Reconstitution of Pilus Assembly Reveals a Bacterial Outer Membrane Catalyst

Abstract

Type 1 pili from uropathogenic Escherichia coli are a prototype of adhesive surface organelles assembled and secreted by the conserved chaperone/usher pathway. We reconstituted type 1 pilus biogenesis from purified pilus proteins. The usher FimD acted as a catalyst to accelerate the ordered assembly of protein subunits independently of cellular energy. Its activity was highly dependent on the adhesin subunit FimH, which triggered the conversion of FimD into a high-efficiency assembly catalyst. Furthermore, a simple kinetic model adequately rationalized usher-catalyzed pilus assembly in vivo. Our results contribute to a mechanistic understanding of protein-catalyzed biogenesis of supramolecular protein complexes at the bacterial outer cell membrane.

Quality control of disulfide bond formation in pilus subunits by the chaperone FimC

“…Recently, we reported the NMR structure of a designed, selfcomplemented FimA variant (FimAa) 28 , in which FimA is artificially extended at its C terminus by a hexaglycine linker followed by the FimA donor strand segment (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. FimAa has the same slow, spontaneous folding rate as wild-type FimA (1.6-h folding half-life) and adopts a conformation in which the C-terminal copy of the donor strand is incorporated into the tertiary structure in an antiparallel orientation relative to the FimA F strand, which corres ponds to the expected donor strand insertion pattern in the quaternary structure of the pilus rod 28 .…”

“…Type 1 pili are assembled in vivo via the 'chaperone-usher pathway' 8 , involving the soluble periplasmic chaperone FimC 9 and the assembly platform ('usher') FimD in the outer membrane [10][11][12] . The pilus subunits enter the periplasm in an unfolded conformation and are recognized by the chaperone FimC, which catalyzes their folding 13 .…”

Molecular mechanism of bacterial type 1 and P pili assembly

“…From these results, it became evident that DSE rates between cognate pairs are much higher than between non-cognate pairs (two-to 50-fold), whereby the DSE rates followed the expected trend according to the established incorporation order in the tip fibrillum: for example, in the presence of an activated usher, FimG undergoes clearly the fastest DSE with FimH (171 min −1 ), followed by FimF with FimG (3 min −1 ), then FimA with FimF (0.03 min −1 ). It is interesting to note that, once the tip fibrillum is assembled in the correct order, the DSE of FimA-FimA is extremely high (960 min −1 ) [55,57].…”

“…Using the known usher-mediated DSE rate [55,57] and the known affinities of the various subunits for the usher NTD [46], a comprehensive mathematical model of pilus biogenesis was generated [57]. It was shown that the mathematically derived ordering of pilus subunit displays a significant dependence on chaperone-subunit concentrations.…”

“…However, pilus subunit ordering is not solely determined by the affinities of the chaperonesubunit complexes for the usher's NTD but depends also on the DSE rate between subunits. In vitro studies have been carried out on the DSE rates between cognate and non-cognate subunit pairs in both the Fim and the Pap systems and both with and without usher [55][56][57]. By 'cognate', we mean here pairs of adjacent subunits interacting with each other as observed in the mature pilus.…”