Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine

Abstract

P pili are prototypical chaperone-usher pathway-assembled pili used by Gram-negative bacteria to adhere to host tissues. The PapC usher contains five functional domains: a transmembrane β-barrel, a β-sandwich Plug, an N-terminal (periplasmic) domain (NTD), and two C-terminal (periplasmic) domains, CTD1 and CTD2. Here, we delineated usher domain interactions between themselves and with chaperone-subunit complexes and showed that overexpression of individual usher domains inhibits pilus assembly. Prior work revealed that the Plug domain occludes the pore of the transmembrane domain of a solitary usher, but the chaperone-adhesinbound usher has its Plug displaced from the pore, adjacent to the NTD. We demonstrate an interaction between the NTD and Plug domains that suggests a biophysical basis for usher gating. Furthermore, we found that the NTD exhibits high-affinity binding to the chaperone-adhesin (PapDG) complex and low-affinity binding to the major tip subunit PapE (PapDE). We also demonstrate that CTD2 binds with lower affinity to all tested chaperone-subunit complexes except for the chaperone-terminator subunit (PapDH) and has a catalytic role in dissociating the NTD-PapDG complex, suggesting an interplay between recruitment to the NTD and transfer to CTD2 during pilus initiation. The Plug domain and the NTDPlug complex bound all of the chaperone-subunit complexes tested including PapDH, suggesting that the Plug actively recruits chaperone-subunit complexes to the usher and is the sole recruiter of PapDH. Overall, our studies reveal the cooperative, active roles played by periplasmic domains of the usher to initiate, grow, and terminate a prototypical chaperone-usher pathway pilus.biolayer interferometry | macromolecular assembly | urinary tract infections | virulence factor | bacterial pathogenesis

Molecular mechanism of bacterial type 1 and P pili assembly

“…In addition, the NTD alone has by far the highest affinity for chaperone-subunit complexes [40,46,47], consistent with the NTD being the initial recruitment site.…”

“…Consistent with this is the fact that, among all chaperone-subunit complexes, the chaperone-adhesin complex exhibits the highest affinity for the usher NTD [49]. However, the rank order of NTD-binding affinities for the other tip fibrillum subunits does not match the incorporation order as observed in the mature pilus [40,[45][46][47], suggesting that other factors play a role (see below).…”

“…One major step in usher activation is the swinging out of the plug from its position within the usher pore lumen to its position in the periplasm where it interacts with the usher NTD. Electrophysiological studies suggested that both the NTD and the CTDs are involved in gating the usher, even in the absence of a translocation substrate [61], with only the FimH adhesin being able to stabilize the plug-NTD interaction in the open state while reaching out for the pore [43,47] ( figure 4, view b). Recently, it was proposed that the lectin domain of FimH actively displaces the plug domain, because the plug domain was calculated to have weaker interactions with the usher lumen than the lectin domain of FimH [27].…”

“…Recently, it was proposed that the lectin domain of FimH actively displaces the plug domain, because the plug domain was calculated to have weaker interactions with the usher lumen than the lectin domain of FimH [27]. Deletion of the plug domain showed that it is essential in pilus biogenesis [61,62], its role in active subunit recruitment becoming recently apparent: the NTD of PapC alone was only able to bind PapD-PapG and PapD-PapE, but not the other chaperone-subunit complexes PapD-PapK/A/H, whereas an NTD-plug complex is able to bind all chaperone-subunit complexes [47]. ( figure 4, view c).…”

“…Thus, after activation and initial recruitment of FimC-FimH to the usher NTD, the FimC-FimH complex must be transferred to the CTDs ( figure 4, step 2). How this 'handover' is managed is still not completely clear, although it was shown that CTD2 is able to destabilize the NTD-FimC-FimH complex, increasing the dissociation rate by one order of magnitude [47]. Except for the terminator complex PapD-PapH, CTD2 was able to bind to all chaperone-subunit complexes, although with much lower affinity than the NTD or the NTD-plug complex [47], which is in line with it being the second station for subunits during pilus growth.…”