The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose-sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D-mannose. The unique site binding pocket occupied by D-mannose was probed using site-directed mutagenesis. All but one of the mutants examined had greatly diminished mannose-binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono-saccharide D-mannose was delineated from this of mannotriose (Man(alpha 1-3)[Man(alpha 1-6)]Man) by generating mutants that abolished D-mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide alpha-D-mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose-binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose-binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.

The initial encounter of a microbial pathogen with the host often involves the recognition of host receptors by different kinds of bacterial adhesive organelles called pili, fimbriae, fibrillae or afimbrial adhesins. The development of over 26 of these architecturally diverse adhesive organelles in various Gram‐negative pathogens depends on periplasmic chaperones that are comprised of two immunoglobulin‐like domains. All of the chaperones possess a highly conserved sheet in domain 1 and a conserved interdomain hydrogen‐bonding network. Chaperone‐subunit complex formation depends on the anchoring of the carboxylate group of the subunit into the conserved crevice of the chaperone cleft and the subsequent positioning of the COOH terminus of subunits along the exposed edge of the conserved sheet of the chaperone. We discovered that the chaperones can be divided into two distinct subfamilies based upon conserved structural differences that occur in the conserved sheet. Interestingly, a subdivision of the chaperones based upon whether they assemble rod‐like pili or non‐pilus organelles that have an atypical morphology defines the same two subgroups. The molecular dissection of the two chaperone subfamilies and the adhesive fibers that they assemble has advanced our understanding of the development of virulence‐associated organelles in pathogenic bacteria.

P pili are important virulence factors in uropathogenic Escherichia coli. The Cpx two‐component signal transduction system controls a stress response and is activated by misfolded proteins in the periplasm. We have discovered new functions for the Cpx pathway, indicating that it may play a critical role in pathogenesis. P pili are assembled via the chaperone/usher pathway. Subunits that go ‘OFF‐pathway’ during pilus biogenesis generate a signal. This signal is derived from the misfolding and aggregation of subunits that failed to come into contact with the chaperone in the periplasm. In response, Cpx not only controls the stress response, but also controls genes necessary for pilus biogenesis, and is involved in regulating the phase variation of pap expression and, potentially, the expression of a panoply of other virulence factors. This study demonstrates how the prototypic chaperone/usher pathway is intricately linked and dependent upon a signal transduction system.

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PapD is an immunoglobulin-like chaperone that mediates the assembly of P pili in uropathogenic strains of Escherichia coli. It binds and caps interactive surfaces on pilus subunits to prevent their premature associations in the periplasm. We elucidated the structural basis of a mechanism whereby PapD also interacts with itself, capping its own subunit binding surface. Crystal structures of dimeric forms of PapD revealed that this self-capping mechanism involves a rearrangement and ordering of the C2-D2 and F1-G1 loops upon dimerization which might ensure that a stable dimer is not formed in solution in spite of a relatively large dimer interface. An analysis of site directed mutations revealed that chaperone dimerization requires the same surface that is otherwise used to bind subunits.

SummaryPapD is the periplasmic chaperone required for the assembly of P pili in pyelonephritic strains of Escherichia coli. It consists of two immunoglobulin-like domains bisected by a subunit binding cleft. PapD is the prototype member of a superfamily of immunoglobulin-like chaperones that work in concert with their respective ushers to assemble a plethora of adhesive organelles including pilus-and non-pilus-associated adhesins. Three highly conserved residue clusters have been shown to play critical roles in the structure and function of PapD, as determined by site-directed mutagenesis. The in vivo stability of the chaperone depended on the formation of a buried salt bridge within the cleft. Residues along the G1 beta strand were required for efficient binding of subunits consistent with the crystal structure of PapD-peptide complexes. Finally, Thr-53, a residue that is part of a conserved band of residues located on the aminoterminal domain surface opposite the subunit binding cleft, was also found to be critical for pilus assembly, but mutations at Thr-53 did not interfere with chaperone-subunit complex formation.

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