Déformation et dynamique de la membrane plasmique endothéliale révélées par les tétraspanines lors de l'adhésion de Neisseria meningitidis

Neisseria meningitidis is a human restricted commensal bacterium found in the nasopharyngeal mucosa. Upon crossing the epithelial barrier and disseminating into the bloodstream, it becomes a pathogen responsible for cerebrospinal meningitis and severe sepsis. Meningococci ability to invade the bloodstream relies on the colonization of endothelial cells thanks to the interaction between bacterial filamentous organelle type IV pili (T4P) and endothelial cell receptors CD147 and β2-Adrenergic. This interaction triggers protective microvilli-like deformation of the plasma membrane, enriched in receptors, cytoskeleton and scaffolding proteins, such as ezrin (actin linker) and tetraspanins CD9, CD81 and CD151 (membrane organisers). T4P retraction forces and ezrin recruitment have both been showed to be critical in the membrane structures stability, presumably through actin polymerisation. This thesis investigates the infection of endothelial cells by N. meningitidis from the perspective of the plasma membrane organisers tetraspanins by (1) exploring the role of tetraspanins in meningococcal adhesion and the subsequent host cell response, (2) studying the mechanisms driving their recruitment beneath bacterial colonies and (3) using tetraspanins as markers of plasma membrane tubular structures to study membrane deformation and protein enrichment in the early meningococcal infection. To carry out this project, we developed several experimental approaches, including the quantification of protein recruitment by immunofluorescence, their imaging using STED and CLEM, preparation of plasma membrane sheets, and the implementation of the TurboID method. We identified CD9, CD81, and CD151 as being robustly recruited by adhesive T4P at bacterial adhesion sites. However, their expression inhibition or suppression in endothelial cells did not affect bacterial adhesion or ezrin recruitment. STED super-resolution microscopy and CLEM correlative microscopy revealed that CD9 accumulates at adhesion sites separately from ezrin. Interestingly, membrane deformation and CD9 recruitment persisted even through the inhibition of the previously described triggering of the host cell response. This suggests the potential involvement of a yet-to-be-identified pathway for receptors recruitment at bacterial adhesion sites, driven by membrane curvature rather than T4P-induced mechanical forces and cell signalling. To investigate the origins and implications of this recruitment pathway, we leveraged CD9's ability to be recruited by membrane curvature at adhesion sites. FRAP experiments suggested that CD9 and other transmembrane receptors accumulate and remain trapped within membrane deformations, even under several complete inhibitions of cellular response. In summary, although we demonstrated that tetraspanins CD9, CD81, and CD151 do not play a critical role in meningococcal adhesion to endothelial cells, we used CD9 as a marker to reveal mechanically induced membrane deformation upon T4P tip-mediated adhesion. This membrane remodelling functions as a parallel receptor recruitment pathway near meningococci, leading to receptor accumulation and enhancing the likelihood of interaction with T4P ligands. Once interaction is established, T4P retraction promotes membrane elongation and stabilisation via ezrin phosphorylation and actin polymerisation, leading to further receptors enrichment that forms an amplification loop.