PMC

CTD2 mediates dissociation of the PapC NTD/chaperone–adhesin complex. Super Streptavidin pins were incubated in 50 μg/mL of biotinylated NTD, washed, and incubated in 0.5 μM PapDG to measure the NTD–PapDG interaction (left side of graph). The pins containing the NTD–PapDG complex were moved to wells containing HBS only, HBS with 0.5 μM CTD, or 0.5 μM Plug domain (right side of graph). Incubation of the bound NTD–PapDG complex with CTD2 (blue, CTD2) causes faster dissociation of chaperone–adhesin from the NTD compared with buffer alone (red, HBS). Incubating NTD–PapDG complex with the Plug domain causes the Plug domain’s association with the bound complex (green, Plug).

Model of pilus biogenesis at the usher. The plug domain resides in the translocation pore in the inactive usher (A). Upon chaperone–adhesin binding to the NTD, the plug domain extends to the periplasm where it stably binds to the NTD (B). CTD2 mediates binding to the chaperone–adhesin complex at the NTD (C) where it catalyzes dissociation of the NTD–PapDG complex and remains bound to PapDG (D). Subsequent interactions of incoming subunits with the CTDs and Plug result in pilus assembly (E–G). Rod and tip components interact with Plug and CTD2, but the terminator complex PapDH is directly targeted to the Plug domain without transfer to the NTD or CTD2 (G).

PapC periplasmic domain CTD2 mediates binding with chaperone–subunit complexes. PapC CTD2 mediates concentration-dependent, micromolar affinity interactions with chaperone–subunit complexes PapDG, PapDE_ntd, PapDA_Knte, and apo-PapD, but has no affinity for PapDH. BSA was used as a binding specificity control. The tips of Super Streptavidin pins were coated with 50 μg/mL of biotinylated PapC CTD2 and washed to remove unbound protein. These pins were dipped in increasing concentrations of chaperone–subunit complexes (shown at 2 μM each) to measure binding of chaperone–subunit complexes to CTD2 (left side of graph) and then moved to wells containing HBS to measure dissociation rates (right side of graph).

In vivo overexpression of usher periplasmic domains interferes with pilus biogenesis. Electron microscopy (A) and hemagglutination assays (B) reveal that overexpression of the PapC domains CTD2, NTD, and Plug in the periplasmic space decreases pilus biogenesis. E. coli C600 cells carrying the pFJ3 plasmid were transformed with pKDC3 (CTD), pKDC5 (Plug), and pKDC16 (NTD). These strains were grown on TSA plates with IPTG for induction of P pili from the pFJ3 plasmid and expression of Plug, NTD, and CTD. A lawn of cells was grown and collected for electron microscopy (A) and hemagglutination assays (HA) (B) to investigate the impact of overexpressed PapC domains on pilus biogenesis. HA titer is the highest dilution of bacteria that still provides agglutination of human type A erythrocytes.

PapC Plug domain mediates a high-affinity, stable interaction with PapC NTD, and this complex is capable of recruiting chaperone–subunit complexes. (A) In a biolayer interferometry assay, Super Streptavidin pins incubated in 50 μg/mL of biotinylated PapC Plug domain were incubated with increasing concentrations of purified PapC NTD for 2 min (0.2, 0.4, 3.2, 6.6, and 13.2 μM) to detect NTD–Plug association. The pins with NTD–Plug complex were then moved to wells containing HBS to measure dissociation for 30 min. A concentration-dependent, stable interaction between NTD and Plug domains of PapC was observed. (B) The stable complex that forms between PapC NTD and Plug domains is active in recruiting all tested chaperone–subunit complexes and apo-PapD. Using biolayer interferometry as in A, a stable complex between NTD and Plug domain was obtained by incubating Super Streptavidin pins coated with 50 μg/mL of Plug domain in 24.6 μM NTD and then washing them. The pins coated with NTD–Plug complex were incubated in chaperone–subunit complexes for 5 min in increasing concentrations of chaperone–subunit complexes or chaperone alone (shown at 1 μM each) and then moved to wells containing HBS for measurement of dissociation rates.