The Dr hemagglutinin of uropathogenic Escherichia coli is a fimbrial homopolymer of DraE subunits encoded by the dra operon. The dra operon includes the draB and draC genes, whose products exhibit homology to chaperone-usher proteins involved in the biogenesis of surface-located polymeric structures. DraB is one of the periplasmic proteins belonging to the superfamily of PapD-like chaperones. It possesses two conserved cysteine residues characteristic of the FGL subfamily of Caf1M-like chaperones. In this study we obtained evidence that DraB cysteines form a disulfide bond in a mature chaperone and have the crucial function of forming the DraB-DraE binary complex. Expression experiments showed that the DraB protein is indispensable in the folding of the DraE subunit to a form capable of polymerization. Accumulation of DraB-DraE n oligomers, composed of head-to-tail subunits and the chaperone DraB, was observed in the periplasm of a recombinant E. coli strain which expressed DraB and DraE (but not DraC). To investigate the donor strand exchange mechanism during the formation of DraE oligomers, we constructed a series of DraE N-terminal deletion mutants. Deletion of the first three N-terminal residues of a potential donor strand resulted in a DraE protein lacking an oligomerization function. In vitro data showed that the DraE disulfide bond was not needed to form a binary complex with the DraB chaperone but was essential in the polymerization process. Our data suggest that assembly of Dr fimbriae requires a chaperone-usher pathway and the donor strand exchange mechanism.

The dra gene cluster of uropathogenic strains of Escherichia coli produces proteins involved in bacterial attachment to and invasion of the eukaryotic host tissues. The crystal structure of a construct of E. coli DraD possessing an additional C‐­terminal extension of 13 amino acids, including a His6 tag, has been solved at a resolution of 1.05 Å. The protein forms symmetric dimers through the exchange of the C‐terminal β‐­strands, which participate in the immunoglobulin‐like β‐­sandwich fold of each subunit. This structure confirms that DraD is able to act as an acceptor in the donor‐strand complementation mechanism of fiber formation but, in contrast to DraE adhesin, its native sequence does not have a donor strand; therefore, DraD can only be located at the tip of the fiber.

The potential of the major structural protein DraE of Escherichia coli Dr fimbriae has been used to display an 11-amino-acid peptide of glycoprotein D derived from herpes simplex virus (HSV) type 1. The heterologous sequence mimicking an epitope from glycoprotein D was inserted in one copy into the draE gene in place of a predicted 11-amino-acid sequence in the N-terminal region of surface-exposed domain 2 within the conserved disulfide loop (from Cys21 to Cys53). The inserted epitope was displayed on the surface of the chimeric DraE protein as evidenced by immunofluorescence and was recognized by monoclonal antibodies to the target HSV glycoprotein D antigen. Conversely, immunization of rabbits with purified chimeric Dr-HSV fimbriae resulted in a serum that specifically recognized the 11-amino-acid epitope of HSV glycoprotein D, indicating the utility of the strategy employed.

The dra gene cluster, expressed by uropathogenic Escherichia coli strains, determines bacterial attachment and invasion. The Dr fimbrial structures formed at the bacterial cell surface are composed of DraE subunits. The Dr fimbriae-coding cluster contains six open reading framesdraA, draB, draC, draD, draP and draE -among which the draE gene encodes the structural fimbrial subunit DraE. Very little is known about E. coli surface expression of the draD gene product. The expression of DraD and its role in the biogenesis of Dr fimbriae were determined by constructing mutants in the dra operon and by immunoblot and immunofluorescence experiments. In this study, DraD was found to be a surface-exposed protein. The expression of DraD was independent of the DraC usher and DraE fimbrial subunits. Polymerization of DraE fimbrial subunits into fimbrial structures did not require expression of the DraD protein.

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