Glycan-dependent mechanical activation of the ß2-adrenergic receptor: a new pathophysiological paradigm – MECAD-R

The present project will investigate several breakthroughs emerging from a previous study, in which we deciphered protein-protein interactions and signalling pathways activated by a bacterial pathogen (N. meningitidis, meningococcus, the agent of cerebrospinal meningitis) to colonize brain vasculature and breach the blood brain barrier.
From the obtained data it appears that, after early adhesion to endothelial cells mediated by CD147, N. meningitidis, induces a specific signalling in host cells by activating the human catecholamine beta-2 adrenoceptor (ß2AR), a member of the G protein-coupled receptor (GPCR) family. The ß2AR is very likely bound and activated via an allosteric interaction established by PilE and PilV, two components of meningococcus type IV pili (Tfp), with sialic acid residues terminating tandem asparagin-branched glycans in the N-terminal region of the receptor, independently of the nature of surrounding amino acid residues. The confirmation of this hypothesis would represent a so far unknown mechanism of GPCR activation. It would also potentially contribute to explain the species selectivity of the pathogen, which only infects humans. In the context of N. meningitidis infection, the pathogen might activate its signaling receptor via mechanical forces. Indeed, the PilE and PilV-containing Tfp are retractable organelles, this property allowing the bacterium to crawl at the surface of endothelial cells. In addition, bacterial colonies growing attached at the luminal surface of endothelial cells are submitted to shear stress forces generated by the blood flow. Thus Pili might well exert traction on the ß2AR via receptor glycan-bound pilins. Preliminary data support the hypothesis of mechano-transducing properties of endothelial cell ß2ARs, triggered by traction forces engaging its glycan chains. Although activation of “classical” GPCRs by mechanical forces directly applied on receptors has been postulated for many years, the present project would demonstrate this hypothesis for the first time.
The project aims also at characterizing the physical forces, which can activate this GPCR, and how these forces induce the activating conformational change of the receptor. To address these issues we have included in our study group a physicist with skills in both soft matter physics and cell biology and a technical background for measuring forces at the molecular level in live cells.
Finally, although occurring in a pathophysiological context, some molecular mechanisms emerging from our study likely reflect unknown physiological aspects or ß2AR signalling. For example, our data demonstrate that the meningococcus adhesion receptor (CD147) and the ß2AR are pre-associated in endothelial cells, but the physiological function of this heterodimer remains unknown. Also, our preliminary data indicate unsuspected potential mechano-transduction properties of the ß2AR, in another context than bacterial infection. The physiological role of this particular mode of receptor activation in the context of cell-endothelium communication/interaction remains totally unexplored and represents one of the aims of our project.
The principal objectives of the proposal are therefore:
To demonstrate that the composition of ß2AR glycans determines the selectivity of N. meningitidis for humans.
To establish the role of the ß2AR receptor activation and of sialyltransferase(s) in the pathophysiology of meningococcal infection in vivo.
To confirm that mechanical forces exerted on ß2AR glycans induce specific signalling pathways in endothelial cells and characterize these forces.
To characterize signalling pathways downstream of mechanically activated ß2AR and their role in physiology.