Role of the disulfide bond in stabilizing and folding of the fimbrial protein DraE from uropathogenic Escherichia coli.

Dr fimbriae are homopolymeric adhesive organelles of uropathogenic Escherichia coli composed of DraE subunits, responsible for the attachment to host cells. These structures are characterized by enormously high stability resulting from the structural properties of an Ig-like fold of DraE. One feature of DraE and other fimbrial subunits that makes them peculiar among Ig-like domain-containing proteins is a conserved disulfide bond that joins their A and B strands. Here, we investigated how this disulfide bond affects the stability and folding/unfolding pathway of DraE. We found that the disulfide bond stabilizes self-complemented DraE (DraE-sc) by ∼50 kJ mol-1 in an exclusively thermodynamic manner, i.e. by lowering the free energy of the native state and with almost no effect on the free energy of the transition state. This finding was confirmed by experimentally determined folding and unfolding rate constants of DraE-sc and a disulfide bond-lacking DraE-sc variant. Although the folding of both proteins exhibited similar kinetics, the unfolding rate constant changed upon deletion of the disulfide bond by 10 orders of magnitude, from ∼10-17 s-1 to 10-7 s-1 Molecular simulations revealed that unfolding of the disulfide bond-lacking variant is initiated by strands A or G and that disulfide bond-mediated joining of strand A to the core strand B cooperatively stabilizes the whole protein. We also show that the disulfide bond in DraE is recognized by the DraB chaperone, indicating a mechanism that precludes the incorporation of less stable, non-oxidized DraE forms into the fimbriae.

Pilicides inhibit the FGL chaperone/usher assisted biogenesis of the Dr fimbrial polyadhesin from uropathogenic Escherichia coli.

BACKGROUND: The global spread of bacterial resistance has given rise to a growing interest in new anti-bacterial agents with a new strategy of action. Pilicides are derivatives of ring-fused 2-pyridones which block the formation of the pili/fimbriae crucial to bacterial pathogenesis. They impair by means of a chaperone-usher pathway conserved in the Gram-negative bacteria of adhesive structures biogenesis. Pili/fimbriae of this type belong to two subfamilies, FGS and FGL, which differ in the details of their assembly mechanism. The data published to date have shown that pilicides inhibit biogenesis of type 1 and P pili of the FGS type which are encoded by uropathogenic E. coli strains. RESULTS: We evaluated the anti-bacterial activity of literature pilicides as blockers of the assembly of a model example of FGL-type adhesive structures--the Dr fimbriae encoded by a dra gene cluster of uropathogenic Escherichia coli strains. In comparison to the strain grown without pilicide, the Dr⁺ bacteria cultivated in the presence of the 3.5 mM concentration of pilicides resulted in a reduction of 75 to 87% in the adherence properties to CHO cells expressing Dr fimbrial DAF receptor protein. Using quantitative assays, we determined the amount of Dr fimbriae in the bacteria cultivated in the presence of 3.5 mM of pilicides to be reduced by 75 to 81%. The inhibition effect of pilicides is concentration dependent, which is a crucial property for their use as potential anti-bacterial agents. The data presented in this article indicate that pilicides in mM concentration effectively inhibit the adherence of Dr⁺ bacteria to the host cells--the crucial, initial step in bacterial pathogenesis. CONCLUSIONS: Structural analysis of the DraB chaperone clearly showed it to be a model of the FGL subfamily of chaperones. This permits us to conclude that analyzed pilicides in mM concentration are effective inhibitors of the assembly of adhesins belonging to the Dr family, and more speculatively, of other FGL-type adhesive organelles. The presented data and those published so far permit to speculate that based on the conservation of chaperone-usher pathway in Gram-negative bacteria , the pilicides are potential anti-bacterial agents with activity against numerous pathogens, the virulence of which is dependent on the adhesive structures of the chaperone-usher type.