Niels Geudens

Biosynthesis, Chemical Structure, and Structure-Activity Relationship of Orfamide Lipopeptides Produced by Pseudomonas protegens and Related Species

Orfamide-type cyclic lipopeptides (CLPs) are biosurfactants produced by Pseudomonas and involved in lysis of oomycete zoospores, biocontrol of Rhizoctonia and insecticidal activity against aphids. In this study, we compared the biosynthesis, structural diversity, in vitro and in planta activities of orfamides produced by rhizosphere-derived Pseudomonas protegens and related Pseudomonas species. Genetic characterization together with chemical identification revealed that the main orfamide compound produced by the P. protegens group is orfamide A, while the related strains Pseudomonas sp. CMR5c and CMR12a produce orfamide B. Comparison of orfamide fingerprints led to the discovery of two new orfamide homologs (orfamide F and orfamide G) in Pseudomonas sp. CMR5c. The structures of these two CLPs were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Mutagenesis and complementation showed that orfamides determine the swarming motility of parental Pseudomonas sp. strain CMR5c and their production was regulated by luxR type regulators. Orfamide A and orfamide B differ only in the identity of a single amino acid, while orfamide B and orfamide G share the same amino acid sequence but differ in length of the fatty acid part. The biological activities of orfamide A, orfamide B, and orfamide G were compared in further bioassays. The three compounds were equally active against Magnaporthe oryzae on rice, against Rhizoctonia solani AG 4-HGI in in vitro assays, and caused zoospore lysis of Phytophthora and Pythium. Furthermore, we could show that orfamides decrease blast severity in rice plants by blocking appressorium formation in M. oryzae. Taken all together, our study shows that orfamides produced by P. protegens and related species have potential in biological control of a broad spectrum of fungal plant pathogens.

Oxidative α,ω-diyne coupling as an approach towards novel peptidic macrocycles

The Glaser-Hay diyne coupling proved to be an efficient cyclisation approach towards diyne containing peptidic macrocycles. A variety of tetrapeptide-based macrocyclic 1,3-diynes were obtained from O-propargylated serine or tyrosine residues using Cu(OAc)2·H2O and NiCl2 under an O2-atmosphere. The effect of the linear 1,3-diyne on peptide conformations was studied by NMR and compared with a macrocycle bearing a saturated linker.

Impact of a stereocentre inversion in cyclic lipodepsipeptides from the viscosin group: a comparative study of the viscosinamide and pseudodesmin conformation and self-assembly.

The viscosin group covers a series of cyclic lipodepsipeptides (CLPs) produced by Pseudomonas bacteria, with a range of biological functions and antimicrobial activities. Their oligopeptide moieties are composed of both L‐ and D‐amino acids. Remarkably, the Leu5 amino acid—centrally located in the nonapeptide sequence—is the sole residue found to possess either an L or D configuration, depending on the producing strain. The impact of this D/L switch on the solution conformation was investigated by NMR‐restrained molecular modelling of the epimers pseudodesmin A and viscosinamide A. Although the backbone fold remained unaffected, the D/L switch adjusted the segregation between hydrophobic and hydrophilic residues, and thus the amphipathicity. It also influenced the self‐assembly capacity in organic solvents. Additionally, several new minor variants of viscosinamide A from Pseudomonas fluorescens DR54 were identified, and an NMR assay is proposed to assess the presence of either an L‐ or D‐Leu5.

Membrane Interactions of Natural Cyclic Lipodepsipeptides of the Viscosin Group

Many Pseudomonas spp. produce cyclic lipodepsipeptides (CLPs), which, besides their role in biological functions such as motility, biofilm formation and interspecies interactions, are antimicrobial. It has been established that interaction with the cellular membrane is central to the mode of action of CLPs. In this work, we focus on the CLPs of the so-called viscosin group, aiming to assess the impact of the main structural variations observed within this group on both the antimicrobial activity and the interaction with model membranes. The antimicrobial activity of viscosin, viscosinamide A, WLIP and pseudodesmin A were all tested on a broad panel of mainly Gram-positive bacteria. Their capacity to permeabilize or fuse PG/PE/cardiolipin model membrane vesicles is assessed using fluorescent probes. We find that the Glu2/Gln2 structural variation within the viscosin group is the main factor that influences both the membrane permeabilization properties and the minimum inhibitory concentration of bacterial growth, while the configuration of the Leu5 residue has no apparent effect. The CLP-membrane interactions were further evaluated using CD and FT-IR spectroscopy on model membranes consisting of PG/PE/cardiolipin or POPC with or without cholesterol. In contrast to previous studies, we observe no conformational change upon membrane insertion. The CLPs interact both with the polar heads and aliphatic tails of model membrane systems, altering bilayer fluidity, while cholesterol reduces CLP insertion depth.

Cyclic Lipodepsipeptides From Pseudomonas spp. – Biological Swiss-Army Knives

Cyclic lipodepsipeptides produced by Pseudomonas spp. (Ps-CLPs) are biosurfactants that constitute a diverse class of versatile bioactive natural compounds with promising application potential. While chemically diverse, they obey a common structural blue-print, allowing the definition of 14 distinct groups with multiple structurally homologous members. In addition to antibacterial and antifungal properties the reported activity profile of Ps-CLPs includes their effect on bacterial motility, biofilm formation, induced defense responses in plants, their insecticidal activity and anti-proliferation effects on human cancer cell-lines. To further validate their status of potential bioactive substances, we assessed the results of 775 biological tests on 51 Ps-CLPs available from literature. From this, a fragmented view emerges. Taken as a group, Ps-CLPs present a broad activity profile. However, reports on individual Ps-CLPs are often much more limited in the scope of organisms that are challenged or activities that are explored. As a result, our analysis shows that the available data is currently too sparse to allow biological function to be correlated to a particular group of Ps-CLPs. Consequently, certain generalizations that appear in literature with respect to the biological activities of Ps-CLPs should be nuanced. This notwithstanding, the data for the two most extensively studied Ps-CLPs does indicate they can display activities against various biological targets. As the discovery of novel Ps-CLPs accelerates, current challenges to complete and maintain a useful overview of biological activity are discussed.

Membrane interactions of natural cyclic lipodepsipeptides

Keywords: azobenzene ; photoswitchable peptide ; light-responsive ligand ; optogenetics ; phage display ; streptavidin Reference EPFL-CONF-202371doi:10.1002/psc.2688View record in Web of Science Record created on 2014-10-23, modified on 2017-05-12Cyclic lipodepsipeptides (CLPs) are a diverse group of secondary metabolites produced by various bacteria with important biological functions, but with yet unresolved molecular mechanisms. Our previous efforts have gone towards characterizing with NMR the conformation and self-assembling properties of a collection of CLPs known as the viscosin group. [1-4] CLPs increasingly attract attention because of their antifungal and antibiotic properties through membrane permeabilization. A full understanding of their membrane interactions is essential to elucidate the exact working mechanism of CLPs. To obtain comprehensive structural information in a membrane environment, we have used liquid-state NMR and model membrane systems, such as micelles and isotropic lipid bicelles. Like micelles, isotropic bicelles display favourable NMR relaxation properties, while possessing structural characteristics of lipid bilayers. [5] The orientation and insertion depth of CLPs in a membrane environment can be investigated using diffusion NMR and paramagnetic relaxation enhancement measurements. The latter is achieved by introducing paramagnetic probes at various locations. By introducing a water-soluble paramagnetic complex to a bicelle sample, NMR signals from nuclei closer to the aqueous phase can be identified. Adding lipid molecules with covalently linked paramagnetic radicals at various positions deliver the orientation and the insertion depth of the peptides in the bilayer. Finally, 31P longitudinal relaxation measurements allow to obtain detailed information regarding the local dynamics of the lipid head groups of the bicelles. [6] The NMR results are complemented with other experimental techniques, including fluorescence spectroscopy, circular dichroism and infrared spectroscopy. We have also performed all-atom molecular dynamics (MD) simulations of CLPs within lipid membranes, which can be confronted with the experimental NMR results. References 1. Sinnaeve, D., Hendrickx, P.M. et al., Chemistry - A European Journal, 2009, 15(46): 12653-12662 2. Sinnaeve, D., Delsuc, M.-A. et al., Chemical Science, 2012, 3: 1284-1292 3. De Vleeschouwer, M., Sinnaeve D. et al., Chemistry - A European Journal, 2014, 20(25): 7766-7775 4. Geudens N., De Vleeschouwer M. et al., ChemBioChem, 2014, 15: 2736-2746 5. Durr, U. H., Gildenberg M. et al., Chem Rev, 2014, 112(11): 6054-6074 6. Bodor, A., Kover, E et al., BBA Biomembranes, 2015, 1848(3): 760-766No abstract is available for this article.