Leaders : Pauline Macheboeuf and Carlos Contreras-Martel
The emergence of multidrug-resistant strains of pathogenic bacteria represents a major challenge in modern healthcare. The incidence of so-called "superbugs"—microorganisms resistant to most clinically available antibiotics—is rising at an alarming rate. In the United States and Europe, five bacterial pathogens are responsible for the majority of hospital-acquired (nosocomial) infections. Collectively referred to as the “ESKAPE” pathogens—Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species—these organisms are so named because of their increasing ability to "escape" the effects of existing antibacterial treatments.
Recent data indicate that in several European countries, over 70% of pathogenic bacterial isolates exhibit resistance to at least one currently available antibiotic. Alarmingly, despite the growing public health threat posed by antimicrobial resistance, most major pharmaceutical companies have significantly reduced or entirely abandoned efforts in antibacterial drug discovery. As a result, there is an urgent and unmet medical need for the development of novel antibacterial agents capable of combating these resistant infections.
The bacterial cell wall is primarily composed of peptidoglycan (PG), a three-dimensional polymer consisting of disaccharide subunits cross-linked by pentapeptide stems. This mesh-like structure provides the bacterial cell with its shape, enables it to withstand osmotic pressure, and plays a crucial role in cell division. Due to its vital function and absence in eukaryotic cells, the PG has long been a prime target for the development of new antibiotics.
Our research focuses on two distinct stages of PG biosynthesis in Gram-negative bacteria :
• The synthesis of the cytoplasmic PG precursor, catalyzed by the Mur ligase enzymatic complex, as well as its dynamic recruitment by the cell wall synthesis machineries, during various stages of the bacterial cell cycle.
• The development of novel inhibitory compounds targeting penicillin-binding proteins (PBPs), which are responsible for the final steps of PG assembly in the periplasm.
Mur ligases
Proteins involved in PG biosynthesis assemble into multiprotein complexes that coordinate cell elongation and division. Inhibition or dysregulation of these proteins can lead to defects in cell shape, often resulting in lysis and cell death. Among them, the cytoplasmic Mur ligases have been proposed to form functional complexes—an idea supported by the observation that, in many bacterial species, mur genes located within highly conserved operons have fused, resulting in the production of chimeric proteins.
A comparative genomic analysis of over 140 bacterial genomes enabled us to characterize, in particular, the MurE–MurF chimera from Bordetella pertussis, using X-ray crystallography and fluorescence polarization. The elongated architecture of the chimera reveals that the two active
sites are in close proximity, and interaction data suggest that MurE–MurF can associate with other Mur ligases via its central domains (Shirakawa et al., PNAS, 2023). Our results point to strong evolutionary constraints to maintain close genomic proximity between gene encoding proteins that physically interact, thereby linking Mur ligase interactions, complex assembly, and genome organization. In addition to revealing the molecular interface between the MurE and MurF subunits, this study lays the groundwork for elucidating the complete structure of the Mur ligase complex.
Furthermore, the Mur ligase complex must be directed either to the elongasome or the divisome, two complexes involved in the elongation and division of bacteria, which compete for PG precursors at different stages of bacterial growth. We discovered that some mur genes are fused to division genes, leading to the formation of chimeric proteins that physically link the Mur complex to the divisome. A deeper understanding of the architecture and dynamics of these complexes within the bacterial cell will ultimately pave the way for the discovery of novel antibacterial compounds.
Penicillin-binding proteins (PBP)
PBPs catalyze the final steps of PG biosynthesis and are the targets of the β-lactam antibiotics, such as penicillin. However, due to the growing issue of antibiotic resistance, there is an urgent need to discover new molecules capable of effectively inhibiting PBPs. The development of such inhibitors relies on a structure-based approach, made possible through close collaboration between chemists and our team. This work involves the chemical synthesis of novel compounds, evaluation of their interactions with PBPs, and crystallographic analysis of the resulting complexes. These structural data help deepen our understanding of the interaction mechanisms and enhance the selectivity of candidate inhibitors.
In previous work, we have significantly contributed to this field by publishing numerous crystal structures of PBPs in complex with various ligands. These structural insights, obtained in part through productive collaborations with several medicinal chemistry groups worldwide, have greatly supported the development of novel inhibitory compounds. Our research has primarily focused on PBP1b from Streptococcus pneumoniae, studied in interaction with various antibiotics as well as biochemical probes such as pseudo-substrates, lactivicins, boronates, and β-lactones.
PBPs are particularly attractive drug targets for several fundamental reasons : (1) they are essential for bacterial viability ; (2) their natural substrate, the PG, is unique to bacteria ; and (3) there have no human homologs, thereby minimizing off-target effects. Furthermore, our laboratory has solved numerous PBP crystal structures, providing a strong foundation for in silico modeling and the rational design of new therapeutic inhibitors.
Our current objective is to design novel antimicrobial agents based on high-resolution experimental structural data of PBP–inhibitor complexes. These complexes originate from both Gram-negative ESKAPE pathogens, and our Gram-positive model organism, S. pneumoniae. This structure-guided approach aims to identify innovative inhibitors capable of bypassing resistance mechanisms by specifically targeting essential PBPs in these clinically relevant bacteria.