Thèse Purification Role And Biological Activities Of Specialized Metabolites From Bacteria Associated With Entomopathogenic Nematodes H/F - Doctorat.Gouv.Fr

Établissement : Université de Montpellier École doctorale : GAIA - Biodiversité, Agriculture, Alimentation, Environnement, Terre, Eau Laboratoire de recherche : DGIMI - Diversité, Génomes & Interactions Microorganismes-Insectes Direction de la thèse : Alyssa CARRé-MLOUKA ORCID 0000000261418115 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-06T23:59:59 Les nématodes entomopathogènes utilisés en biocontrôle sont porteurs d'un microbiote bactérien dont les membres peuvent présenter des activités biologiques d'intérêt, et parmi lesquels les bactéries des genres Xenorhabdus et Pseudomonas sont connues pour leurs propriétés insecticides, antimicrobiennes et promotrices de la croissance des plantes. Ce projet multidisciplinaire mêlant des approches de microbiologie, biochimie et chimie analytique s'attachera à en identifier, quantifier et caractériser les supports moléculaires (métabolites spécialisés ou secondaires) dans un contexte de santé des plantes cultivées, en accord avec l'approche « One Health ».
Ce projet s'appuiera sur des analyses métabolomiques ciblées et non ciblées couplées à des approches de spectrométrie de masse à haute résolution afin d'explorer la diversité des métabolites spécialisés bactériens associés aux nématodes entomopathogènes, en utilisant comme référence les lipocyclopeptides PAXs de Xenorhabdus, pour lesquels des outils sont déjà en place. La mise en place de réseaux moléculaires permettra d'identifier de nouveaux métabolites potentiellement impliqués dans les interactions entre ces bactéries, les nématodes et leur environnement hôte. Certains de ces composés seront ensuite caractérisés structurellement et fonctionnellement afin d'évaluer leur rôle dans la niche écologique des nématodes et leur potentielles applications. En complément, une approche intégrative combinant des outils bioinformatiques d'analyse génomique et des essais biologiques in vitro et in vivo permettra d'établir des corrélations entre la production de ces métabolites et leurs effets contre agents phytopathogènes, insecticides ou promoteurs de la croissance des plantes.
En plus des retombées scientifiques sur la compréhension du rôle des métabolites spécialisés de Xenorhabdus et Pseudomonas en contexte symbiotique, ce projet pourrait ouvrir la voie à de nouvelles stratégies de biocontrôle basées sur des bactéries ou des composés naturels bactériens. L'identification de ces molécules et la caractérisation de leurs activités permettraient d'envisager leur valorisation dans un contexte agroécologique, en lien avec les enjeux actuels de réduction des intrants chimiques et de développement de solutions biologiques durables pour la protection des cultures. Healthy plants are essential for ensuring abundant, high-quality harvests. Biocontrol methods, considered environmentally friendly, protect plants using living organisms or natural substances, in line with the 'One Health' approach. Among biocontrol agents, nemato-entomopathogenic complexes (NEPs) combine nematodes (Steinernema) and bacteria (notably Xenorhabdus and Pseudomonas) that live in a three-phase cycle culminating in the death of crop pest larvae. During their free-living phase in the soil, they seek out insect prey, which they infect by penetrating through natural openings. The bacteria are then released, causing the insect's death by septicemia (pathogenic phase). The carcasses are then used as a nutrient source (necrotrophic phase) by nematodes and bacteria, which produce specialized metabolites, of which more than 130 compounds (including PAX lipocyclopeptides) have already been described for Xenorhabdus1. Finally, bacteria and nematodes reassociate to form NEP complexes once again. These complexes leave the insect carcass in search of new prey.
The symbiotic association between bacteria and a nematode represents an ecological niche selected during the evolutionary history of the various partners. Bacterial strains isolated from nematodes are therefore likely to exhibit unique adaptations compared to strains living freely in the soil. This could pave the way for the identification of new specialized metabolites with original structures and biological activities of interest, particularly in the context of plant health, but also for understanding microbial interactions within the insect larval carcass, which are essential for the proper progression of the nematode cycle. Work conducted between the two partner laboratories has demonstrated the relevance of these approaches by focusing more specifically on particular metabolites. For example, we have shown that stilbene could function as a signaling molecule involved in the establishment of diverse bacterial phenotypes, such as motility or the production of pigments like anthraquinones.2 In Xenorhabdus nematophila, PAX lipocyclopeptides are produced only at certain stages of the nematode-bacterial cycle and appear to play a role in biofilm formation and interaction with the nematode partner.3
Metabolomics is a key analytical tool for exploring and understanding the chemical diversity of low molecular weight metabolites produced by these bacteria. By enabling the comprehensive analysis of metabolome variations according to the different phases of the nematobacterial life cycle, environmental conditions, and the biological interactions involved, metabolomics offers an integrative approach to linking metabolic production, biological functions, and observed phenotypes.4 The untargeted approach aims to exhaustively analyze all detectable metabolic signals, without prior assumptions about their identity, in order to generate metabolic signatures characteristic of the different stages of the parasite life cycle and the experimental conditions studied. These signatures, true snapshots of the metabolome, will allow the identification of specific variations associated with the production of specialized metabolites, paving the way for the discovery of new or poorly characterized compounds. In addition, targeted metabolomics will be used for the validation, quantification and monitoring of selected metabolites of interest, particularly based on knowledge acquired on PAX lipocyclopeptides, which will serve as a methodological reference. This project aims to discover and characterize novel specialized metabolites from the microbiota of nematodes, possessing biological activities of interest in the context of microbial ecology of carcasses (antimicrobial, synergistic, influencing biofilm formation) and/or plant health (insecticides, antimicrobials directed against phytopathogenic microorganisms, and/or growth-promoting activities in model crops (rice, wheat, peas).
In Thesis A, available genomes of Xenorhabdus and Pseudomonas strains will be analyzed using bioinformatics tools (antiSMASH, NaPDos, among others) to identify gene clusters involved in the biosynthesis of specialized metabolites. Algorithms for predicting the final metabolites will be used to guide extraction methods based on the predicted structures; these predictions will be compared with the results of untargeted metabolomics analysis from the thesis project B. Culturomic approaches will enable the optimization of the production of these metabolites, for which the genes encoding biosynthetic enzymes are often cryptic. In thesis A, bacterial strains will be cultured under various conditions (medium composition, pH, temperature, co-cultures, addition of sub-inhibitory concentrations of antibiotics, etc.) to promote the production of maximum diversity of specialized metabolites. Differential expression profiles of these specialized metabolites, including their quantification, can thus be established based on the work of PhD project B, enabling the selection of the most suitable culture media. For each chosen condition, the culture supernatants and corresponding bacterial pellets will be extracted using different solvents and tested for biological activity (thesis A). Positive extracts will be purified by liquid chromatography. Once obtained in sufficient quantities, the purified active molecules will be characterized by tandem mass spectrometry (MS/MS) in connection with PhD project B. Depending on the detected activities, these metabolites can then be localized (MALDI imaging) and/or quantified in vivo (LC-MS) in the decomposing insect larva following infestation by entomopathogenic nematodes.
Particular attention will be paid to optimizing experimental conditions to ensure efficient extraction of metabolites present in complex matrices such as decomposing tissues and soils colonized by entomopathogenic nematodes. Appropriate sample pretreatment protocols will be implemented to improve the sensitivity and reproducibility of metabolomic analyses. Purification of crude extracts by liquid chromatography before injection into mass spectrometry will minimize interferences and improve the detection sensitivity of the compounds of interest.
In parallel, comparative analyses will be conducted (thesis project B), using a rigorous statistical approach, on samples from various biological conditions, defined by thesis project A, in order to characterize metabolic differences depending on the presence or absence of nematodes and insects. The integration of the two thesis projects will strengthen the interpretation of the results and allow for the development of functional hypotheses regarding the role of the identified metabolites. Finally, experimental validation of the metabolites of interest will be carried out through biological tests to evaluate their activity and their involvement in the interactions between nematodes, symbiotic bacteria, and the host environment

The ideal candidate will hold a Master's degree (or equivalent) in microbiology, biochemistry or biotechnology, with a strong interest in microbial chemical ecology and the discovery of specialized metabolites and natural compounds using analytical chemistry tools. They must possess a solid foundation in experimental microbiology (bacterial culture) and ideally have initial experience in the extraction, purification, and characterization of metabolites (liquid chromatography, biological assays), as well as some knowledge of mass spectrometry and/or bioinformatics (genome analysis, antiSMASH tools). The candidate must be rigorous, autonomous, and possess critical thinking skills, while also being able to integrate into an interdisciplinary environment at the interface between microbiology and analytical chemistry. A strong motivation for research, an interest in applications in biocontrol and agroecology, and a good level of scientific English are also expected.