Katia Gindro

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.

Histological study of the responses of two Vitis vinifera cultivars (resistant and susceptible) to Plasmopara viticola infections

Leaves of Vitis vinifera L. cvs. Chasselas (susceptible) and Solaris (resistant) were inoculated with Plasmopara viticola. Samples were then examined by scanning electron microscopy, light and epifluorescence microscopy. On the resistant cv. Solaris, stomatal deposits, identified as callose, were visible around the germinating zoospores 7 h after inoculation. Twenty-four hours after inoculation, infected stomata exhibited secretions that enveloped the zoospores. At this time, infected stomata were surrounded by necrotic tissues. At 120 h after inoculation, undefined material was deposited on the cuticle in the necrotic areas. Stomata in the vicinity of the infection sites contained callose deposits in and around the stomatal openings, but no necrotic zones were observed. On the sensitive cv. Chasselas neither secretion nor callose deposits were observed. Sporangiophores emerged about 96 h after inoculation and were fully developed 24 h later. Sporulation through small stomata-like apertures present all along the primary vein was also observed on the upper leaf surface. The role of stomatal callose deposits in the defense reactions of the Solaris grapevine cultivar against P. viticola is discussed.

Integration of Molecular Networking and In-Silico MS/MS Fragmentation for Natural Products Dereplication

Dereplication represents a key step for rapidly identifying known secondary metabolites in complex biological matrices. In this context, liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is increasingly used and, via untargeted data-dependent MS/MS experiments, massive amounts of detailed information on the chemical composition of crude extracts can be generated. An efficient exploitation of such data sets requires automated data treatment and access to dedicated fragmentation databases. Various novel bioinformatics approaches such as molecular networking (MN) and in-silico fragmentation tools have emerged recently and provide new perspective for early metabolite identification in natural products (NPs) research. Here we propose an innovative dereplication strategy based on the combination of MN with an extensive in-silico MS/MS fragmentation database of NPs. Using two case studies, we demonstrate that this combined approach offers a powerful tool to navigate through the chemistry of complex NPs extracts, dereplicate metabolites, and annotate analogues of database entries.

De Novo Production of Metabolites by Fungal Co-culture of Trichophyton rubrum and Bionectria ochroleuca

The co-cultivation of fungi has recently been described as a promising strategy to induce the production of novel metabolites through possible gene activation. A large screening of fungal co-cultures in solid media has identified an unusual long-distance growth inhibition between Trichophyton rubrum and Bionectria ochroleuca. To study metabolite induction in this particular fungal interaction, differential LC-MS-based metabolomics was performed on pure strain cultures and on their co-cultures. The comparison of the resulting fingerprints highlighted five de novo induced compounds, which were purified using software-oriented semipreparative HPLC-MS. One metabolite was successfully identified as 4″-hydroxysulfoxy-2,2″-dimethylthielavin P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated form, as well as three other related compounds, were found in the pure strain culture of B. ochroleuca.

Detection of metabolite induction in fungal co-cultures on solid media by high-throughput differential ultra-high pressure liquid chromatography–time-of-flight mass spectrometry fingerprinting

Access to new biological sources is a key element of natural product research. A particularly large number of biologically active molecules have been found to originate from microorganisms. Very recently, the use of fungal co-culture to activate the silent genes involved in metabolite biosynthesis was found to be a successful method for the induction of new compounds. However, the detection and identification of the induced metabolites in the confrontation zone where fungi interact remain very challenging. To tackle this issue, a high-throughput UHPLC-TOF-MS-based metabolomic approach has been developed for the screening of fungal co-cultures in solid media at the petri dish level. The metabolites that were overexpressed because of fungal interactions were highlighted by comparing the LC-MS data obtained from the co-cultures and their corresponding mono-cultures. This comparison was achieved by subjecting automatically generated peak lists to statistical treatments. This strategy has been applied to more than 600 co-culture experiments that mainly involved fungal strains from the Fusarium genera, although experiments were also completed with a selection of several other filamentous fungi. This strategy was found to provide satisfactory repeatability and was used to detect the biomarkers of fungal induction in a large panel of filamentous fungi. This study demonstrates that co-culture results in consistent induction of potentially new metabolites.

The effectiveness of stilbenes in resistant Vitaceae: ultrastructural and biochemical events during Plasmopara viticola infection process

Leaves of different Vitis vinifera L. cultivars, susceptible or resistant to downy mildew, Chasselas, Solaris, IRAC 2091 (cvs. Gamaret x Bronner) and Muscadinia rotundifolia were inoculated with Plasmopara viticola. Samples were then examined by scanning and transmission electron microscopy, by light microscopy and for their ability to synthesise stilbenes. These phytoalexins were strictly analysed at infection sites. In the susceptible Chasselas, P. viticola colonises, at 72h post-infection (hpi), all of the spongy mesophyll with functional haustoria and produces mainly the non toxic piceide. No necrotic zone was observed on Chasselas leaves. The ultrastructural response to downy mildew infection is different in each of the other three resistant grape cultivars. In Solaris, where leaf necrosis are rapidly induced, the infection is restricted to the upper part of the loose spongy mesophyll, and associated with a rapid cell wall disruption and the dispersion of cytoplasmic content along with the production of viniferins. In IRAC 2091, leaf necrosis are quite similar to those observed on Solaris but the infected plant cell, as well as the haustoria, show high electron dense cellular particles without any recognisable organelles, probably related to the effect of the toxic compound pterostilbene, which is synthesised in this grape cultivar. In M. rotundifolia leaf necrosis are much more scarce and smaller than in other cultivars, but pathogen and plant cells are both strongly affected, with concomitant expulsion of cytoplasmic materials through the stomata after P. viticola penetration. In this cultivar, the concentration of all identified stilbenes exceeds 1×10³ μmol mg(-1) FW. The critical role of stilbenes in the resistance of Vitis spp. is discussed.

Induction of defence mechanisms in grapevine leaves by emodin- and anthraquinone-rich plant extracts and their conferred resistance to downy mildew.

The ability of two plant extracts, Rheum palmatum root extract (RPRE) and Frangula alnus bark extract (FABE), to protect Vitis vinifera leaves from Plasmopara viticola infection was evaluated. These natural products are toxic to the pathogen and induce defence reactions in a susceptible cultivar of V. vinifera (V. vinifera cv. Chasselas), including stilbenic phytoalexin accumulation, enhanced peroxidase (EC 1.11.1.7) activity, and a hypersensitive reaction. Inhibition of the first stage of biotrophic hyphal development of P. Viticola by the two plant extracts was observed. HPLC-DAD-MS analysis showed that these two natural extracts contain many phenolic compounds belonging to the anthraquinone family, such as rhein, frangulin A, emodin, aloe-emodin, chrysophanol, and physcion. Emodin alone is able to impair P. viticola development and to stimulate viniferins and the accumulation of pterostilbene.

Differential Analysis of Mycoalexins in Confrontation Zones of Grapevine Fungal Pathogens by Ultrahigh Pressure Liquid Chromatography/Time-of-Flight Mass Spectrometry and Capillary Nuclear Magnetic Resonance

An original approach was developed for the chemical and biological investigation of zone lines formed by the confrontation of fungi growing in confined spaces. Two wood-decaying fungi involved in esca disease, Eutypa lata and Botryosphaeria obtusa, were grown in Petri dishes. Metabolic profiles of pure fungal strains and confrontation zones were differentially analyzed by ultrahigh pressure liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC/TOFMS). Selected metabolites induced by the confrontation were isolated and characterized by capillary NMR (CapNMR) at the submilligram level. Fungitoxic and phytotoxic assays were applied to the crude extracts and isolated molecules. While the extracts of pure strains were inactive, the extract from confrontation zones exhibited significant activities. A very strongly induced compound, O-methylmellein, may explain these toxic properties. The developed approach demonstrates the use of fungal confrontations as an original source of bioactive molecules and gives new insights into the study of esca disease.

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.

Identification of Infectious Agents in Onychomycoses by PCR-Terminal Restriction Fragment Length Polymorphism

ABSTRACT A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na2S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5′-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.