Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin.

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha-D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (Kd = 0.15 microM) than mannose (Kd = 2.3 microM). Exploration of the binding affinities of alpha- d-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.

Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: preserved folding energy drives fiber formation.

Most gram-negative pathogens express fibrous adhesive virulence organelles that mediate targeting to the sites of infection. The F1 capsular antigen from the plague pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and serves as a prototype for nonpilus organelles assembled via the chaperone/usher pathway. Genetic data together with high-resolution X-ray structures corresponding to snapshots of the assembly process reveal the structural basis of fiber formation. Comparison of chaperone bound Caf1 subunit with the subunit in the fiber reveals a novel type of conformational change involving the entire hydrophobic core of the protein. The observed conformational change suggests that the chaperone traps a high-energy folding intermediate of Caf1. A model is proposed in which release of the subunit allows folding to be completed, driving fiber formation.

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the F1 antigen Caf1M-Caf1 chaperone-subunit pre-assembly complex from Yersinia pestis.

The F1 capsular antigen of the plague-causing pathogen Yersinia pestis is assembled from monomeric Caf1 subunits via the Caf1M/Caf1A chaperone/usher system. Y. pestis Caf1M-Caf1 chaperone-subunit complex was purified from the periplasm of Escherichia coli cells overexpressing Caf1M and Caf1 and was crystallized in PEG 4000 solution using hanging-drop vapour diffusion. The crystals diffract to a minimum Bragg spacing of 1.8 A and belong to space group P2(1), with unit-cell parameters a = 36.0, b = 69.2, c = 69.1 A, beta = 93.0 degrees. SeMet-labelled Caf1M-Caf1 complexes were purified and crystallized under the same conditions. The SeMet crystals were identical to the native crystals and diffracted to 1.9 A. Heavy-atom derivative crystals were prepared by soaking in 10 mM K(2)PtCl(4), giving two Pt sites per complex. The experimental electron-density map was obtained by a combination of MAD and MIR methods using both Se- and Pt-derivative crystals.