Structure, Biosynthesis, and Biological Activity of the Cyclic

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Structure, Biosynthesis, and Biological Activity of the Cyclic Lipopeptide Anikasin Sebastian Götze, Regine Herbst-Irmer, Martin Klapper, Helmar Görls, Kilian R. A. Schneider, Robert Barnett, Thomas Burks, Ursula Neu, and Pierre Stallforth ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00589 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 29, 2017

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ACS Chemical Biology

Structures of anikasin and all CLPs belonging to the amphisin group. Amino acids are represented in three letter code and refer to the proteinogenic L-enantiomer if not indicated otherwise. Chemical moieties labeled in black are conserved within this group. 94x45mm (300 x 300 DPI)

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Biosynthetic genes and proposed biosynthesis of anikasin. 237x154mm (300 x 300 DPI)


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Structure of one of the independent molecules of anikasin. Anisotropic displacement parameters are depicted at the 50% probability level. Hydrogen atoms are omitted for clarity. 205x167mm (300 x 300 DPI)



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Phenotypic analysis of P. fluorescens HKI0770 wt, ∆ani, and ∆pys with respect to amoebal predation. Klebsiella pneumoniae served as control organism (edible bacterium for both amoebae). P. violaceum and D. discoideum show opposite phenotypes regarding edibility of P. fluorescens HKI0770 mutants. While P. violaceum can feed on ∆ani (fruiting bodies can form on lawns of ∆ani) it cannot feed on ∆pys (no fruiting bodies observed). 176x111mm (300 x 300 DPI)


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81x26mm (300 x 300 DPI)



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Structure, Biosynthesis, and Biological Activity of the Cyclic Lipopeptide Anikasin Sebastian Götze,† Regine Herbst-Irmer,‡ Martin Klapper,† Helmar Görls,# Kilian R. A. Schneider,† Robert Barnett,† Thomas Burks,† Ursula Neu,§ Pierre Stallforth*,† †

Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Junior Research Group Chemistry of Microbial Communication, Beutenbergstraße 11a, 07745 Jena, Germany ‡ Universität Göttingen, Insitute of Inorganic Chemistry, Tammannstraße 4, 37077 Göttingen, Germany #

Friedrich-Schiller-Universität Jena, Institute for Inorganic and Analytical Chemistry, Humboldtstraße 8, 07743 Jena, Germany

§

Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Arnimallee 22, 14195 Berlin, Germany.

Supporting Information Placeholder

[Paste publication-size TOC graphic here – see end of document] ABSTRACT: The class of cyclic lipopeptide natural products consists of compounds with a diverse range of bioactivities. In this study, we elucidated the structure of the cyclic lipopeptide anikasin using X-ray crystallography, analyzed its biosynthetic gene cluster, and investigated its natural role in the interaction between the producer strain Pseudomonas fluorescens HKI0770 and protozoal predators. These results led to the conclusion that anikasin has dual functionality enabling swarming motility and as a niche amoebicide, which effectively inhibits the social amoeba Polysphondylium violaceum and protects the producer strain from protozoal grazing.

Cyclic lipopeptides (CLPs) constitute a structurally diverse family of natural products,1 which are predominantly biosynthesized by bacteria of the genera Streptomyces,2 Bacillus,3 and Pseudomonas.4 Much of their biological activity is attributed to the amphiphilic character of CLPs, which renders them ideal biosurfactants. Hence, many CLPs are crucial for bacterial swarming motility and biofilm formation.5 Structurally, CLPs consist of an oligopeptide with a peptidically linked, N-terminal fatty acid. The linear or branched lipid tail can vary in length (typically C6–C18) and in the degree of oxidation.6 The C-terminus of the oligopeptide (up to 25 amino acids) forms a lactone or lactam with a hydroxyl, phenol, or amino functional group that is either present in the side chains of the peptide or part of the lipid moiety, thus giving rise to macrocycles of varying sizes (typically 4–16 amino acids).7 Given that CLPs are biosynthesized by nonribosomal peptide synthetases (NRPS), both non-proteinogenic (e.g., Dconfigured or β-amino acids) and modified amino acids (e.g., 4-chloro-threonine) can be present in the peptide.8 Whilst the biological activity is often reduced to their biosurfactant properties, CLPs can also display potent and selective antibacterial, antifungal, and cytotoxic activities. A recent success story highlights the evolving role of CLPs as important pharmaceutical leads.7 Daptomycin, developed by Cubist Pharmaceuticals is one of the few newly approved antibiotics used for the treatment of Gram-positive bacterial infections.9,10 Additionally, some Bacillus strains are even applied as biocontrol agents in crop protection, since CLPs can increase overall plant fitness.3

Although CLPs have been investigated for a couple of decades, research mainly focused on their biosynthesis and potential mechanism of action against microbial pathogens. However, the ecological benefit that bacteria gain from the production of these complex natural products has received less attention.11 In addition, structure elucidation of isolated CLPs often lacked stereochemical information.4 This can be explained by the complexity of the three-dimensional structure of these natural products. Only an interdisciplinary approach combining techniques ranging from bioinformatics over molecular biology to analytical chemistry is capable of solving the complex structures of CLPs.12 Unfortunately, a lack of structural insight precludes any detailed structure-activity relationship studies. We previously isolated a CLP during a study devoted to understanding bacterial defense mechanisms against predators.13 In this earlier study, bioassay-guided fractionation of Pseudomonas fluorescens HKI0770 extracts revealed multiple amoebicidal alkaloids, namely the pyreudiones, as well as a CLP that was not investigated in detail. Unlike the pyreudiones, the CLP only showed modest activity (IC50 = 94 µg mL-1) against Dictyostelium discoideum14 – a model predator of bacteria. In this work, we present the structure and the biological activity of this CLP, named anikasin (1, Figure 1). Unambiguous structure determination was achieved by a combination of chemical degradation, X-ray diffraction, and molecular biology approaches (Table 1). Anikasin was shown to belong to the amphisin family of CLPs and to be structurally related to arthrofactin.15 Furthermore, we could attribute a biological


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activity to anikasin, which is complementary to the pyreudiones in defense against predators. Table 1. Structures of anikasin and all CLPs belonging to the amphisin group. Amino acids are represented in three letter code and refer to the proteinogenic L-enantiomer if not indicated otherwise. Chemical moieties labeled in black are conserved within this group. 1617,18 OH O Anikasin

(R)

N H

D-Leu

N H

D-Leu

N H

D-Leu

N H

D-Leu

D-Asp

D-allo-Thr

Amphisin16

(R)

D-Leu D-Leu D-Ser

Leu

D-Ser

Leu

Ile Asp

Leu

D-Gln

Leu

Ile Asp

Leu

D-Gln

Leu

Ile

Glu

D-Leu

D-Ser

D-Leu

Ile

D-Asp

Leu

D-Ser

Ile

O

OH O D-Asp

D-allo-Thr

D-Leu D-Leu D-Ser

O

OH O Tensin17

(R)

D-Asp

D-allo-Thr

D-Leu D-Leu D-Ser

O

OH O Pholipeptin20

(?)

Asp

Thr

Arthrofactin15,18

(?)

D-Leu D-Leu D-Ser

O

OH O N H

D-Leu

D-Asp

D-allo-Thr

D-Leu D-Leu D-Ser

Ile Asp

O

High-resolution mass spectrometry of anikasin revealed a pseudo-molecular-ion peak at m/z 1354.8079 [M+H]+. To narrow down the number of possible molecular formulae, we cultured the producer strain in minimal medium containing (15NH4)2SO4 and subsequently isolated 15N-labeled anikasin. Comparison of the m/z values [M+H]+ (1365.7793 for 15Nlabeled anikasin) revealed a difference of Δm/z = 11, indicating that this CLP contains 11 nitrogen atoms. Analysis of the 1 H- and 13C-NMR spectra further revealed that anikasin contains at least 50 carbon and more than 100 hydrogen atoms. Using these constraints for the number of atoms the molecular formula of anikasin (C64H111N11O20) was finally deduced. A literature search revealed that three known CLPs, arthrofactin15, lokisin,19 and pholipeptin20 also share this molecular formula. While the absolute configuration of arthrofactin and

pholipeptin are known (Table 1), only incomplete stereochemical information of lokisin is reported. Comparison of the NMR spectra of anikasin with either lokisin or pholipeptin did not show large deviations in chemical shift values between the CLPs. This strongly suggested that anikasin was a known compound. Unfortunately, we were unable to obtain samples of lokisin or pholipeptin for comparative studies and [α]25 values have not been reported for either 𝐷 of the compounds.21 Therefore, we decided to perform a detailed structure elucidation of anikasin. Acid hydrolysis using 6 N HClaq. followed by derivatization with Marfey's reagent revealed the amino acid composition of anikasin: 2 x D-Ser; 1 x L-Asp; 1 x D-Asp; 1 x D-allo-Thr; 1 x L-Ile; 2 x L-Leu, and 3 x D-Leu.22 This result is in contrast to the amino acid composition of pholipeptin, which contains 5 x D-Leu and 1 x L-Thr, but it is identical to that of lokisin (yet for lokisin the exact position of the individual amino acids within the CLP are not known). In order to confirm the amino acid sequence and the respective stereochemistry, we turned our attention to the identification of the biosynthetic genes responsible for the production of anikasin. Bioinformatic analysis of the genome sequence of P. fluorescens HKI077013 using antiSMASH 3.023 led to the identification of three clustered NRPS genes namely aniA (2 modules, 6.4 kbp), aniB (4 modules, 13.0 kbp), and aniC (5 modules, 17.8 kbp). Each module consists of a condensation (C), adenylation (A), and thiolation (T) domain, which activates a specific amino acid, condenses it with the nascent peptide chain, and controls the stereochemistry of the respective building block.24 In total the three subunits contain 11 modules including a terminal tandem thioesterase (TE) domain catalyzing the macrolactonization

Figure 1. Biosynthetic genes and proposed biosynthesis of anikasin.



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and release of anikasin (Figure 1).25 This gene architecture is similar to that of the arthrofactin biosynthetic gene cluster in Pseudomonas sp. MIS38.26 The predicted amino acid sequence matched the proposed constitutional formula of anikasin. A bioinformatics analysis of the C domains allowed tentatively assigning the stereochemistry of each amino acid. The overall predicted amino acid composition was in excellent agreement with the results obtained from the Marfey's reagent derivatization experiments (Figure 1 and SI). Whilst being a reliable tool, in silico prediction of amino acid sequence or configurations can fail at times. To unambiguously determine the structure of anikasin the CLP either had to be analyzed via X-ray crystallography or synthesized. While methods for the synthesis of CLPs belonging to the viscosin group, which is closely related to the amphisin group, are available, it was not feasible to modify these procedures to develop a total synthesis for anikasin.27 Therefore, we turned our attention to finding conditions to obtain crystals suitable for X-ray analysis. Two CLPs from the amphisin group were already successfully crystallized using solvent mixtures containing high concentrations of water.16,17 In our hands these mixtures did not lead to the formation of suitable crystals. Hence, we screened various mixtures of aprotic solvents and finally found an appropriate condition (acetonitrile/chloroform 50 % v/v) that led to the crystallization of anikasin. It is important to note that we only obtained crystals with 15N-labeled CLP preparations. This can be explained by the fact that we isolated 15N-labeled anikasin from bacteria grown in minimal medium. This probably resulted in a CLP sample containing fewer impurities that could impede crystallization. X-ray data were collected at BESSY II at beamline BL14.328 at 0.89429 Å on a non-merohedral twin29. Data were processed with XDSAPP.30 The structure was solved by SHELXD31 and refined on F2 using SHELXL32 in the graphical user interface ShelXle.33

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Anikasin crystallizes in space group P21 with two molecules and several water molecules in the asymmetric unit.34 The configuration of all stereo centers given in Figure 1 can be confirmed (see Figure 2). Both molecules (see Figure S1 and S2) show very similar conformations. After determination of the structure of anikasin, we turned our attention to investigating the ecological function of this CLP. We could already show that anikasin is responsible for the swarming motility of P. fluorescens HKI0770.13 It is known that some CLPs can also function as chelators for metal cations.11,35 Therefore, we investigated the capability of anikasin to chelate metal ions using a simple thin layer chromatography (TLC) experiment (Figure S3).36 This qualitative assay indicated that none of the tested cations (Ca2+, Mg2+, Co2+, Fe2+, Fe3+, Cu2+, and Mn2+) formed a strong complex with anikasin suggesting that this CLP is not involved in trace metal chelation. Further studies, for example chemical shift perturbation experiments, are, however, required to confirm these initial results.37 P. fluorescens HKI0770 was isolated from forest soil samples. Soil bacteria are exposed to strong selection pressures by a complex ecological environment, which contains bacterial and fungal competitors as well as predators of bacteria such as amoebae or nematodes. Consequently, we tested if anikasin is cytotoxic or has any antimicrobial activity. The CLP did not show antibacterial activity against a variety of different species and was only weakly active against Enterococcus faecium (Table S1). In addition, no antifungal properties were observed for anikasin (Table S2). The CLP was also barely cytotoxic or antiprolific (>80 µg mL-1) when tested against human cell lines (Table S3). As previously mentioned, the producer strain P. fluorescens HKI0770 also biosynthesizes tetramic acids, namely the pyreudiones, which display amoebicidal activity and prevent amoebal grazing. In order to assess the ability of anikasin to prevent predation by amoebae, we used already reported gene inactivation mutants of P. fluorescens HKI0770, which either lack the ability to produce anikasin (Δani, previously known as Δclp) or the pyreudiones (Δpys).13 After screening different social amoebae (see the SI for a full list) we observed that Polysphondylium violaceum38 can graze and develop on mutants producing pyreudiones but not on those producing anikasin (Figure 3). P. violaceum stands in stark contrast to all other amoebae screened, because it is inhibited by a CLP and not by the tetramic acids, which are generally considered highly bioactive compounds.39 In addition, P. violaceum is a common inhabitant of German forest soils and was already isolated in Thuringia40 as was P. fluorescens HKI0770. Pseudomonas spp. capable of producing members of the viscosin CLP group were also reported to evade predation by the amoebo-flagellate Naegleria americana in the ecological context of the wheat rhizosphere.41 Taken together, these results indicate that besides enabling swarming, anikasin can also serve as a niche amoebicide that protects bacteria from protozoal predators.

Figure 2. Structure of one of the independent molecules of anikasin. Anisotropic displacement parameters are depicted at the 50% probability level. Hydrogen atoms are omitted for clarity.


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ACS Chemical Biology The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT

Figure 3. Phenotypic analysis of P. fluorescens HKI0770 wt, Δani, and Δpys with respect to amoebal predation. Klebsiella pneumoniae served as control organism (edible bacterium for both amoebae). P. violaceum and D. discoideum show opposite phenotypes regarding edibility of P. fluorescens HKI0770 mutants. While P. violaceum can feed on Δani (fruiting bodies can form on lawns of Δani) it cannot feed on Δpys (no fruiting bodies observed). In conclusion, we unambiguously determined the structure of anikasin, a CLP produced by P. fluorescens HKI0770, using a range of analytical techniques, most importantly X-ray crystallography. Furthermore, we shed light on its biosynthesis using bioinformatics tools. Although we cannot exclude that anikasin has already been described (in the form of lokisin), our study is the first that provides clear evidence for its threedimensional structure. In addition, we investigated the biological activity of anikasin by means of phenotypic analysis using gene inactivation mutants of the producing strain. We could show that anikasin is not only responsible for the swarming motility of P. fluorescens HKI0770, which is itself a mechanism to escape predators, but it can also act as an amoebicide, which inhibits protozoan grazing. These data underline the importance of CLPs as defense compounds in the ecologically important bacteria-protozoan relationship.

We thank A. Perner, and H. Heinecke for MS and NMR measurements. We would also like to thank H.-M. Dahse and C. Weigel for performing cytotoxicity and antimicrobial assays. We would like to thank M. S. Weiss and M. Gerlach of BESSY II for support with X-ray measurements. In addition, we also want to thank F. Kloss for useful discussions. We are grateful for financial support from the Leibniz Association. We thank the Daimler and Benz Foundation (Fellowship to P.S.) and the Dr. Illing Stiftung for financial support. An Aventis Foundation Ph.D. fellowship (M.K.) is acknowledged. This work was also supported by the DFG-funded graduate school of excellence Jena School for Microbial Communication (R.B.).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the internet at http://pubs.acs.org. Figure S1 and S2 (conformation of anikasin), Table S1-S3 (antimicrobial and cytotoxicity assays); Isolation of 15N-labeled anikasin; Crystallization of anikasin; Determination of the stereochemistry of the amino acids using Marfey’s reagent; Plaque assay; NMR spectra of anikasin (PDF). Crystallographic information (CIF).

TOC

AUTHOR INFORMATION Corresponding Author

REFERENCES

* Email: [email protected]

Author Contributions

1

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(2016) Twinning in chemical crystallography - a practical guide. Z. Kristallogr. 231, 573-581. 30 Sparta, K. M.; Krug, M.; Heinemann, U.; Mueller, U.; Weiss, M. S.; (2016) XDSAPP 2.0. J. Appl. Cryst. 49, 1085-1092. 31 Sheldrick, G. M. (2008) A short history of SHELX. Acta Crystallogr. A64, 112-122. 32 Sheldrick, G. M. (2015) Crystal structure refinement with SHELXL. Acta Crystallogr. C71, 3-8. 33 Hübschle, C. B.; Sheldrick, G. M.; Dittrich, B. (2011) ShelXle: a Qt graphical user interface for SHELXL. J. Appl. Crystallogr. 44, 1281-1284. 34 CCDC 1566928 contains the supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures. 35 Grangemard, I.; Wallach, J.; Maget-Dana, R.; Peypoux, F. (2001) Lichenysin: a more efficient cation chelator than surfactin. Appl. Biochem. Biotechnol. 90, 199-210. 36 Kloss, F.; Pidot, S.; Goerls, H.; Friedrich, T.; Hertweck C. 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Structure, Biosynthesis, and Biological Activity of the Cyclic

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