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IntroductionIn 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.MethodUsing 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 discussionThis 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.

Chemical UV filters are increasingly used in cosmetics to protect skin from UV radiation. As a consequence, they are released into the aquatic environment via recreational activities and wastewaters. In aquatic ecosystems, fish eggs in contact with sediment can be affected by organic and lipophilic pollutants such as UV filters. The present study aims to evaluate the toxicity of six individual UV filters, diethylhexyl butamido triazone (DBT), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), ethylhexyl triazone (ET), 2-ethylhexyl salicylate (ES), homosalate (HS), and octocrylene (OC), in the embryo-larval stages of zebrafish Danio rerio. Contamination of fish eggs and larvae with UV filters occurred through contact with spiked sediment for 96 h at a concentration of 10 μg g−1. Among the six UV filters tested, OC delayed hatching success, whereas ES significantly increased the heartbeat rate of embryo–larvae after sediment exposure, probably as a stress response.

Organic UV filters are continuously released in aquatic ecosystems due to their widespread use, especially in touristic coastal environments. Generally, organic UV filters are poorly soluble in water and tend to accumulate in the sediment compartment. This represents a conceivable risk for sediment-dwelling organisms and a potential for transfer of the UV filters up the food chain. This study aimed to assess the potential transfer of seven UV filters including benzophenone-3 (BP3), bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT), butyl methoxydibenzoylmethane (BM), methylene bis-benzotriazolyl tetramethylbutylphenol (MBBT), 2-ethylhexyl salicylate (ES), diethylhexyl butamido triazone (DBT), and octocrylene (OC) from artificial spiked sediment (10 µg.g−1 dry weight) to sediment-dwelling worms. All UV filters were detected in the worms after 28 days of exposure, but only BP3 was apparently bioaccumulated, with a biota sediment accumulation factor (BSAF) of 12.38 ± 4.65. However, metabolomic profiling revealed that OC was metabolized by the worms into 11 fatty acid conjugates, demonstrating that OC did also accumulate in the worms in the form of OC–fatty acid conjugates. Here, the sole quantification of the parent organic UV filter underestimated the accumulation factor and the exposure of organisms. In general, it is therefore important to pair the conventional method (BSAF calculus) with other techniques, such as metabolomics, to assess the actual potential for bioaccumulation of xenobiotics including transformed xenobiotics.

Multi-omic approaches have recently made big strides toward the effective exploration of microorganisms, accelerating the discovery of new bioactive compounds. We combined metabolomic, molecular networking, and genomic-based approaches to investigate the metabolic potential of the Streptomyces sp. RO-S4 strain isolated from the polluted waters of Bejaia Bay in Algeria. Antagonistic assays against methicillin-resistant Staphylococcus aureus with RO-S4 organic extracts showed an inhibition zone of 20 mm by using the agar diffusion method, and its minimum inhibitory concentration was 16 μg/ml. A molecular network was created using GNPS and annotated through the comparison of MS/MS spectra against several databases. The predominant compounds in the RO-S4 extract belonged to the angucycline family. Three compounds were annotated as known metabolites, while all the others were putatively new to Science. Notably, all compounds had fridamycin-like aglycones, and several of them had a lactonized D ring analogous to that of urdamycin L. The whole genome of Streptomyces RO-S4 was sequenced to identify the biosynthetic gene cluster (BGC) linked to these angucyclines, which yielded a draft genome of 7,497,846 bp with 72.4% G+C content. Subsequently, a genome mining analysis revealed 19 putative biosynthetic gene clusters, including a grincamycin-like BGC with high similarity to that of Streptomyces sp. CZN-748, that was previously reported to also produce mostly open fridamycin-like aglycones. As the ring-opening process leading to these compounds is still not defined, we performed a comparative analysis with other angucycline BGCs and advanced some hypotheses to explain the ring-opening and lactonization, possibly linked to the uncoupling between the activity of GcnE and GcnM homologs in the RO-S4 strain. The combination of metabolomic and genomic approaches greatly improved the interpretation of the metabolic potential of the RO-S4 strain.

The intensive use of sunscreen products has raised concerns regarding their environmental toxicity and the adverse impacts of ultraviolet (UV) filters on ecologically important coral communities. Prior metabolomic analyses on symbiotic coral Pocillopora damicornis exposed to the UV filter butyl methoxydibenzoylmethane (BM, avobenzone) revealed unidentified ions in the holobiont metabolome. In the present study, follow-up differential metabolomic analyses in BM-exposed P. damicornis detected 57 ions with significantly different relative concentrations in exposed corals. The results showed an accumulation of 17 BM derivatives produced through BM reduction and esterification. The major derivative identified C16:0-dihydroBM, which was synthesized and used as a standard to quantify BM derivatives in coral extracts. The results indicated that relative amounts of BM derivatives made up to 95% of the total BM (w/w) absorbed in coral tissue after 7 days of exposure. Among the remaining metabolites annotated, seven compounds significantly affected by BM exposure could be attributed to the coral dinoflagellate symbiont, indicating that BM exposure might impair the photosynthetic capacity of the holobiont. The present results suggest that the potential role of BM in coral bleaching in anthropogenic areas should be investigated and that BM derivatives should be considered in future assessments on the fate and effects of BM in the environment.

An endophytic fungal strain isolated from a seagrass endemic to the Mediterranean Sea (Posidonia oceanica) was studied in order to identify its antimicrobial constituents and further characterize the composition of its metabolome. It was identified as Fusarium petroliphilum by in-depth phylogenetic analyses. The ethyl acetate extract of that strain exhibited antimicrobial activities and an ability to inhibit quorum sensing of Staphylococcus aureus. To perform this study with a few tens of mg of extract, an innovative one-step generic strategy was devised. On one side, the extract was analyzed by UHPLC-HRMS/MS molecular networking for dereplication. On the other side, semi-preparative HPLC using a similar gradient profile was used for a single-step high-resolution fractionation. All fractions were systematically profiled by 1H-NMR. The data were assembled into a 2D contour map, which we call “pseudo-LC-NMR,” and combined with those of UHPLC-HRMS/MS. This further highlighted the connection within structurally related compounds, facilitated data interpretation, and provided an unbiased quantitative profiling of the main extract constituents. This innovative strategy led to an unambiguous characterization of all major specialized metabolites of that extract and to the localization of its bioactive compounds. Altogether, this approach identified 22 compounds, 13 of them being new natural products and six being inhibitors of the quorum sensing mechanism of S. aureus and Pseudomonas aeruginosa. Minor analogues were also identified by annotation propagation through the corresponding HRMS/MS molecular network, which enabled a consistent annotation of 27 additional metabolites. This approach was designed to be generic and applicable to natural extracts of the same polarity range.

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.

Social insects are in mutualism with microorganisms, contributing to their resistance against infectious diseases. The fungus Pseudallescheria boydii SNB-CN85 isolated from termites produces ovalicin derivatives resulting from the esterification of the less hindered site of the ovalicin epoxide by long-chain fatty acids. Their structures were elucidated using spectroscopic analysis and semisynthesis from ovalicin. For ovalicin, these compounds displayed antiprotozoal activities against Plasmodium falciparum and Trypanosoma brucei, with IC50 values of 19.8 and 1.1 µM, respectively, for the most active compound, i.e., ovalicin linoleate. In parallel, metabolomic profiling of a collection of P. boydii strains associated with termites made it possible to highlight this class of compounds together with tyroscherin derivatives in all strains. Finally, the complete genome of P. boydii strains was obtained by sequencing, and the cluster of potential ovalicin and ovalicin biosynthesis genes was annotated. Through these metabolomic and genomic analyses, a new ovalicin derivative named boyden C, in which the 6-membered ring of ovalicin was opened by oxidative cleavage, was isolated and structurally characterized.

The chemical composition of Lebanese Hypericum scabrum essential oil (EO) was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GG-MS). Its antimicrobial activity was evaluated by determining its minimal inhibitory concentrations (MICs) against a Gram-negative and a Gram-positive bacterium, one yeast, and five dermatophytes. H. scabrum EO was most active on filamentous fungi (MIC values of 32–64 µg/mL). Synergy within the oil was investigated by testing each of the following major components on Trichophyton rubrum: α-pinene, limonene, myrcene, β-pinene and nonane, as well as a reconstructed EO. The antifungal activity of the natural oil could not be reached, meaning that its activity might be due, in part, to minor constituent(s). The interactions between H. scabrum EO and commercially available antifungals were assessed by the checkerboard test. A synergistic effect was revealed in the combination of the EO with amphotericin B.

Leaf-inhabiting fungal and bacterial endophytes are at their most diverse in tropical rainforest plant hosts, with some influencing host plant fitness as either symbionts or pathogens. Endophyte activity and community composition is thought to depend on competition amongst co-occurring species for resources. Here, we reveal the strength of competitive interactions between endophytes by combining e-DNA metabarcoding to characterize the community with culturing assays to ascertain their potential activity. The endophyte community associated with the understory palm Astrocaryum sciophilum was determined by extracting eDNA from 120 leaflets of eight plants located in a lowland rainforest in French Guiana. This revealed 516 fungal and 606 bacterial Operational Taxonomic Units (OTUs). Co-occurrence analysis of the most abundant OTUs revealed that direct fungal-fungal interactions were overall more negative than bacterial-bacterial interactions. This was confirmed by C-score calculations for the whole endophyte community, revealing significantly greater levels of species segregation for fungi but not bacteria when compared with simulated random communities. Following the culturing of 131 fungal, and 66 bacterial Astrocaryum endophytes collected from the same leaves, Minimal Inhibitory Concentrations of ethyl acetate culture extracts were evaluated against the fungus Trichophyton rubrum and the bacterium Staphylococcus aureus. Overall, a minority of extracts demonstrated antipathogen activity, with greater antifungal activity expression for both fungi and bacteria when compared with antibacterial activity. In order to explore whether this activity translated into competitive structuring of the community, a BLAST was conducted to match metabarcoding sequences with Sanger derived culture sequences. This demonstrated that cultures displaying antipathogen activity were significantly more likely to co-occur with fungi if they were bacteria, and bacteria if they were fungi. Nevertheless, overall correlation values averaged around zero. These results indicate that for Astrocaryum palms, fungal endophytes are likely to play a greater role in determining colonization success of subsequent endophytes whether these be fungal or bacterial, but that overall endophyte communities do not display strong patterns of community structuring through competition. Endophyte communities are thus likely to reach relative stasis in mature leaves, where the diverse community of fungi and bacteria amongst other factors prevent the subsequent establishment of pathogens.

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.

With the occurrence of antibiotic-resistant Staphylococcus aureus strains, identification of new anti-staphylococcal drugs has become a necessity. It has long been demonstrated that plants are a large and diverse source of antibacterial compounds. Psiloxylon mauritianum, an endemic medicinal plant from Reunion Island, was chemically investigated for its reported biological activity against S. aureus. Aspidin VB, a phloroglucinol derivative never before described, together with Aspidin BB, were first isolated from the ethyl acetate extract of P. mauritianum leaves. Their structures were elucidated from spectroscopic data. Aspidin VB exhibited strong antibacterial activity against standard and methicillin-resistant S. aureus strains, with a minimal inhibition concentration (MIC) of 0.25 μg/mL, and no cytotoxicity was observed at 10−5 M in MRC5 cells. Due to its biological activities, Aspidin VB appears to be a good natural lead in the fight against S. aureus.

The presence of pharmaceutical and personal care product (PPCP) residues in the aquatic environment is an emerging issue due to their uncontrolled release through gray water, and accumulation in the environment that may affect living organisms, ecosystems and public health. The aim of this study is to assess the toxicity of benzophenone-3 (BP-3), bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT), butyl methoxydibenzoylmethane (BM), methylene bis-benzotriazolyl tetramethylbutylphenol (MBBT), 2-ethylhexyl salicylate (ES), diethylaminohydroxybenzoyl hexyl benzoate (DHHB), diethylhexyl butamido triazone (DBT), ethylhexyl triazone (ET), homosalate (HS) and octocrylene (OC) on marine organisms from two major trophic levels, including autotrophs (Tetraselmis sp.) and heterotrophs (Artemia salina). In general, results showed that both HS and OC were the most toxic UV filters for our tested species, followed by a significant effect of BM on Artemia salina due to BM—but only at high concentrations (1 mg/L). ES, BP3 and DHHB affected the metabolic activity of the microalgae at 100 µg/L. BEMT, DBT, ET, MBBT had no effect on the tested organisms, even at high concentrations (2 mg/L). OC toxicity represents a risk for those species, since concentrations used in this study are 15–90 times greater than those reported in occurrence studies for aquatic environments. For the first time in the literature, we report HS toxicity on a microalgae species at concentrations complementing those found in aquatic environments. These preliminary results could represent a risk in the future if concentrations of OC and HS continue to increase.

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This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC50) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC50: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis.

Posidonia oceanica (L.) Delile is an endemic Mediterranean seagrass angiosperm. The taxonomic diversity of the Posidonia-associated microbiota has previously been inventoried using culture-based and culture-independent approaches. However, little is known about the type of chemical interactions that occur between the cells within this microbiota. In this study, we isolated 60 bacterial strains from the leaves and rhizomes of P. oceanica and evaluated their capacity to communicate through quorum sensing. We found 6 strains able to emit 8 different types of quorum sensing acyl homoserine lactones, identified through UHPLC-HRMS/MS after bioguided fractionation of large cultures. We also identified 19 strains able to produce furanosyl diester borate, another type of quorum sensing autoinducer. Also, 5 strains were found to produce quorum quenching compounds. Finally, as quorum sensing is known to be involved in biofilm production, we also tested whether strains positive to our biotests were able to produce biofilms. Collectively, these data reveal the importance of examining chemical interactions within this bacterial consortium associated with P. oceanica that could have an impact on the physiology of this key Mediterranean seagrass.

Quorum sensing (QS) is a density-dependent mechanism allowing bacteria to synchronize their physiological activities, mediated by a wide range of signaling molecules including N-acyl-homoserine lactones (AHLs). Production of AHL has been identified in various marine strains of Proteobacteria. However, the chemical diversity of these molecules still needs to be further explored. In this study, we examined the diversity of AHLs produced by strain MOLA 401, a marine Alphaproteobacterium that belongs to the ubiquitous Rhodobacteraceae family. We combined an original biosensors-based guided screening of extract microfractions with liquid chromatography coupled to mass spectrometry (MS), High Resolution MS/MS and Nuclear Magnetic Resonance. This approach revealed the unsuspected capacity of a single Rhodobacteraceae strain to synthesize 20 different compounds, which are most likely AHLs. Also, some of these AHLs possessed original features that have never been previously observed, including long (up to 19 carbons) and poly-hydroxylated acyl side chains, revealing new molecular adaptations of QS to planktonic life and a larger molecular diversity than expected of molecules involved in cell–cell signaling within a single strain.

The democratization of sequencing technologies fostered a leap in our knowledge of the diversity of marine phytoplanktonic microalgae, revealing many previously unknown species and lineages. The evolutionary history of the diversification of microalgae can be inferred from the analysis of their genome sequences. However, the link between the DNA sequence and the associated phenotype is notoriously difficult to assess, all the more so for marine phytoplanktonic microalgae for which the lab culture and, thus, biological experimentation is very tedious. Here, we explore the potential of a high-throughput untargeted metabolomic approach to explore the phenotypic−genotypic gap in 12 marine microalgae encompassing 1.2 billion years of evolution. We identified species- and lineage-specific metabolites. We also provide evidence of a very good correlation between the molecular divergence, inferred from the DNA sequences, and the metabolomic divergence, inferred from the complete metabolomic profiles. These results provide novel insights into the potential of chemotaxonomy in marine phytoplankton and support the hypothesis of a metabolomic clock, suggesting that DNA and metabolomic profiles co-evolve.