Thèse Protéines de Liaison à la Pénicilline Pbp2 et Pbp3 chez les Entérobactéries Épidémiologie Fonction et Rôle des Polymorphismes dans la Résistance aux -Lactamines de Dernier Recours H/F - Doctorat.Gouv.Fr

Établissement : Université Paris-Saclay GS Santé et médicaments École doctorale : Innovation thérapeutique : du fondamental à l'appliqué Laboratoire de recherche : IDMIT - Immunological Diseases, Microbiology and Innovative Therapies Direction de la thèse : Thierry NAAS ORCID 0000000199379572 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-30T23:59:59 Les protéines liant les pénicillines (PLPs) sont des enzymes essentielles impliquées dans la synthèse de la paroi cellulaire bactérienne et constituent les principales cibles des antibiotiques -lactamines. Parmi elles, les PLP2 et PLP3 jouent des rôles clés dans l'élongation cellulaire et la division bactérienne, respectivement. Les entérobactéries, notamment Escherichia coli, Klebsiella pneumoniae et le complexe Enterobacter cloacae, sont des pathogènes majeurs responsables d'un large éventail d'infections, en particulier des infections urinaires et des bactériémies.
La résistance aux antimicrobiens chez ces bactéries représente un enjeu majeur de santé publique à l'échelle mondiale et est principalement liée à la production de -lactamases, incluant les -lactamases à spectre étendu (BLSE) et les carbapénémases. Cependant, les modifications de la cible, notamment via des mutations des PLPs, sont de plus en plus reconnues comme un mécanisme contribuant à la résistance, en particulier vis-à-vis des nouvelles -lactamines et des associations -lactamine/inhibiteur de -lactamase.
Les mutations de la PLP3 sont particulièrement bien décrites chez E. coli, notamment l'insertion de quatre acides aminés (YRIN/YRIK), qui modifie le site de liaison de l'antibiotique et diminue l'affinité pour les -lactamines. En revanche, les mutations de la PLP2 sont moins fréquentes mais peuvent contribuer à la résistance, en particulier aux carbapénèmes. Bien que ces mutations aient souvent un effet modéré prises isolément, elles peuvent augmenter significativement le niveau de résistance lorsqu'elles sont associées à d'autres mécanismes, notamment la production de -lactamases. Des différences spécifiques selon les espèces sont observées, E. coli présentant une fréquence plus élevée de mutations de la PLP3, tandis que le rôle des PLPs chez K. pneumoniae et le complexe E. cloacae reste encore insuffisamment exploré.
L'objectif de ce projet est de caractériser les polymorphismes des PLP2 et PLP3 chez ces trois espèces à partir de bases de données génomiques publiques, d'isolats producteurs de carbapénémases adressés au Centre National de Référence des entérobactéries productrices de carbapénémases (CNR CPE), ainsi que d'une collection d'isolats de CPE provenant de Colombie. Ces isolats seront testés vis-à-vis de nouveaux antibiotiques, tels que ceftazidime/avibactam, aztréonam/avibactam et céfidérocol, ainsi que de combinaisons en cours de développement comme céfépime/zidebactam, céfépime/taniborbactam et céfidérocol/xeruborbactam.
Les mutations localisées à proximité du site actif de ces PLPs seront introduites dans des souches sauvages par mutagenèse de type TN-MAGE ou par des approches basées sur CRISPR-Cas, afin d'étudier la contribution individuelle de chaque mutation dans un fond génétique isogénique. Ces PLPs mutées seront caractérisées à l'aide d'approches phénotypiques, moléculaires, biochimiques et de modélisation moléculaire. Une meilleure compréhension du rôle des mutations des PLPs dans les profils de résistance est essentielle pour optimiser les stratégies thérapeutiques et orienter le développement de nouveaux antibiotiques et outils diagnostiques.
Antimicrobial resistance (AMR) among Gram-negative bacteria has become one of the most pressing challenges in modern medicine. Members of the order Enterobacterales, particularly Escherichia coli, Klebsiella pneumoniae, and the Enterobacter cloacae complex, are major causes of both community-acquired and healthcare-associated infections. These organisms are responsible for urinary tract infections, bloodstream infections, pneumonia, and intra-abdominal infections, contributing significantly to global morbidity and mortality [1,2].-lactam antibiotics remain a cornerstone of treatment for these infections due to their broad spectrum of activity, their safety, reliable killing properties and clinical efficacy. These antibiotics exert their effect by targeting penicillin-binding proteins (PBPs), enzymes involved in the synthesis of peptidoglycan, a critical component of the bacterial cell wall [3]. Among these, PBP2 and PBP3 are essential high-molecular-weight PBPs responsible for cell elongation and cell division, respectively.

However, the usefulness of ß-lactam is threatened by the global proliferation of -lactamases (BL) with broad hydrolytic capacities, especially in multi-resistant (MDR) gram-negative bacteria. These BLs are divided into 4 classes based on their sequence identities1. Classes A, C, and D contain active-site serine enzymes whose reaction pathways involve acylenzyme adducts while class B represents metallo--lactamases (MBLs) do not form such intermediates. Currently, BL-mediated resistance does not spare even the more potent -lactams (i.e., carbapenems), whose activity is challenged by MBLs as well as by serine carbapenemases (classes A and D) [4,5]. The overproduction of efflux pumps and the production of AmpC or ESBL type -lactamase which weakly hydrolyze carbapenems, together with the reduction in the expression of porin (defect of penetration) would also play a major role in resistance to beta- lactams and carbapenems [5]. The spread of carbapenem-resistant Enterobacterales (CRE) is of concern, as these bacteria are generally resistant to all families of antibiotics and a major cause of nosocomial and community-acquired infections [6] and carbapenems are key players in the treatment of these infections [7].

While -lactamase production has long been recognized as the dominant resistance mechanism in Enterobacterales, increasing evidence highlights the importance of target modification, particularly mutations in PBP2 and PBP3. These alterations reduce antibiotic binding affinity and contribute to clinically significant resistance, especially when combined with other mechanisms [8-11].

Historically, treatment options for carbapenem-resistant Enterobacterales (CRE) infections have included polymyxins, tigecycline, aminoglycosides, and fosfomycin, used either as monotherapy or in combination regimens [8]. However, several of these agents are associated with significant toxicity, limited clinical efficacy, and high rates of resistance among CRE isolates. The introduction of novel antibiotics has therefore renewed therapeutic options, enabling effective treatment of infections that were previously considered therapeutic dead ends due to pan-drug-resistant bacteria [8]. Newer ß-lactam agents include ceftazidime-avibactam, aztreonam-avibactam, meropenem-vaborbactam, imipenem-relebactam, and cefiderocol are approved for the treatment of patients with CRE infections when the isolates are susceptible to these agents [8-10]. However, for some of these molecules, resistance has been described even before their clinical use. In particular, aztreonam/avibactam and cefiderocol resistant Escherichia coli isolates producing NDM-5 are increasingly described globally, even in countries were these antibiotics are not in clinical use [9-11]. In silico analysis of more than 1,500 E. coli genomes from the French National Reference Center together with susceptibility testing to cefiderocol and aztreonam-avibactam revealed an association between reduced susceptibility to these antibiotics and a mutated penicillin-binding protein 3 (PLP3) (insertion of 4 amino acids) (T. Naas, personal data). Very recently PBP2 variants have also been described in E. coli responsible of Zidebactam resistance [12]. However, data on K. pneumoniae and E. cloacae are very scarce The current research proposal is aimed at better understanding the role of mutations in PBP2 and PBP3 in E. coli, Klebsiella pneumoniae and Enterobacter cloacae complex. We plan to develop tools to study the activity of PBPs (a topic that has been neglected in the last decades) towards new ß-lactams, and to better understand their evolvability by mutations, expression and contribution in the resistance to these molecules. To achieve these goals, the project will be divided into four complementary workpackages WP1: Construction of a Comprehensive Database of PLPs in E. coli, K. pneumoniae and Enterobacter cloacae complex
Objective: To establish a curated and comprehensive database of PLP types 2, and 3 in E. coli, K. pneumoniae and Enterobacter cloacae complex
A systematic in silico screening will be conducted across publicly available genomic and metagenomic resources, including EnteroBase. This large-scale analysis will enable comprehensive identification of PLP variants on a global level without any a priori' on the resistance phenotype. This will allow an estimation of the global distribution of PBP2 and PBP3 polymorphims.
WP2: Distribution of PBP2 and PBP3 polymorphisms in carbapenem-resistant isolates from France and Colombia
Objective: To establish a comprehensive database of PLP types 2, and 3 in E. coli, K. pneumoniae and Enterobacter cloacae complex present in carbapenem-resistant bacteria in two University hospitals from France and Colombia.
Hundred and fifty CREs of each species (450 in total) will be collected in each country (Hopital Bicetre, and the French National Reference Center of CPEs, France) and from the Don Bosque hospital in Bogota (Colombia). Phenotypic characterization will be performed by determining minimum inhibitory concentrations (MICs) for 32 antibiotics (including novel molecules still in phase 1 and 2 of clinical development) using Sensititre microdilution technology. Whole-genome sequencing of the 900 isolates will be conducted using long read technology (nanopore) [10], ensuring robust representation of PLP diversity at both national levels, to allow acquired resistome, MLST, genetic relatedness and plasmid characterization. Particular attention will be paid PBP2 (mdrA) and PBP3 (ftsI) and their associated carbapenemase. This will allow to compare the prevalence of these variants identified in WP1 to those in MDR isolates from both countries. In addition, during this WGS approach, novel carbapenemases, or novel variants may be discovered. These variants will be systematically identified, curated, and integrated into the Beta-Lactamase Database (BLDB), thereby strengthening its scope and utility [13].
WP3: Contribution of PLPs to -Lactam Resistance
Objective: To elucidate the role of mutations in penicillin-binding proteins (PBPs) in E. coli, K. pneumoniae and Enterobacter cloacae complex -lactam resistance.
Allelic replacement of mutant PBP variants will be performed in an isogenic background using genome editing approaches (TM-MAGE or CRISPR-Cas9) [14]. Resulting strains will be phenotypically characterized through antibiotic susceptibility testing and MIC determination, alone or in combination with various ß-lactamases including ESBLs and carbapenemases, to identify PLP/-lactamase combinations that may confer resistance to last-resort therapies such as ceftazidime/avibactam, aztreonam/avibactam and newer molecules.
This approach will enable systematic evaluation of naturally occurring mutations and polymorphisms in PLPs and their impact on -lactam inhibition, a critical step toward the development of predictive in silico antibiograms.

WP4: In vitro investigation of PBP activities
WP4 is built on the complementary expertise of our team in medical microbiology, -lactam resistance mechanisms, molecular biology, and biochemistry, together with the group of Bogdan Iorga (CNRS, ICSN, Gif-Sur Yvette, France), which brings expertise in medicinal chemistry, structural biology, molecular modeling, and molecular dynamics simulations.
We will develop innovative tools to investigate PLP activity-an area largely neglected by the pharmaceutical industry in recent decades-and to better understand their evolutionary potential under antibiotic pressure. We will establish quantitative assays to measure substrate affinity and competition, including binding assays using fluorescent -lactams (biocillin, carbapenems, and aztreonam) [15] and transpeptidase activity assays based on FRET technology. PBPs displaying the most significant phenotypic effects will be overexpressed and purified to determine enzymatic constants, particularly inhibition parameters (Ki) in the presence of different -lactams. Structural and computational analyses will be performed to model the impact of key mutations, including the insertion of four amino acids in PBP3, on protein function and drug interaction.

Expertise en Microbiologie, Résistance aux antibiotiques, biologie moléculaire