Ramifications of kinetic partitioning on usher-mediated pilus biogenesis

Abstract

The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperoneadhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperoneadhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly.

Molecular mechanism of bacterial type 1 and P pili assembly

“…Indeed, FimD binds to different FimC-subunit complexes with different affinities, which would be a key element for correct initiation of pilus assembly and for the correct ordering of the subunit incorporation into the pilus. 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).…”

“…Although the FimC-FimH complex is known to be needed to activate the usher [49,59,60], the usher activation mechanism is still unclear. 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.…”

Molecular Strategies for Fimbrial Expression and Assembly

“…The strucfimbriae resemi ture of Type 1 fimbriae has been determined by the use of genetorder, organizati ics, biochemistry, electron microscopy, x-ray crystallography, and al., 1998). The P NMR techniques (Brinton, 1965;Mitsui et al, 1973;Kuehn et al, resents a parad 1994; Saulino et al, 1998). At least nine gene products are composite fiber required for expression ( Fig.…”

“…The chaperone, subunits, and usher interactions then lead to the formation and translocation of a linear pilus fiber across the outer membrane through the usher channel. Interaction of the FimC-FimH complex with FimD secures the usher FimD in an assembly-competent conformation (Saulino et al, 1998). Formation of the usher channel has been demonstrated by in vitro reconstitution of PapC (or FimD) into liposomes (Thanassi et al, 1998).…”

“…Interaction of the FimC-FimH complex with FimD secures the usher FimD in an assembly-competent conformation (Saulino et al, 1998). Formation of the usher channel has been demonstrated by in vitro reconstitution of PapC (or FimD) into liposomes (Thanassi et al, 1998). The 2-to 3-nm-diameter channel allows the tip fimbrillum and linear pilus rod to pass through and form the final helical rod on the cell surface.…”