Pseudopilus assembly and substrate recognition factors in the Klebsiella type II secretion system ; Facteurs impliqués dans l'assemblage du pseudopilus et dans la reconnaissance du substrat dans le système de sécrétion de type II chez Klebsiella

Gram-negative bacteria employ several types of complex machineries to secrete specific proteins across their cell envelope to the cell surface or to their milieu. The Klebsiella Type II secretion system (T2SS), our experimental model, is dedicated to secretion of pullulanase (PulA), an enzyme of the amylase family. PulA secretion relies on the assembly of periplasmic filaments called pseudopili, a process which is thought to drive the folded T2SS substrates from the periplasm across the outer membrane. Pseudopili, closely related to bacterial type IV pili, are composed of four minor tip subunits (PulHIJK) and a major core subunit PulG. They are polymerized at the inner membrane assembly platform subcomplex of the T2SS, composed of PulF, PulL and PulM inner membrane proteins. The mechanism of pseudopilus assembly and their role in substrate recognition are still poorly understood. PulA is a 117 kDa lipoprotein secreted in a folded form by the Klebsiella T2SS. In this work we aimed to identify T2SS components that interact directly with PulA to promote its transport across the outer membrane. Towards this goal, we performed systematic bacterial two-hybrid (BACTH) analysis, chemical cross-linking and co-purification. We also set up and in vivo UV crosslinking approach by introducing the unnatural amino acid pBPA in the PulA regions previously identified as putative secretion signals. The BACTH analysis identified the assembly platform components PulF and PulM as putative interacting partners of PulA, as well as the pseudopilins PulG, PulI and PulH. In the second part of the work, we focused on the assembly platform components PulL and PulM that promote pseudopilus assembly and PulA secretion. This work was done in collaboration with the team of N. Izadi-Pruneyre, who determined the structure of the C-terminal domains (CTDs) of PulL and PulM and of their complex. We tested mutations of residues mapping in the PulLCTD -and PulMCTD interface and found that they affect PulA secretion. We also investigated the ...