Structural basis for adhesin secretion by the outer-membrane usher in type 1 pili

Abstract

Gram-negative bacteria produce chaperone-usher pathway pili, which are extracellular protein fibers tipped with an adhesive protein that binds to a receptor with stereochemical specificity to determine host and tissue tropism. The outer-membrane usher protein, together with a periplasmic chaperone, assembles thousands of pilin subunits into a highly ordered pilus fiber. The tip adhesin in complex with its cognate chaperone activates the usher to allow extrusion across the outer membrane. The structural requirements to translocate the adhesin through the usher pore from the periplasm to the extracellular space remains incompletely understood. Here, we present a cryoelectron microscopy structure of a quaternary tip complex in the type 1 pilus system from Escherichia coli, which consists of the usher FimD, chaperone FimC, adhesin FimH, and the tip adapter FimF. In this structure, the usher FimD is caught in the act of secreting its cognate adhesin FimH. Comparison with previous structures depicting the adhesin either first entering or having completely exited the usher pore reveals remarkable structural plasticity of the two-domain adhesin during translocation. Moreover, a piliation assay demonstrated that the structural plasticity, enabled by a flexible linker between the two domains, is a prerequisite for adhesin translocation through the usher pore and thus pilus biogenesis. Overall, this study provides molecular details of adhesin translocation across the outer membrane and elucidates a unique conformational state adopted by the adhesin during stepwise secretion through the usher pore. This study elucidates fundamental aspects of FimH and usher dynamics critical in urinary tract infections and is leading to antibiotic-sparing therapeutics.

Keywords: adhsein FimH; outer-membrane usher FimD; pilus biogenesis; type 1 pilus.

Fig. 1.

Type 1 pilus system and structure of a tip complex FimD–FimCFH. (A) Domains of the FimD usher, adhesin FimH, subunit FimF, and chaperone FimC. FimD consists of five functional domains: NTD, plug domain, β-barrel translocation domain, and two CTDs. FimH has a lectin domain FimH_L and a pilin domain FimH_P. (B) Cryo-EM density and structure of FimD–FimCFH. The outer-membrane boundaries are indicated. Each subunit is uniquely colored (FimD in blue, FimH in green, FimC in yellow, and FimF in purple).

Fig. 2.

Stepwise secretion of the FimH adhesin. (A) Structure of the initiation complex FimD–FimCH (PDB: 3RFZ) (B) Structure of FimD–FimCFH. (C) Structure of the elongation complex FimD–FimCFGH (PDB: 6E14). The FimD usher is colored from light blue to dark blue from the amino- to the carboxy-terminus. Other subunits are similarly colored for comparison. (D) Domain movements in the stepwise secretion of the adhesin FimH. (E) Superposition of the three distinct conformations of adhesin FimH during transport. Structures are aligned on the basis of the pilin domain (FimH_P).

Fig. 3.

Structural plasticity between FimH lectin and pilin domains is a requirement for adhesin secretion through the FimD usher. pBAD FimC_His–FimH with either wild-type FimH, FimH(G159A/G160A), or FimH(ΔG159) was expressed in UTI89 E. coli with locked-on type 1 pili with a deletion of the fimH gene. (A) Pili on the surface of the cell were counted and classified as bald (no pili), low (1 to 20 pili/cell), moderate (20 to 200 pili/cell), or abundant (>200 pili/cell). Three hundred bacterial cells were counted for each strain. (B) Representative negative-stain electron microscopy images showing FimC_His–FimH_WT (abundant), FimC_His–FimH(G159A/G160A) (moderate), and FimC_His–FimH(ΔG159) (low) pili. (C) Outer-membrane purification of FimD–FimC_His–FimH with the indicated mutations in FimH.

Fig. 4.

Domain movements in the usher FimD. (A) Superposition of FimD–FimCF structures from FimD–FimCFH and FimD–FimCFGH (PDB: 6E14). Protein components are colored in the same schemes as those in (B) and (C). (B) FimD–FimCF in FimD–FimCFH. (C) FimD–FimCF in FimD–FimCFGH.