Maalej, Meriem (2021) Deciphering the recognition patterns between Lipopolysaccharides and human lectins using Nuclear Magnetic Resonance spectroscopy. [Tesi di dottorato]

Preview

Text Maalej_Meriem_33.pdf Download (23MB) | Preview

Collection description

For an equilibrated survival of humans against bacterial infections, immune cells have a well- defined function to distinguish between self and non-self structures. Escherichia coli is a Gram- negative bacterium that reside in the gut microbiota, either as a beneficial or harmful microorganism. The tolerance of pathogenic strains or their clearance is covered by many immune cell receptors such as Pathogen Recognition Receptors (PRRs) including C-type lectin receptors (CLRs) that specifically interact with carbohydrates moieties. Bacterial Lipopolysaccharide (LPS) is one of the bacterial signatures and it is the hallmark of Gram-negative bacteria. The molecular diversity of both LPS (i.e. various carbohydrates, lengths and heterogeneity levels) and lectins (variable binding sites architectures) would confer them the particularity of controlling variable situations and targeting specific interactions. The understanding of the mechanisms of LPS-Lectins interactions is challenging and requires an interdisciplinary approach. On the above basis, we sought to investigate the interaction between a C-type lectin i.e. human Macrophage Galactose-type Lectin MGL and LPSs (from E. coli R1, R3 mutants and O157:H7 strain). Bacterial LPSs were extracted, purified, and thereafter considered for the investigation of this large and complex interaction system. The difficult experimental handling of such native biomolecules directed the use of a divided set of approaches. In fact, our scientific strategy includes the use of Nuclear Magnetic Resonance NMR spectroscopy, fluorescence microscopy, and molecular binding essays. By combining these methods, we studied two distinct Lectin-LPS interaction systems: i) the molecular interaction between the Carbohydrate Recognition Domain (CRD) of MGL and soluble LPS versions by using NMR titrations and computational methods; ii) the recognition of E. coli mutants and LPS glycoconjugates by the Extracellular Domain (ECD) of MGL at both molecular and cellular levels by STD-NMR combined with computational analyses, Biolayer Interferometry (BLI), Electron Microscopy (EM) and fluorescence microscopy coupled to flow Cytometry. When only the CRD of MGL is considered, all tested E. coli LPS were found to bind in the millimolar affinity range, to an extended interacting area that includes a putative secondary binding region, in addition to the canonical calcium binding site, common in C-type lectins. Spectroscopic and microscopic investigations provided promising results about the recognition between ECD MGL and E. coli LPS/LOS. Trimeric human MGL interacts specifically with E. coli R1 LOS mainly through binding to the terminal di-galactose moiety. In addition, the dissociation constant (Kd) was estimated to be in the nanomolar range thus indicating a strong molecular binding. Moreover, the specific interaction between fluorescently labelled human MGL and E. coli R1 bacteria was investigated by single-cell essays. MGL-bound E. coli R1 bacteria were fluorescent (up to 40% of the bacterial population), exclusively at stationary phase, whereas E. coli R3 were not. Here again, binding specificity and selectivity was confirmed for ECD MGL-R1 interaction system. We showed that MGL strongly and specifically interacts with E. coli R1 glycoconjugates at the surface of bacteria through its terminal di-galactose motif. The contribution of a putative secondary binding site on MGL in glycoconjugates recognition remains to be investigated. This PhD work showed that, despite the difficulties that such large system studies may encounter, many findings are attainable by using our scientific strategy. The possibility of investigating data from atomic to cellular scale on LPS-lectin interactions in either modified or native states, opens prominent horizons for the study of bacterial infections.

Downloads

Actions (login required)

View Item