methanol was used to dissolve F7 and mixed with 20 g of silica gel. After evaporation of the solvent, the charged silica gel was applied to a silica gel column (30 cm × 6.5 cm, flow rate: 1.5 mL/min). Using a gradient (chloroform−methanol (v/ v): 0% to 50%) we obtained 12 fractions. All fractions were partitioned by solid phase extraction and open column chromatography using Sephadex LH-20 (flow rate: 4 mL/min). Final purification of all described natural products was conducted by semipreparative HPLC on a C8 column (flow rate: 4 mL/min). Antimicrobial Tests of the Isolated Phenazines 1−5. For the antimicrobial tests, the respective compound was dissolved in DMSO (1 μg/μL), and 50 μL of the solution was applied on a paper disk (d = 6 mm). The disks were then placed onto an agar plate previously inoculated with Bacillus subtilis ATCC 6633, Staphylococcus aureus SG 511, Escherichia coli SG 458 B4, Pseudomonas aeruginosa SG 137, Pseudomonas aeruginosa K 799/61, Staphylococcus aureus 134/94 (MRSA), Enterococcus faecalis 1528 (VRE), Mycobacterium vaccae IMET 10670, Sporobolomyces salmonicolor SBUG 549, Candida albicans H 8, or Penicillium notatum JP 36. Ciprofloxacin and amphotericin B were used as positive controls against bacteria and

fungi, respectively. DMSO was used as a negative control. After incubation at 37 °C for 24 h, growth inhibition zones (in mm) were recorded as antimicrobial activity. The MIC assay using the test strains Staphylococcus aureus SG 511 (MRSA), Mycobacterium fortuitum IMET 10667, Mycobacterium aurum SB 66, Mycobacterium vaccae IMET 10670, and Mycobacterium smegmatis SG 987 was done by the broth dilution method according to the NCCLS (National Committee for Clinical Laboratory Standards).33,34

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ASSOCIATED CONTENT

S Supporting Information *

Analytical data of compounds 1−5, 1H NMR, 13C NMR, and 2D NMR spectra, and antimicrobial test results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support and a grant to D.H. from the “Studienstiftung des deutschen Volkes” is gratefully acknowledged. Further we would like to thank H. Heinecke for recording all NMR spectra, A. Perner for HRMS measurements, Dr. H. M. Dahse for cytotoxicity tests, K. Perlet for strain handling and important discussions, and C. Weigel for antimicrobial tests. We also appreciate the contribution of K. Menzel and M. Steinacker (Bio Pilot Plant, HKI), who have done much of the work within the fermentation and extraction processes.

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REFERENCES

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