ED Sciences de la Vie et de la Santé
Resistance to last-resort antibiotics in Pseudomonas aeruginosa and new diagnostic and therapeutic strategies
by Léa BIENTZ (Microbiologie fondamentale et Pathogénicité)
The defense will take place at 9h30 - « Salle de conférence » Le Centre d'Appui à la Recherche et à la Formation (CARF) Site Carreire de l'Université de Bordeaux 146, rue Léo Saignat 33076 Bordeaux cedex.
in front of the jury composed of
- Véronique DUBOIS - Professeure des universités - praticienne hospitalière - Université de Bordeaux - Directeur de these
- Katy JEANNOT - Professeure des universités - praticienne hospitalière - Université de Franche-Comté - Rapporteur
- Thierry NAAS - Maître de conférences - praticien hospitalier - Université Paris-Saclay - Rapporteur
- Marie-Cécile PLOY - Professeure des universités - praticienne hospitalière - Université de Limoges - Examinateur
- Laurent STECKEN - Praticien hospitalier - CHU Bordeaux - Examinateur
Antibiotic (ATB) resistance is a major public health concern. In this context, Pseudomonas aeruginosa, a mainly nosocomial and multi-resistant opportunistic bacterium, is considered a high-priority pathogen for surveillance, research and the development of new ATBs. The aim of this study was to investigate the mechanisms of resistance in P. aeruginosa to ceftolozane-tazobactam (C/T) and carbapenems, which are last-resort ATBs, and to investigate new diagnostic and therapeutic strategies. This work was supported by high-throughput sequencing data from the international MAGITICS (MAchine learning for diGItal diagnosTICS of antimicrobial resistance) project. A total of 319 P. aeruginosa strains were selected for their various resistance profiles. Eighty-five different sequence types (STs) were found, and 38.9% of the strains belonged to a high-risk epidemic clone. Eleven chromosomal genes involved in resistance were analysed, in particular the oprD gene, involved in resistance to carbapenems, which was modified in 43.9% of strains, including 35% by the presence of an insertion sequence. Genes encoding a β-lactamase and/or an aminoglycoside-modifying enzyme were acquired in 119 strains, 87% of which were associated with an integron. Preliminary results of MIC (minimum inhibitory concentration) prediction based on genomic data showed an accuracy between 64.6% and 85.1% according to the antibiotic (ceftazidime, imipenem, amikacin and ciprofloxacin). Metabolomic analysis revealed a significant difference in the presence of metabolites between a susceptible strain and an isogenic resistant strain. Genomic data enabled us to monitor the emergence of resistance in 9 out of 50 patients treated with C/T (18%) over a 13-month period. This resistance was always associated with presence of mutations (T70I, F121L, P154L, G157D, P217Q, E221K and del210-216) in the active site of the chromosomal cephalosporinase AmpC. Some patients carried different isolates with various mutations associated with hypermutator strains. Cloning experiments in an AmpC-deficient strain were used to evaluate the level of C/T resistance for each mutation, and their impact on new ATBs. All the mutations, except F121L and P154L, conferred a high level of resistance to C/T and restored susceptibility to imipenem. Another study of 8 clinical strains producing the IMP-13 carbapenemase enabled us to show for the first time that the blaIMP-13 gene was carried by a functional ICE (integrative and conjugative element) using conjugation experiments. This ICE of 88589 bp, belonging to the ICEclc family, was inserted in the 3' of tRNAGly PA0729.1. This mobilisation was associated with variability in the 3' of the ICE with transfer of adjacent genes present in the donor strain. So, even if the spread of blaIMP-13 is mainly due to the ST621 epidemic clone, the mobilisation of the ICE is possible and should not be underestimated. Finally, we studied a new therapeutic approach using nanobodies (Nbs) targeted against the OprM protein involved in several efflux systems leading to resistance. Some Nbs stopped bacterial growth and restored the activity of the pumped-out ATB. Peptidomimetics based on the Nbs paratope could represent a new class of efflux pump inhibitors. This work has contributed to improved knowledge of resistance mechanisms, both chromosomal and by horizontal gene transfer, and has also allowed us to approach promising new diagnostic and therapeutic strategies in tackling antibiotic resistance.
Directed evolution and engeneering of molecular mechanisms of proteins in synaptic transmission
by Ivana TRIVUNOVIC (Institut Interdisciplinaire de Neurosciences)
The defense will take place at h00 - Amphi Centre Broca Nouvelle - Aquitaine Centre Broca Nouvelle-Aquitaine 146 rue Leo Saignat 33077 Bordeaux France
in front of the jury composed of
- Matthieu SAINLOS - Directeur de recherche - Université de Bordeaux - Directeur de these
- Nathalie SANS - Directrice de recherche - Université de Bordeaux - Examinateur
- Gerti BELIU - Chargé de recherche - University of Würzburg - Rapporteur
- Arnaud GAUTIER - Professeur des universités - University of Sorbonne - Rapporteur
Over the last decades, technological advancements have allowed to generate a more accurate vision of the complex organization of neuronal cytoskeleton and synapses. These advancements span from significant development of different imaging techniques, as well as the molecular tools currently used to probe different cellular compartments and specific proteins in excitatory synapses. Despite these progresses, our understanding of a number of structural and functional elements of these specialized neuronal compartments are still incomplete. Previously developed tools, such as synthetic antibodies and fusion proteins, need further improvement to achieve precise structural and functional probing and modulation of endogenous proteins. The work of this thesis was devoted to exploiting protein engineering techniques to achieve highly diversified genomic libraries using site-directed mutagenesis and high-throughput screening. This allows for applying evolutional pressure on libraries containing billions of sequences to identify the best candidate binders based on the human 10th fibronectin domain. Intrabody library design and selections were focused on two cytoskeletal proteins, talins and spectrins, in charge of neuronal development and synaptic formation and stabilization. Talin plays a critical role in cellular communication with the extracellular matrix and maintaining the overall cellular stability and integrity. It contains two distinguishable functional domains, the FERM N-term and the talin rod domain. In the context of this work, we report three highly specific binders to talin rod-subdomains R1R2, R9 and R11. Complexes of these binders with their target domains have been characterized with 2D NMR, providing detailed insight into the target domains' binding epitopes. Spectrins create a highly regular protein network with actin rings across the cell, spanning from the axon initial segment to dendritic spines. In this context, small domains of αII-spectrin and β(II-IV)-spectrins were the targets in our selections, resulting in several promising candidates for structural and functional probing of spectrins. Protein-protein interaction modulation was focused on inhibiting several proteins in the post-synaptic density region, involved in glutamate receptor trafficking, synapse stabilization and signal transduction from binding to the PDZ domains of PSD95. This approach in protein-protein inhibition of the PDZ domain will allow for the blocking of only one of many interactions of the PDZ domain and shed more light on the contribution of each PDZ domain-mediated interaction in the complex regulation of synaptic plasticity. Upon full characterization, these intrabodies and affinity clamps will provide a new set of genetically encoded tools for endogenous protein labelling and modulation.