DEVELOPMENT OF PILUS ORGANELLE SUBASSEMBLIES IN-VITRO DEPENDS ON CHAPERONE UNCAPPING OF A BETA-ZIPPER

The major subassemblies of virulence-associated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were recons tituted from purified chaperone-subunit complexes in vitro. Subunits a re held in assembly-competent conformations in chaperone-subunit compl exes prior to their assembly into mature pili. The PapD chaperone bind s, in part, to a conserved moth present at the C terminus of the subun its via a beta zippering interaction. Amino acid residues in this cons erved motif were also found to be essential for subunit-subunit intera ctions necessary for the formation of pill, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit-su bunit interactions in the periplasm. Uncapping of the chaperone-protec ted C terminus of PapA and PapE was mimicked in vitro by freeze-thaw t echniques and resulted in the formation of pilus rods and tip fibrilla e, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimutha l disorder. This change in the number of subunits per turn of the heli x most likely reflects involvement of the leading edge of the beta zip per in forming a right-handed helical cylinder, Organelle development is a fundamental process in all living cells, and these studies shed n ew light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.