title: Donor strand complementation governs intersubunit interaction of fimbriae of the alternate chaperone pathway.
abstract: 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 beta-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 >or= 12 residues were recoverable from periplasmic fractions. Genetic disruption of the donor beta-strand reduced CfaE recovery. A hexahistidine-tagged variant of dsc19CfaE formed soluble monomers, folded into beta-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.
authors.affiliation: Enteric Diseases Department, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Poole
authors.forename: Annette L
authors.initials: AL
authors.lastname: McVeigh
authors.forename: Ravi P
authors.initials: RP
authors.lastname: Anantha
authors.forename: Lanfong H
authors.initials: LH
authors.lastname: Lee
authors.forename: Yasemin M
authors.initials: YM
authors.lastname: Akay
authors.forename: Emily A
authors.initials: EA
authors.lastname: Pontzer
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Scott
authors.forename: Esther
authors.initials: E
authors.lastname: Bullitt
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Savarino
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000737:chemistry; D006385:Hemagglutination Inhibition Tests; D016253:Microscopy, Immunoelectron; D011485:Protein Binding; D017510:Protein Folding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C022924:colonization factor antigens; D036781:Fimbriae Proteins