Léonie Pellissier

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.

Herbal preparations (HPs) used in folk medicine are complex mixtures of natural products (NPs). Their efficacy in vivo after ingestion depends on the uptake of the active ingredient, and, in some cases, their metabolites, in the gastrointestinal tract. Thus, correlating bioactivities measured in vitro and efficacy in vivo is a challenge. An extract of Pueraria lobata rich in different types of isoflavones was used to evaluate the capacity of viable porcine small intestine ex vivo to elucidate the absorption of HP constituents, and, in some cases, their metabolites. The identification and transport of permeants across the jejunum was monitored by liquid chromatography-mass spectrometry (LC-MS), combining targeted and untargeted metabolite profiling approaches. It was observed that the C-glycoside isoflavones were stable and crossed the intestinal membrane, while various O-glycoside isoflavones were metabolized into their corresponding aglycones, which were then absorbed. These results are consistent with human data, highlighting the potential of using this approach. A thorough investigation of the impact of absorption and biotransformation was obtained without in vivo studies. The combination of qualitative untargeted and quantitative targeted LC-MS methods effectively monitored a large number of NPs and their metabolites, which is essential for research on HPs.

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 palmAstrocaryum sciophilum. The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strainLasiodiplodia venezuelensiswas 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 ofL. venezuelensis.

Community assembly theory assumes that ecological communities are spatially delimited into patches. Within these patches, coexistence results from environmental filtering, competition and immigration. Truly delineated communities exist in laboratory studies of microbial cultures in Petri dishes, yet empirical tests conducted in continuous environments often use patches defined by convention as opposed to realized boundaries. Here we perform a test of ecological community assembly rules using foliar endophyte communities from a tropical rainforest, where leaves are considered as patches for both fungal and bacterial communities. We determined the diversity of fungal and bacterial endophytes using environmental DNA sequencing of 365 top‐canopy leaves, collected from 38 host trees belonging to 22 different species across a 4‐ha research plot. Three leaves were collected from three or more branches within each tree crown. We tested the effect of host tree species and their level of phylogenetic relatedness on community composition as well as the contribution of geographic distance between leaves to endophyte community diversity. Endophyte diversity significantly differed across host tree species, as did community composition. Within certain endophytic orders (Xylariales, Rhizobiales) species assemblages significantly differed across host tree species, but this trend was weaker or non‐existent in other orders known to contain pathogens and saprotrophs (Polyporales, Solirubrobacterales). Phylogenetically related host tree species displayed more similar endophyte communities than expected by chance, but geographically close trees did not. Consistent with the finding of host‐specificity, nearby leaves tended to host more similar communities than distantly positioned ones. These findings demonstrate that foliar endophytes are structured by dispersal across small spatial scales, but at the scale of the canopy they display patterns of neutral filtering, with only a small part of variation described by host tree differences. Endophyte communities thus act as a model system in evoking the rules predicted by theoretical community ecology.

A library of 197 endophytic fungi and bacteria isolated from the Amazonian palm tree Astrocaryum sciophilum was extracted and screened for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Four out of five antibacterial ethyl acetate extracts were also cytotoxic for the MRC-5 cells line. Liquid chromatography coupled to tandem mass spectrometry (UPHLC-HRMS/MS) analyses combined with molecular networking data processing were carried out to allow the identification of depsipeptides and cyclopeptides responsible for the cytotoxicity in the dataset. Specific ion clusters from the active Luteibacter sp. extract were also highlighted using an MRSA activity filter. A chemical study of Luteibacter sp. was conducted leading to the structural characterization of eight fatty acid exhibiting antimicrobial activity against MRSA in the tens of µg/mL range.

A biological evaluation of a library of extracts from entomopathogen strains showed that Pantoea sp. extract has significant antimicrobial and insecticidal activities. Three hydroxyacyl-phenylalanine derivatives were isolated from this strain. Their structures were elucidated by a comprehensive analysis of their NMR and MS spectroscopic data. The antimicrobial and insecticidal potencies of these compounds were evaluated, and compound 3 showed 67% mortality against Aedes aegypti larvae at a concentration of 100 ppm, and a minimum inhibitory concentration (MIC) of 16 µg/mL against methicillin-resistant Staphylococcus aureus. Subsequently, hydroxyacyl-phenylalanine analogues were synthesized to better understand the structure-activity relationships within this class of compounds. Bioassays highlighted the antimicrobial potential of analogues containing saturated medium-chain fatty acids (12 or 14 carbons), whereas an unsaturated long-chain fatty acid (16 carbons) imparted larvicidal activity. Finally, using a molecular networking-based approach, several close analogues of the isolated and newly synthesized lipoamino acids were discovered in the Pantoea sp. extract.

Entomopathogenic microorganisms (principally fungi and bacteria) are natural pathogens of insects involved in the regulation of host populations, representing an underexplored field of natural products (NPs) research. They have been shown to produce a wide range of bioactive secondary metabolites and thus appear as a promising source of new agents of biological, pharmaceutical and ecological interests (1). Our objective was to explore the chemodiversity and simultaneously investigate antimicrobial and insecticidal compounds within a unique collection of 57 enthomopathogenic strains collected on living infected insects. Recently, molecular networking (MN) has been established as a useful tool for the chemical exploration of complex extracts, since it enables the clustering of detected metabolites as structural families according to the similarity of their fragmentation patterns (tandem MS² spectra) (2). Here, a multi-informative MN was built by overlaying different information layers (taxonomy, bioactivity) on top of the MN generated from the full collection of entomopathogenic fungi. This method allowed the organisation of 203'928 MS² spectra into 821 clusters containing 5'784 nodes. By annotating the constructed MN against experimental and in silico spectral NPs databases (3), we could identify molecular families previously described in those organisms like beauveriolides or eniatins. Additionally, the exploration of the MN via the bioactivity mapping allowed to highlight clusters containing nodes from bioactive extracts. Compounds found in some of these clusters could be linked to previously isolated larvicidal and antibiotic secondary metabolites. The approach permitted to easily pinpoint unknown analogues of those compounds that are likely to be bioactive and the data generated will be used to target their isolation. [1] Beemelmanns et al. Beilstein J Org Chem. 2016;12:314 – 27. [2] Wang M et al. Nat Biotech. 2016;34:828 – 37. [3] Allard PM et al. Anal Chem. 2016;88:3317 – 23. Photo credits: ©Andreas Kay, 2016, https://www.flickr.com/photos/andreaskay/31115643631 (accessed March 2017)