Sylvain Schnee

Metabolite induction via microorganism co-culture: A potential way to enhance chemical diversity for drug discovery

Microorganisms have a long track record as important sources of novel bioactive natural products, particularly in the field of drug discovery. While microbes have been shown to biosynthesize a wide array of molecules, recent advances in genome sequencing have revealed that such organisms have the potential to yield even more structurally diverse secondary metabolites. Thus, many microbial gene clusters may be silent under standard laboratory growth conditions. In the last ten years, several methods have been developed to aid in the activation of these cryptic biosynthetic pathways. In addition to the techniques that demand prior knowledge of the genome sequences of the studied microorganisms, several genome sequence-independent tools have been developed. One of these approaches is microorganism co-culture, involving the cultivation of two or more microorganisms in the same confined environment. Microorganism co-culture is inspired by the natural microbe communities that are omnipresent in nature. Within these communities, microbes interact through signaling or defense molecules. Such compounds, produced dynamically, are of potential interest as new leads for drug discovery. Microorganism co-culture can be achieved in either solid or liquid media and has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. Because of the complexity of microbial extracts, advanced analytical methods (e.g., mass spectrometry methods and metabolomics) are key for the successful detection and identification of co-culture-induced metabolites. This review focuses on co-culture studies that aim to increase the diversity of metabolites obtained from microbes. The various strategies are summarized with a special emphasis on the multiple methods of performing co-culture experiments. The analytical approaches for studying these interaction phenomena are discussed, and the chemical diversity and biological activity observed among the induced metabolites are described.

Vitis vinifera Canes, a New Source of Antifungal Compounds against Plasmopara viticola, Erysiphe necator, and Botrytis cinerea

Methanolic and ethanolic crude extracts of Vitis vinifera canes exhibited significant antifungal activity against the three major fungal pathogens affecting grapevines, Plasmopara viticola, Erysiphe necator and Botrytis cinerea. The active extracts were analyzed by LC-PDA-ESI-MS, and selected compounds were identified. Efficient targeted isolation using medium-pressure liquid chromatography afforded six pure constituents in one step. The structures of the isolated compounds were elucidated by NMR and HRMS. Six identified compounds (ampelopsin A, hopeaphenol, trans-resveratrol, ampelopsin H, ε-viniferin, and E-vitisin B) presented antifungal activities against P. viticola. ε-Viniferin also exhibited a low antifungal activity against B. cinerea. None of the identified compounds inhibited the germination of E. necator. The potential to develop a novel natural fungicide against the three major fungal pathogens affecting V. vinifera from viticulture waste material is discussed.

Study of Leaf Metabolome Modifications Induced by UV-C Radiations in Representative Vitis, Cissus and Cannabis Species by LC-MS Based Metabolomics and Antioxidant Assays

UV-C radiation is known to induce metabolic modifications in plants, particularly to secondary metabolite biosynthesis. To assess these modifications from a global and untargeted perspective, the effects of the UV-C radiation of the leaves of three different model plant species, Cissus antarctica Vent. (Vitaceae), Vitis vinifera L. (Vitaceae) and Cannabis sativa L. (Cannabaceae), were evaluated by an LC-HRMS-based metabolomic approach. The approach enabled the detection of significant metabolite modifications in the three species studied. For all species, clear modifications of phenylpropanoid metabolism were detected that led to an increased level of stilbene derivatives. Interestingly, resveratrol and piceid levels were strongly induced by the UV-C treatment of C. antarctica leaves. In contrast, both flavonoids and stilbene polymers were upregulated in UV-C-treated Vitis leaves. In Cannabis, important changes in cinnamic acid amides and stilbene-related compounds were also detected. Overall, our results highlighted phytoalexin induction upon UV-C radiation. To evaluate whether UV-C stress radiation could enhance the biosynthesis of bioactive compounds, the antioxidant activity of extracts from control and UV-C-treated leaves was measured. The results showed increased antioxidant activity in UV-C-treated V. vinifera extracts.

Evidence for a Constitutive Cutinase in Ungerminated Conidia of Erysiphe necator Schwein

Soluble proteins of ungerminated conidia of Erysiphe necator exhibited esterase and cutinase activities, and such hydrolytic activities were measured in the parietal protein fraction. However, histochemical localisation of esterase activity was detected upon further fungal development, as well in elongating germ tubes and in appressoria. The esterase activity was spectrophotometrically quantified using para-nitrophenylbutyrate as a substrate, and cutinase activity was determined using 3H-labelled cutin. Histochemical localisation was determined using indoxyl acetate as a substrate. Diisopropyl fluorophosphate was used as inhibitor of serine hydrolase. The role of a putative constitutive parietal cutinase in the ungerminated conidia of E. necator in adhesion to the host and differentiation of infective structures, as well as implications for successful penetration, are discussed.

Generation of Antifungal Stilbenes Using the Enzymatic Secretome of Botrytis cinerea

The protein secretome of Botrytis cinerea was used to perform the biotransformation of resveratrol, pterostilbene, and a mixture of both. Metabolite profiling by UHPLC-HRMS revealed the presence of compounds with unusual molecular formula, suggesting the existence of new products. To isolate these products, the reactions were scaled-up, and 21 analogues were isolated and fully characterized by NMR and HRESIMS analyses. The reaction with pterostilbene afforded five new compounds, while the reaction with a mixture of pterostilbene and resveratrol afforded seven unusual stilbene dimers. The antifungal properties of these compounds were evaluated using in vitro bioassays against Plasmopara viticola. The cytological effects of the isolated antifungal compounds on the ultrastructure of P. viticola were also evaluated.

Investigations on the role of cuticular wax in resistance to powdery mildew in grapevine

Cuticular wax on the plant epidermis inhibits or enhances prepenetration events of powdery mildew (Erysiphe necator Schwein). We examined the role of cuticular leaf and berry waxes as a resistance mechanism in four grapevine genotypes (Italia × Mercan-174, Gürcü, Isabella, Özer Karası) resistant to powdery mildew after natural infection and inoculation. To understand cuticular wax properties, we determined the amount of wax and antifungal effects of thin layer chromatography (TLC) fractions from cuticular leaf and berry waxes, then assessed the chemical composition of fractions with different antifungal activities using gas chromatography/mass spectrometry (GC/MS). Susceptible genotypes Cabernet Sauvignon and Italia were used for comparison. Resistant and sensitive genotypes did not differ significantly in the total amount of wax on leaves and berries; however, 27 fatty acids, 26 alkanes, 6 terpenes, 4 indole derivatives and 4 ketones, and 3 amides, 3 phenols and 3 steroids were detected in fractions with high antifungal activity (≥75% inhibition of germination) in leaf and/or berry cuticular waxes of resistant genotypes only. These compounds may be evaluated as markers for powdery mildew resistance during genotype selection in a grapevine breeding program.