Bacteria have evolved sophisticated membrane channels, called secretins, specifically adapted for the assembly or secretion of bulky extracellular structures such as type 4 pili (T4P), needle complexes of type 3 secretion systems (T3SS) or large fully folded effectors in type 2 secretion systems (T2SS) (Review by Barbat et al., 2023b). We have recently shown that T2SS secretins must adopt different conformations to allow the passage of large effectors while maintaining the integrity of the bacterial envelope (Barbat et al., 2023a). In this project, we plan to develop and implement an ambitious in vivo experimental approach aimed not only at demonstrating the need for flexibility of the XcpD secretin during the secretion process, but also at identifying the partners that trigger these conformational changes. Specifically, we will further investigate the protein dynamics that exist between two adjacent secretin subunits by performing in-cell EPR experiments on spin-labelled proteins in their native environment (Review by Pierro et al., 2024) as well as in different genetic backgrounds lacking potential partners. With this approach, we hope to gain a better understanding of the different chronological steps that occur during the secretion process mediated by this singular nanomachine.
References:
Barbat, B., Douzi, B., Ball, G., Tribout, M., El Karkouri, K., Kellenberger, C., and Voulhoux, R. (2023a). Insights into dynamics and gating properties of T2SS secretins. Sci Adv 9, eadg6996.
Barbat, B., Douzi, B., and Voulhoux, R. (2023b). Structural lessons on bacterial secretins. Biochimie 205, 110-116.
Pierro, A., Bonucci, A., Magalon, A., Belle, V., and Mileo, E. (2024). Impact of Cellular Crowding on Protein Structural Dynamics Investigated by EPR Spectroscopy. Chem Rev 124, 9873-9898.