The rapid rise of antibiotic resistance threatens the effectiveness of bacterial infection treatments worldwide. Among the most problematic pathogens, Pseudomonas aeruginosa remains particularly challenging. In response, researchers are exploring “pathoblockers” anti-virulence compounds that interfere with pathogenic mechanisms such as adhesion and biofilm formation without directly killing bacteria. By reducing selective pressure for resistance development, pathoblockers, alongside bacteriophage therapy, antimicrobial peptides, and quorum-sensing inhibitors, offer promising alternatives against multidrug-resistant infections.
A key aspect of Pseudomonas aeruginosa pathogenicity lies in its ability to establish stable interactions with host tissues through specific molecular recognition. Human glycans are major targets of virulence factors produced by pathogenic microorganisms, including soluble lectins from Pseudomonas aeruginosa. Among these, LecA is a calcium-dependent galactose-binding lectin involved in tissue adhesion and biofilm formation, making it a promising anti-adhesion therapeutic target. Here, deuterated galactose was produced via hydrogen–deuterium exchange and co-crystallized with fully perdeuterated LecA, and the complex structure was determined by neutron diffraction.
Neutron macromolecular crystallography (NMX) enables direct visualization of hydrogen atoms in biological macromolecules, with its power significantly enhanced by perdeuteration, allowing unambiguous localization of both exchangeable and non- exchangeable hydrogens as deuterium. Joint X-ray and neutron refinement was performed to optimize data quality; the neutron dataset was collected on LADI-III at the Institut Laue-Langevin (ILL), and the X-ray dataset on beamline BM07/FIP2 at the European Synchrotron Radiation Facility(ESRF).
Beyond demonstrating an efficient chemical approach for deuterated carbohydrate production via hydrogen isotope exchange, joint X-ray/neutron co-refinement revealed key structural insights into LecA–galactose recognition:
- The protonation state and orientation of His50, critical for galactose binding (Figure 1A–B);
- A buried, structurally conserved water molecule bridging two LecA residues and the ligand via hydrogen bonds (Figure 1C);
- The precise orientation of galactose O3 and O4 hydroxyl hydrogens, positioned to minimize electrostatic repulsion with the coordinating calcium ion (Figure 1A–B).
These findings highlight the added value of NMX in resolving hydrogen-bond geometry and protonation states inaccessible to X-ray methods alone, providing atomic-level insights directly relevant to the rational design of glycomimetics targeting Pseudomonas aeruginosa.
T. Arnaud (ILL), J. Devos (ILL), L. Gajdos (ILL), M. Blakeley (ILL)
Arnaud T, Tatol C, Devos J, Gajdos L et al. (2026) ChemistryEurope, 4, e202500424
Figure 1: Neutron and X-ray structure of LecA in complex with deuterated galactose, focused on the binding site. X-ray electron density maps (2Fo – Fc) are contoured in blue at 1σ. Neutron 2Fo – Fc nuclear density maps are contoured in gray at 0.7σ. Carbon atoms are represented in black, nitrogen in blue, oxygen in red, hydrogen in white, and deuterium in yellow. (A) Superposition of X-ray and neutron maps highlighting the additional information provided by neutron macromolecular crystallography (NMX). (B) Interactions revealed by NMX; continuous nuclear density is observed at strong hydrogen bonds. (C) Water coordination network at the LecA–galactose binding interface