Soutenance de thèse : Investigation of LPS recognition by immunity C-type lectin receptors in a cell-surface mimicking environment

Par Massilia Abbas (Groupe Membrane et pathogènes )

The envelope of Gram-negative bacteria is decorated with LipoPolySaccharides (LPSs) representing the main glycolipid component of its surface. LPSs are constituted of three main parts : lipid A linked to an ligoSaccharide chain (OS), which in turn is linked to an O-antigen polysaccharide portion. Given their dense packing and tructural variability, LPSs are key elements in antimicrobial resistance and virulence. As surface exposed components, they are potent activators of the immune system of plants, animals, and humans. Their lipid A moiety is detected by the immune system either extracellularly by the TLR4 cascade or intracellularly by the caspase system. Whereas, their glycan part is found to be recognized by C-type Lectin Receptors (CLRs) present on Antigen Presenting Cells (APC). A protein family to which key roles have been attributed in host defence and homeostasis.
In this study, we investigated the interactions involving human Macrophage Galactose-type Lectin MGL and E. coli surface glycans. We demonstrated the ability of MGL to bind E. coli R1 core OS by integrative approaches spanning from the cellular to the atomic level. Fluorescence microscopy and flow cytometry primarily revealed the strong ability of MGL to bind E. coli R1 type core surfaces, while SPR provided an estimation of the interaction affinity. Nevertheless, this interaction was found to occur regardless of the canonical calcium-dependent glycan binding site. NMR spectroscopy was used to identify a novel carbohydrate binding site on opposite surface of the canonical interaction site within MGL Carbohydrate Recognition Domain (CRD). A model of the trimeric MGL was built using a combination of small-angle X-ray scattering (SAXS) and AlphaFold modelling. This model showed the convenient 3D arrangement of MGL CRDs presenting up to six accessible glycan binding sites (2 per CRD) favourable to bind LPSs at the bacterial surface with enhanced affinity.
MGL interaction with bacterial glycans was further monitored in a cell surface mimicking model, employing Styrene-Maleic Acid (SMA) copolymer LPS nanodiscs. This approach was based on previous studies demonstrating the ability of these polymers to isolate patches of native bilayers from biological membranes. Accordingly, a protocol for the preparation of LPS nanodiscs originating from various strains ranging from laboratory to pathogenic E. coli strains was established. This protocol was successfully applied on purified LPS and LPS extracted from outer membranes. The resulting membrane-mimetic models were studied and proved suitable for several biophysical methods ; their size distribution and thickness were assessed by Atomic Force Microscopy. The different components of bacterial outer membranes could be observed at atomic scale by solid-state NMR. LPS nanodiscs were also successfully used to monitor interactions with MGL immunity C-type lectin by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and BioLayer Interferometry (BLI). These models will now be used to build a model depicting the arrangement of MGL ECD on LPS membrane nanodiscs.
In summary, investigating the binding sites and functional roles of MGL and other lectins, alongside advancements in LPS nanodisc technology, holds significant promise for understanding pathogen recognition by the host immune system. Ongoing improvements in this technology will undoubtedly enhance our ability to study complex host-pathogen interactions, paving the way for the development of potential therapeutic agents.