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Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colonization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1 Å resolution and displayed the symmetry of space group P21. CfaBB exhibited a crystal diffraction limit of 2.3 Å resolution and had the symmetry of space group P21212. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-rays to 2.3 Å resolution. These structures were determined using the molecular-replacement method. [ABSTRACT FROM AUTHOR]

Fimbrial filaments assembled by distinct chaperone pathways share a common mechanism of intersubunit interaction, as elucidated for colonization factor antigen I (CFA/I), archetype of enterotoxigenic Escherichia coli (ETEC) Class 5 fimbriae. We postulated that a highly conserved β-strand at the major subunit N-terminus represents the donor strand, analogous to interactions within Class I pili. We show here that CFA/I fimbriae utilize donor strand complementation to promote proper folding of and interactions between CFA/I subunits. We constructed a series of genetic variants of CfaE, the CFA/I adhesin, incorporating a C-terminal extension comprising a flexible linker and 10–19 of the N-terminal residues of CfaB, the major subunit. Variants with a donor strand complement (dsc) of ≥ 12 residues were recoverable from periplasmic fractions. Genetic disruption of the donor β-strand reduced CfaE recovery. A hexahistidine-tagged variant of dsc19CfaE formed soluble monomers, folded into β-sheet conformation, displayed adhesion characteristic of CFA/I, and elicited antibodies that inhibited mannose-resistant haemagglutination by ETEC expressing CFA/I, CS4 and CS14 fimbriae. Immunoelectron microscopy indicated that CfaE was confined to the distal fimbrial tip. Our findings provide the basis to elucidate structure and function of this class of fimbrial adhesins and assess the feasibility of an adhesin-based vaccine. [ABSTRACT FROM AUTHOR]

Understanding of pilus bioassembly in Gram-negative bacteria stems mainly from studies of P pili and type 1 fimbriae of uropathogenic Escherichia coli, which are mediated by the classic chaperone-usher pathway (CUP). However, CFA/I fimbriae, a class 5 fimbria and intestinal colonization factor for enterotoxigenic E. coli (ETEC), are proposed to assemble via the alternate chaperone pathway (ACP). Both CUP and ACP fimbrial bioassembly pathways require the function of a periplasmic chaperone, but their corresponding proteins share very low similarity in primary sequence. Here, the crystallization of the CFA/I periplasmic chaperone CfaA by the hanging-drop vapor-diffusion method is reported. X-ray diffraction data sets were collected from a native CfaA crystal to 2 Å resolution and to 1.8 and 2.8 Å resolution, respectively, from a lead and a platinum derivative. These crystals displayed the symmetry of space group C2, with unit-cell parameters a = 103.6, b = 28.68, c = 90.60 Å, β = 119.7°. Initial phases were derived from multiple isomorphous replacement with anomalous scattering experiments using the data from the platinum and lead derivatives. This resulted in an interpretable electron-density map showing one CfaA molecule in an asymmetric unit. Sequence assignments were aided by anomalous signals from the heavy-atom derivatives. Refinement of the atomic model of CfaA is ongoing, which is expected to further understanding of the essential aspects and allowable variations in tertiary structure of the greater family of chaperones involved in chaperone-usher mediated bioassembly. [ABSTRACT FROM AUTHOR]