Function of the usher N-terminus in catalysing pilus assembly

Area of Science:

• Microbiology
• Bacterial cell surface structures
• Protein secretion systems

Background:

• The chaperone/usher (CU) pathway is essential for assembling bacterial pili (or fimbriae).
• This pathway involves periplasmic chaperones and outer membrane (OM) ushers, which act as assembly platforms.
• The precise mechanism by which ushers catalyze pilus assembly remains largely unknown.

Purpose of the Study:

• To elucidate the mechanism of usher-catalyzed pilus assembly.
• To identify specific regions and residues within ushers critical for catalytic activity.
• To differentiate the roles of usher catalytic activity in pilus initiation versus extension.

Main Methods:

• Utilized uropathogenic Escherichia coli P and type 1 pilus systems.
• Generated mutants in conserved N-terminal disulfide regions of PapC and FimD ushers.
• Investigated the role of FimD residue F4.
• Employed in vivo co-purification and in vitro pilus polymerization assays.

Main Results:

• Conserved N-terminal disulfide regions and FimD residue F4 are essential for usher catalytic activity.
• PapC mutants with altered disulfide loops initiated pilus assembly but failed to extend fibers.
• FimD mutants with altered disulfide loops or F4 initiated pilus assembly but showed defects in fiber extension.

Conclusions:

• The catalytic activity of the PapC usher is required for initiating pilus biogenesis.
• The catalytic activity of the FimD usher is essential for the extension of pilus fibers.
• These findings reveal distinct functional roles for usher catalytic activity in different stages of pilus assembly.