Xenortide Biosynthesis by Entomopathogenic ... - ACS Publications

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Xenortide Biosynthesis by Entomopathogenic Xenorhabdus nematophila Daniela Reimer,† Friederike I. Nollmann,† Katharina Schultz,† Marcel Kaiser,‡ and Helge B. Bode*,† †

Merck Stiftungsprofessur für Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, 60438 Frankfurt am Main, Germany ‡ Swiss Tropical and Public Health Institute, Parasite Chemotherapy Socinstraße 57, P.O. Box, CH-4002 Basel, Switzerland S Supporting Information *

ABSTRACT: The biosynthesis gene cluster of the xenortides and a new derivative, xenortide D, which is produced in only trace amounts, was identified in Xenorhabdus nematophila. The structure of xenortide D was elucidated using a combination of labeling experiments followed by MS analysis and was confirmed by synthesis. Bioactivity tests revealed a weak activity of tryptaminecarrying xenortides against Plasmodium falciparum and Trypanosoma brucei.

B

methylated rhabdopeptides that act as insect virulence factors.12 Construction of an XNC1_2300 insertion mutation allowed the correlation of this two-gene cluster to the already known xenortides6,13 (Figure 1), and therefore the genes were named xndAB. In addition to the known xenortides A−C (1−3),6,13 a new derivative named xenortide D (4, C26H35N4O2, m/z 435.2755 [M + H]+), which is produced in only trace amounts, was detected. Using the well-established approach for structure elucidation based on labeling experiments with deuterated amino acid building blocks and by growth of the producing strain in 13C or 15N medium with the addition of nonlabeled building blocks in inverse labeling experiments,8,10,12,14−16 its composition could be elucidated (Table S1). Briefly, comparison of the MS2 fragmentation pattern of xenortides A−C with 4 indicated valine and phenylalanine as building blocks of 4, w hic h c ould b e confirmed by labeling with L [2,3,4,4,4,5,5,5- 2 H 8 ]valine and L -[2,3,3,5,6,7,8,9- 2 H 8 ]phenylalanine fed to a culture of X. nematophila HGB081 in LB medium (Figure S1, Table S1b). The feeding of L[methyl-2H3]methionine suggested an N-methylation of valine as in the case of xenortide C and an additional methylation of phenylalanine as it is known from xenortides A−C. Tryptamine, which is present in xenortide B, was identified as the C-

acteria of the genus Xenorhabdus nematophila, a symbiont of the entomopathogenic nematode Steinernema carpocapsae, produce within the insect host a cocktail of toxins, enzymes, and small molecules, resulting in the death of its host within 48 h.1−4 Several classes of structurally diverse secondary metabolites with a broad spectrum of bioactivity including insecticidal, antifungal, antibacterial, nematicidal, and cytotoxicity have been isolated from different Xenorhabdus strains.5 Nonribosomally produced secondary metabolites are a major class produced by Xenorhabdus spp., and diverse compounds have been described, e.g., depsipeptides such as xenematides,6,7 linear peptides such as xenoamicin,8 and cyclic xenotetrapeptide9 and xentrivalpeptides.10 The great potential of these bacteria to produce a diversity of novel natural products is underlined by the analyses of their genome sequences,11 showing up to 23 different biosynthesis gene clusters.4 Here we identify the biosynthesis gene cluster of the known xenortide class of natural products6 being structurally similar to the rhabdopeptides, previously described as virulence factors against insects.12 Detailed analysis of the genome sequence of X. nematophila ATCC19061 revealed a large number of biosynthesis gene clusters responsible for the production of secondary metabolites11 and resulted in the identification of two genes, XNC1_2299 and XNC1_2300, with a high homology to genes from the biosynthesis gene cluster of the highly © 2014 American Chemical Society and American Society of Pharmacognosy

Received: May 7, 2014 Published: July 31, 2014 1976

dx.doi.org/10.1021/np500390b | J. Nat. Prod. 2014, 77, 1976−1980

Journal of Natural Products

Note

Table 1. NMR Spectroscopic Data (400 MHz (1H), 125 MHz (13C) in CDCl3) of 4, δ in ppm subunit MeVal

MePhe

Figure 1. HPLC ESIMS analysis of xenortides produced by X. nematophila strains. Base peak chromatogram (BPC) and extracted ion chromatogram (EIC) traces specific for xenortide A (1, m/z 410 [M + H]+) are shown. Depicted are (a) the BPC of HGB081 wild-type after 3 days of incubation, (b) the EIC of HGB081 wild-type, (c) the EIC of xndA::cat, and (d) the EIC of HGB081wild-type injected into G. mellonella. All chromatograms are scaled by the same intensity.

tryptamine

terminal amine in an inverse feeding experiment (Table S1b, Figure S1). In order to confirm the proposed structure, 4 was synthesized (Scheme S1, Figure S2) and compared to natural 4 regarding MS2 fragmentation pattern and retention time (Figure S3). NMR analysis of 4 (Table 1) showed high similarity to that of xenortides A−C.6,13

a

The biosynthesis gene cluster for the xenortides consists of two NRPS genes (xndA and xndB) (Figure 2, Table S2). Two additional genes coding for an NADH flavin reductase and a Daminopeptidase were identified upstream, although predicted to be in a separate transcriptional unit. Deletion of the gene XCN1_2303 encoding the D-aminopeptidase did not affect the production of the xenortides (data not shown), indicating that this gene is not involved in xenortide biosynthesis. XndA (XNC1_2300) shows high similarity at the protein level to RdpB from the structurally similar but longer rhabdopeptides12 (66/79 identity/similarity [in %]). XndA consists of a condensation, adenylation, methylation, and thiolation domain and might be responsible for loading N-methylvaline or Nmethylleucine. XndB (XNC1_2299) shows 83.2% and 80.1% similarity at the protein level to RdpC and RdpA, respectively, and might be responsible for the elongation with Nmethylphenylalanine. The additional terminal C-domain of XndB might catalyze the termination of the enzyme-bound peptide intermediate by condensation with the decarboxylated

position

δC

1 2 3 4a 5a HN-CH3 NH 1 2 3a/b 4 5/9 6/8 7 N-CH3 1 2 3 4 5 6 7 8 9 11 NH NHindol

168.2 (qC) 58.52 (CH) 30.5 (CH) 17.9 (CH3) 18.9 (CH3) 32.6 (CH3) 169.53 (qC) 58.5 (CH) 34.2 (CH2) 137.1 (qC) 128.9 (CH) 127.3 (CH) 122.3 (CH) 32.6 (CH3) 39.9 (CH2) 25.0 (CH2) 112.3 (qC) 136.7 (qC) 118.9 (CH) 119.8 (CH) 122.8 (CH) 111.5 (CH) 136.7 (qC) 119.5 (CH)

δH, mult (J in Hz) 3.70, 1.88, 0.84, 0.82, 1.92, 4.85,

d (4.9) m d (6.9) d (6.9) s bs

5.49, dd (5.6, 11.3) 3.16−3.08, m 7.22, m 7.22, m 7.17, m 3.03, s 3.59, m 3.01−2.89, m

7.58, 7.09, 7.11, 7.35,

d (7.9) m dd d

7.04, bs nd 8.26, s

Interchangeable.

Figure 2. Domain organization of the biosynthetic gene cluster corresponding to the production of the xenortides in X. nematophila. Enzyme-bound intermediates of xenortide A (1) and a possible termination mechanism catalyzed by the C-terminal C-domain are shown. C: condensation domain, A: adenylation domain, MT: methyltransferase domain, PCP: peptidyl carrier protein domain.

amino acid during the release mechanism, as already described for the rhabdopeptides,12 bicornutin,17 fimsbactin,18 acinetobactin,19 and pseudomonine.20 Prediction of the amino acid specificity21 for the A-domains XndA_A1 (DALLMGAVCK) and XndB_A2 (DAFTVAGVCK) revealed phenylalanine as the most likely incorporated amino acid, although this could only be detected in vivo for XndB. The incorporation of leucine or valine catalyzed by XndA is surprising, as in particular the similar A-domain of RdpB differs noticeable in the important 1977



Note

residues (DALVLAVSIK),12 although the same amino acids were incorporated. Furthermore, the xenortide biosynthesis seems to be not as flexible as the rhabdopeptide biosynthesis. No longer derivatives were produced, and an iterative usage of several modules should be excluded. A possible explanation for this might be provided by the starter C-domain of XndA. Compared to the starter C-domain of RdpA, in the XndA Nterminus about 130 amino acids and four out of seven catalytically important motifs22 (motifs C1−C4) are missing and only motif C6 harbors the conserved residues of the consensus sequence described for C-domain23 activity, indicating an inactivity for this domain. Whereas the insect prey environment did influence the production of the rhabdopeptides,12 this was not the case for the xenortides. Comparison of the relative amounts of xenortides A−C and 4 produced in larvae of the greater waxmoth Galleria mellonella and in LB medium under standard conditions (Figure S4) indicated no significant difference from xenortide A as the main xenortide produced under both conditions. These results are congruent with previous results showing no insecticidal activity of xenortides against G. mellonella.6 However, as sufficient amounts of xenortides A−D were available from their isolation or synthesis, all xenortides were tested against the causative agents of sleeping sickness (Trypanosoma brucei rhodesiense), chagas disease (Trypanosoma cruzi), leishmaniasis (Leishmania donnovani), and malaria (Plasmodium falciparum) (Table 2). Although the activity of

Hz. HPLC ESIMS and MSn analysis were carried out on an UltiMate 3000 system (Dionex) coupled to an AmaZonX mass spectrometer (Bruker Daltonics) and a C18 RP column (Acquity UPLC BEH, 1.7 μm, Waters). Compounds were eluted using a CH3CN/H2O gradient containing 0.1% HCOOH (5−95% CH3CN in 15 min, flow rate 0.6 mL/min) and recorded on MS in positive ionization mode at a scan range of m/z = 100−1200. MSn was performed using a manual isolation and fragmentation mode. HPLC HRESIMS were recorded on a Thermo LTQ Orbitrap Hybrid FT mass spectrometer and a C18 RP column (XBridge, 1.7 μm, Waters) using a similar gradient in 20 min, flow rate 0.4 mL/min. Strain Cultivation. Xenorhabdus nematophila ATCC 19061 was cultivated in LB medium at 30 °C. E. coli strains were grown on LB medium at 37 °C. For plasmid selection in E. coli, chloramphenicol (34 μg/mL) was added. X. nematophila HGB081 (ATCC 19061) mutants were selected on LB containing rifampicin (40 μg/mL) and chloramphenicol (34 μg/mL) at 30 °C. Structure elucidation of 4 was done via feeding experiments in combination with detailed HPLC ESIMS and HPLC HRESIMS analysis.15,24 Plasmid and General DNA Procedures. General cloning methods (plasmid isolation, PCR, restriction digests, ligations, gel electrophoresis, and DNA transformations) were carried out according to standard methods.25 Agarose gel extraction of PCR fragments and DNA isolation were performed with GeneJET gel extraction kit (Fermentas) and Puregene yeast/bact. kit B (Qiagen) according to the manufacturer’s instructions. Plasmids and strains constructed and oligonucleotides used are listed in Tables S3 and S4, respectively. Construction of xnd Mutant Strain. For the construction of the xenortide insertion mutant xndA::pDS132, XNC1_2300 was disrupted via plasmid integration. An internal fragment of 719 bp was amplified from chromosomal DNA with primers Xn362fw and Xn365rv and cloned into pDS13226 via the SphI and SacI restriction site. The resulting plasmid was introduced into E. coli S17-1 λ pir by electroporation and introduction into a rifampicin-resistant X. nematophila HGB081 strain27 by biparental conjugation, yielding xndA::pDS132. The genotype of the mutant was confirmed by PCR using primers v364f and v364r lying outside the amplified region and two primers, pDS132fw and pDS132rv, specific for the vector backbone. Structure Elucidation of 4. For structure elucidation, feeding experiments with L-[methyl-2H3]methionine, L-[2,3,4,4,4,5,5,5-2H8]valine, L-[2,3,3,4,5,5,5,6,6,6-2H10]leucine, and L-[2,3,3,5,6,7,8,9-2H8]phenylalanine to LB medium and for an inverse feeding approach with L-tryptamine (TRA) to X. nematophila cultivated in uniformly labeled [U-13C]medium were performed. Briefly, cultures were inoculated with 0.1% (v/v) of a preculture grown overnight in LB medium, washed twice with the final cultivation medium, and grown at 30 °C and 180 rpm in Erlenmeyer flasks containing ISOGRO-13C (5 mL, Sigma-Aldrich) or ISOGRO-15N medium containing K2HPO4 (10 mM), KH2PO4 (10 mM), MgSO4·7H2O (8 mM), and CaCl2·H2O (90 μM) or LB medium, respectively. Precursors were added at 4, 24, and 48 h after inoculation in equal portions to a final concentration of 3 mM. All cultures were harvested after 72 h of cultivation, and metabolites were extracted with ethyl acetate (5 mL), evaporated to dryness, redissolved in MeOH (1 mL), and diluted 1:10 for detailed HPLC ESIMS and HRESIMS analysis. The structure of 4 was elucidated by analysis of the MS2 fragmentation pattern in positive ionization mode [M + H]+ compared to the pattern of xenortides A− C and the identified mass shifts resulting from incorporation of the structure building blocks. Sum formulas of xenortides A−C and D (4) were verified by HRESIMS analysis. In Vivo Production of Xenortides. To study the influence of an in vivo system on the production of xenortides A−D (1−4), X. nematophila wild-type strain was injected into larvae of the greater waxmoth Galleria mellonella (PetShop Haindl, Frankfurt am Main, Germany). X. nematophila HGB081 was cultivated, injected into G. mellonella insect larvae, and extracted as described previously.28 Extracts were analyzed by HPLC ESIMS with a rate of three injections per sample to minimize the error rate of the instrument.

Table 2. Bioactivity of Xenortides A−D (1−4) against Different Protozoa and Cytotoxicity (IC50 in μM)a 1 T. brucei rhodesiense STIB900 T. cruci Tulahuen C4 L. donnovani P. falciparum NF 54 mammalian L6 cells

4

ref

89.2

4.92

0.01

131.5 25.0 >126 >253

52.7 17.3 7.66 85.6

1.73 0.459 0.006 0.02

2

3

22.5

1.57

46.4 124.5 19.2 115.5

11.7 111.2 0.76 13.69

a The positive reference (ref) is different for each target organism: melarsoprol for Trypanosoma brucei rhodesiense, benznidazole for Trypanosoma cruzi, miltefosin for Leishmania donnovani, chloroquine for Plasmodium falciparum NF 54, and podophyllotoxin for mammalian L6 cells.

all compounds was much less than the reference compounds, some interesting observations can be made regarding the structure−activity relationships of the compounds: The tryptamides xenortide B and 4 are more active than the phenylethylamides xenortide A and C, with xenortide B having the highest activity and a potency of less than 1.6 μM against T. brucei rhodiense and P. falciparum. However, xenortide B is also the most toxic against mammalian L6 cells, which weakens it as a lead compound. Nevertheless the biological activity of the xenortides indicates that they also might play a role in the complex life cycle of the bacteria and might participate in insect virulence or protection of the insect cadaver as previously suggested for the rhabdopeptides12 and the fabclavines,14 respectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded on a Bruker NMR spectrometer at 400 MHz at room temperature in CD3OD. Chemical shifts are given in ppm, and coupling constants in 1978



Note

Bioinformatic Analysis and Biosynthetic Gene Cluster Annotation. Identification of the biosynthetic gene cluster corresponding to the production of the NRPS-derived xenortides was done by in silico analysis of all identified NRPS biosynthetic gene clusters in X. nematophila ATCC 19061.11 The biosynthetic gene cluster was analyzed as described previously,23 following a frame plot 4.0beta analysis29 and the PKS/NRPS analysis Web site (http://nrps.igs. umaryland.edu/nrps/).30 For the analysis of all NRPS domains, sequence alignments were constructed using ClustalW,31 and all conserved and catalytic residues were characterized as described in the literature.23,32,33 Amino acid specificity of adenylation domains were predicted on the basis of the 10 amino acid code using NRPSpredictor2.21 Synthesis of 4. For the synthesis of 4 [(N-Me)-L-valinyl-(N-Me)L-phenylalanyltryptamide], tert-butyloxycarbonyl-(N-Me)-PheOH·dicyclohexylamine (100 mg, 0.217 mmol) was dissolved in 1 mL of dichloromethane (CH2Cl2). The solution was cooled to 0 °C and TBTU (77 mg, 0.239 mmol) was added followed by DIPEA (0.148 mL, 0.868 mmol). After 15 min the mixture was warmed to room temperature, and tryptamine hydrochloride (43 mg, 0.217 mmol) was added. The reaction was stirred overnight to give Boc-(N-Me)-Phetryptamide (78 mg, 0.185 mmol, 85%) after flash chromatography. HRESIMS m/z 422.2437 [M + H]+ (calcd for C25H32N3O3, 422.2438); Rf (hexane/ethyl acetate, 50:50) 0.30. The Boc protecting group was removed with trifluoroacetic acid, and the resulting (NMe)-Phe-tryptamide (58 mg, 0.180 mmol) was reacted with Cbz-(NMe)-ValOH (52 mg, 0.198 mmol) in 2 mL of CH2Cl2 at −10 °C with the addition of 2-bromo-1-ethylpyridinium tetrafluoroborate (54 mg, 0.198 mmol) and DIPEA (0.092 mL, 0.540 mmol). After 20 min the mixture was warmed to room temperature and stirred overnight. Flash chromatography using a 12−100% ethyl acetate in n-hexane gradient for 10 column volumes (flow 12 mL/min, 254 nm, silica cartridge 12 M with a pore size of 40−63 μm, 60 Å) of the crude product yielded 67 mg (0.118 mmol) of Cbz-(N-Me)-Val-(N-Me)-Phe-tryptamine (65%). Then 60 mg (0.106 mmol) of Cbz-protected 4 was deprotected with 0.250 mL of 33% HBr/HOAc solution to yield 4 (38 mg, 0.081 mmol, 82%) as a yellowish solid, Rf (CHCl3/MeOH, 90:10) 0.30. Xenortide D (4): yellowish solid; for NMR data see Table 1; HRESIMS m/z 435.2747 [M + H]+ (calcd for C26H35N4O2, 435.2755). Bioactivity Tests. Xenortides A−D (1−4) were tested against the protozoan parasites P. falciparum NF54, T. cruzi Tulahuen C4, L. donnovani MHOM-ET-67/L82, and T. b. rhodesiense STIB900 and for cytotoxicity determination against rat skeletal myoblasts (L6 cells) as described previously.34

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supported M.K. The authors thank P. Grün for help with the preparation of the compounds for the bioactivity testing.

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ASSOCIATED CONTENT

S Supporting Information *

HRMS data for all compounds, LCMS data for natural and synthetic 4, detailed information on the xnd gene cluster, supplementary experimental procedures, NMR data for 4, and in vivo data of 1−4 production. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +49 69 798 29557. Fax: +49 69 798 29527. E-mail: h. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a European Research Council starting grant under grant agreement no. 311477 and the European Community’s Seventh Framework Program (FP7/ 2007-2013) under grant agreement no. 602773, which also 1979



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