Thèse Substrats Enzymatiques Innovants pour la Caractérisation Environementale de D-Peptidases Sanitise H/F - Doctorat.Gouv.Fr

Établissement : Université de Perpignan Via Domitia
École doctorale : Energie et Environnement
Laboratoire de recherche : Centre de recherches insulaires et observatoire de l'environnement
Direction de la thèse : Nicolas INGUIMBERT ORCID 0000000203099048
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-06-12T23:59:59

Des études récentes montrent que les D-peptidases, des enzymes spécialisées dans la dégradation de peptides contenant des D-aminoacides, menacent l'efficacité d'antibiotiques lipopeptidiques tels la polymyxin B, la daptomycine et plus largement des antibiotiques peptidiques contenant des D-aminoacides. Etudier ce type d'enzyme est important, car il constitue un nouveau mécanisme
d'antibiorésistance.
Ces enzymes, produites par des bactéries pour réguler la concentration des antibiotiques qu'elles synthétisent, ont été identifiées par criblage in-silico de génomes bactériens sans que leur importance environnementale n'ait été étudiée. Ce projet de thèse vise à développer une banque de substrats innovants permettant de détecter, marquer ces D-peptidases par une étiquette fluorescente pour les
isoler dans des échantillons environnementaux. Leur identification pourrait aussi révéler de nouveaux composés antibiotiques renforçant ainsi les thérapies existantes menacées par l'antibiorésistance.

Several factors contribute to the development of antibiotic resistance, including the overuse and misuse of antibiotics in human and veterinary medicine. This favors the emergence of bacterial mechanisms that alter the biological targets of antibiotics, reduce their penetration into bacterial cells (via efflux pumps or membrane modifications), or cause their enzymatic degradation. Antimicrobial peptides are a promising alternative to traditional antibiotics, but their efficacy is threatened by a resistance mechanism involving bacterial D-peptidases.
To anticipate and better understand this recently described phenomenon,1 it is essential to study the environmental occurrence of these enzymes. Accordingly, the main objectives of the SANITISE multidisciplinary project are divided into three main tasks:
1- Development and validation of an innovative enzyme substrate for trapping/visualizing/collecting a target enzyme. To ensure the success of the project, commercial bacterial enzymes will be used as study models.
2- Application of the above approach to a D-peptidase currently being characterized at CRIOBE.
3- Development of an environmental D-peptidase substrate library to better characterize their environmental occurrence, particularly in the xylem of Xylella fastidiosa host plants. X. fastidiosa is one of the most important emerging plant pathogens. It is a xylem-limited bacterium, transmitted via insect vectors that causes a variety of diseases in a wide range of crops, including grapevine, almonds and olive trees, with huge economic impact on agriculture and environment. Recently, our group has identified antimicrobial peptides active against X. fastidiosa. Since their activity could be hampered by their degradation in the plant xylem, studying the presence of D-peptidases would provide valuable information and serve to design more stable peptides.
These three complementary tasks will answer the following question: Do D-peptidases endanger the use of antimicrobial peptides in public health and agri-food, and can this problem be avoided?The development of this thesis project follows on from the theses carried out at CRIOBE, which studied the detoxification of cyclic lipopeptides by a herbivorous mollusc. These studies revealed a D-peptidase that remains to be characterized, and the preliminary results provide a solid foundation for the development of the proposed subject.

We will implement an experimental procedure specifically designed to test the following hypotheses:
1. the substrate concept (trap/view/collect) is applicable to several enzyme classes.
2. the D-peptidase on which work has been initiated can be isolated from a complex matrix using this approach
3. This approach can also be applied to environmental samples.
Task 1: Development and validation of the enzyme substrate concept (trap/visualization/collection). 1 publication. Location Perpignan
Two bacterial model enzymes representative of the metallopeptidase class, thermolysin, and of the serine proteases, subtilisin A, will be used. The known substrates of these enzymes will be modified to adapt them to the concept (trap/visualize/collect). The known substrates of these enzymes are suitable for testing this concept, as they are of sufficient length to allow the insertion of the three chemical entities
required, namely a fluorophore derived from a coumarin, a quencher containing a 2,4-dinitrophenyl nucleus, and a diazirine as the group allowing conjugation to the enzyme. Amino acids containing these chemical entities are commercially available.
Substrates will be synthesized by solid-phase peptide synthesis using a state-of-the-art automatic EMC synthesizer available at CRIOBE.
After purification by HPLC, these substrates will be characterized by spectroscopic studies using the NMR and HPLC/MS equipment available on the MSXM platform of the Bio2Mar platform.
The stability of the substrates under UV irradiation will then be studied by HPLC/MS to determine the sensitivity of the photoactivatable probe and its half-life under experimental conditions with and without enzyme.
For experiments carried out with enzyme, electrophoresis gel experiments performed at LGDP under the responsibility of J. Sáez-Vásquez
will verify the labelling of the enzyme by the fluorescent probe.
Task 2: Application to a D-peptidase. 1 publication. Location Perpignan
Based on the analogues of the cleavage products we had previously identified,3 we have recently developed a peptide substrate, containing a 7-methoxycoumarin-4-acetyl group whose fluorescence is quenched by radiative transfer (FRET) to a 2,4-dinitrophenyl (Dnp) quencher. Enzymatic cleavage by a D-peptidase isolated from a marine organism2 after D-leucine (D-leu) induces separation of the quencher from the fluorophore, whose concentration could be quantified by fluorescence. Thus, this pre-optimized substrate 1 is completely cleaved by D-peptidase after 2h of reaction and constitutes a proof of concept. A similar result was obtained for substrate 2, showing that the position of the fluorophore and quencher was not limiting.
Preliminary synthesis tests have enabled us to introduce a photoactivatable diazirine probe into this substrate, and the substrates are now available for initial testing on cell extracts containing D-peptidase.
In these lipopeptide-like substrates, the lipid moiety is constituted by a decanoic acid (Adc), the quencher by a lysine bearing a 2,4-dinitrophenyl group on its side chain (LysDnp), the fluorophore by a lysine bearing a coumarin on its side chain (LysMca), and a D-series leucine after which enzymatic cleavage takes place. For substrates 3 and 4, a photoactivatable group, photoLeucine (PhotolLeu), has been introduced to enable binding to the enzyme.
These substrates form a solid basis for the continuation of the project and should enable D-peptidase to be isolated from pre-concentrated cell extracts. Labeled D-peptidase will then be isolated using standard biochemical separation techniques (size exclusion chromatography, gel electrophoresis). To carry out this work, we will draw on the skills of J. Sáez-Vásquez of the LGDP laboratory, who specializes in enzyme studies. Isolated D-peptidase will be digested with trypsin and the fragments analyzed by Maldi-MS to identify the enzyme class of D-peptidase by BLAST on the NCBI protein library. This proteomics approach will be carried out in collaboration with S Cantel from the IBMM laboratory at the University of Montpellier.
Task 3: Extension to in situ detection of D-peptidase. 1 publication. Location Girona
This is the riskiest part of the project, since it involves applying the concept developed to the in situ detection of D-peptidase. To this end,
the xylem of host plants of X. fastidiosa will be extracted (Plant Pathology Research Group). The resulting samples will be treated with
peptides designed and synthesized at Laboratory of Innovation in Processes and Products of Organic Synthesis Research Group
(LIPPSO) according to the above strategy.

Le/la candidat(e) titulaire d'un master 2 ou d'un diplôme d'ingénieur chimiste possèdera une forte expérience en synthèse organique qui pourra être complétée par des connaissances en synthèse peptidique. Le candidat maîtrisera les techniques usuelles de purification et d'analyse (RMN, LC/MS). Le développement du projet nécessitera un investissement particulier en chimie analytique pour le suivi et l'interprétation des réactions de marquage enzymatique.