Comparative metabolomic study of fungal foliar endophytes and their long-lived host Astrocaryum sciophilum: a model for exploring the chemodiversity of host-microbe interactions.

Introduction: In contrast to the dynamics observed in plant/pathogen interactions, endophytic fungi have the capacity to establish enduring associations within their hosts, leading to the development of a mutually beneficial relationship that relies on specialized chemical interactions. Research indicates that the presence of endophytic fungi has the ability to significantly modify the chemical makeup of the host organism. Our hypothesis proposes the existence of a reciprocal exchange of chemical signals between plants and fungi, facilitated by specialized chemical processes that could potentially manifest within the tissues of the host. This research aimed to precisely quantify the portion of the cumulative fungal endophytic community's metabolome detectable within host leaves, and tentatively evaluate its relevance to the host-endophyte interplay. The understory palm Astrocaryum sciophilum (Miq.) Pulle was used as a interesting host plant because of its notable resilience and prolonged life cycle, in a tropical ecosystem. Method: Using advanced metabolome characterization, including UHPLC-HRMS/MS and molecular networking, the study explored enriched metabolomes of both host leaves and 15 endophytic fungi. The intention was to capture a metabolomic "snapshot" of both host and endophytic community, to achieve a thorough and detailed analysis. Results and discussion: This approach yielded an extended MS-based molecular network, integrating diverse metadata for identifying host- and endophyte-derived metabolites. The exploration of such data (>24000 features in positive ionization mode) enabled effective metabolome comparison, yielding insights into cultivable endophyte chemodiversity and occurrence of common metabolites between the holobiont and its fungal communities. Surprisingly, a minor subset of features overlapped between host leaf and fungal samples despite significant plant metabolome enrichment. This indicated that fungal metabolic signatures produced in vitro remain sparingly detectable in the leaf. Several classes of primary metabolites were possibly shared. Specific fungal metabolites and/or compounds of their chemical classes were only occasionally discernible in the leaf, highlighting endophytes partial contribution to the overall holobiont metabolome. To our knowledge, the metabolomic study of a plant host and its microbiome has rarely been performed in such a comprehensive manner. The general analytical strategy proposed in this paper seems well-adapted for any study in the field of microbial- or microbiome-related MS and can be applied to most host-microbe interactions.

Characterization of Pseudomonas aeruginosa Quorum Sensing Inhibitors from the Endophyte Lasiodiplodia venezuelensis and Evaluation of Their Antivirulence Effects by Metabolomics.

The opportunistic pathogen Pseudomonas aeruginosa is one of the "critical priority pathogens" due to its multidrug resistance to a wide range of antibiotics. Its ability to invade and damage host tissues is due to the use of quorum sensing (QS) to collectively produce a plethora of virulence factors. Inhibition of QS is an attractive strategy for new antimicrobial agents because it disrupts the initial events of infection without killing the pathogen. Highly diverse microorganisms as endophytes represent an under-explored source of bioactive natural products, offering opportunities for the discovery of novel QS inhibitors (QSI). In the present work, the objective was to explore selective QSIs within a unique collection of fungal endophytes isolated from the tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted, and screened for their antibacterial and specific anti-QS activities against P. aeruginosa. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation for its selective activity, leading to the isolation of eight compounds in a single step. Among them, two pyran-derivatives were found to be responsible for the QSI activity, with an effect on some QS-regulated virulence factors. Additional non-targeted metabolomic studies on P. aeruginosa documented their effects on the production of various virulence-related metabolites.

Isolation and Identification of Isocoumarin Derivatives With Specific Inhibitory Activity Against Wnt Pathway and Metabolome Characterization of Lasiodiplodia venezuelensis.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as (R)-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of L. venezuelensis.

Generation of Stilbene Antimicrobials against Multiresistant Strains of Staphylococcus aureus through Biotransformation by the Enzymatic Secretome of Botrytis cinerea.

The biotransformation of a mixture of resveratrol and pterostilbene was performed by the protein secretome of Botrytis cinerea. Several reaction conditions were tested to overcome solubility issues and to improve enzymatic activity. Using MeOH as cosolvent, a series of unusual methoxylated compounds was generated. The reaction was scaled-up, and the resulting mixture purified by semipreparative HPLC-PDA-ELSD-MS. Using this approach, 15 analogues were isolated in one step. Upon full characterization by NMR and HRMS analyses, eight of the compounds were new. The antibacterial activities of the isolated compounds were evaluated in vitro against the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The selectivity index was calculated based on cytotoxic assays performed against human liver carcinoma cells (HepG2) and the human breast epithelial cell line (MCF10A). Some compounds revealed remarkable antibacterial activity against multidrug-resistant strains of S. aureus with moderate human cell line cytotoxicity.

Dynamics of Metabolite Induction in Fungal Co-cultures by Metabolomics at Both Volatile and Non-volatile Levels.

Fungal co-cultivation has emerged as a promising way for activating cryptic biosynthetic pathways and discovering novel antimicrobial metabolites. For the success of such studies, a key element remains the development of standardized co-cultivation methods compatible with high-throughput analytical procedures. To efficiently highlight induction processes, it is crucial to acquire a holistic view of intermicrobial communication at the molecular level. To tackle this issue, a strategy was developed based on the miniaturization of fungal cultures that allows for a concomitant survey of induction phenomena in volatile and non-volatile metabolomes. Fungi were directly grown in vials, and each sample was profiled by head space solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS), while the corresponding solid culture medium was analyzed by liquid chromatography high resolution mass spectrometry (LC-HRMS) after solvent extraction. This strategy was implemented for the screening of volatile and non-volatile metabolite inductions in an ecologically relevant fungal co-culture of Eutypa lata (Pers.) Tul. & C. Tul. (Diatrypaceae) and Botryosphaeria obtusa (Schwein.) Shoemaker (Botryosphaeriaceae), two wood-decaying fungi interacting in the context of esca disease of grapevine. For a comprehensive evaluation of the results, a multivariate data analysis combining Analysis of Variance and Partial Least Squares approaches, namely AMOPLS, was used to explore the complex LC-HRMS and GC-MS datasets and highlight dynamically induced compounds. A time-series study was carried out over 9 days, showing characteristic metabolite induction patterns in both volatile and non-volatile dimensions. Relevant links between the dynamics of expression of specific metabolite production were observed. In addition, the antifungal activity of 2-nonanone, a metabolite incrementally produced over time in the volatile fraction, was assessed against Eutypa lata and Botryosphaeria obtusa in an adapted bioassay set for volatile compounds. This compound has shown antifungal activity on both fungi and was found to be co-expressed with a known antifungal compound, O-methylmellein, induced in solid media. This strategy could help elucidate microbial inter- and intra-species cross-talk at various levels. Moreover, it supports the study of concerted defense/communication mechanisms for efficiently identifying original antimicrobials.