The process of CFA/I assembly requires four functional/structural components: CfaE is a tip-located minor subunit and functions as an adhesive component. CfaB polymerizes into the stalk of a pilus and is therefore named major pilin. CfaA functions as a periplasmic chaperone escorting CfaB or CfaE to their assembly site at the bacterial outer membrane. CfaC, named the usher protein, is an integral outer membrane protein forming the assembly site for pilus biogenesis. As part of the NIHs Biodefense program, my lab has been working on structure determination of CFA/I of ETEC. We have determined the full-length structure of CfaE;a putative receptor-binding site on the CfaEad centered on a cluster of positively charged residues (R181, R182 and R67) was located. To confirm the role of this site, R181, R182, and R67 were each mutated to alanines and the mutant proteins failed to agglutinate human erythrocytes, implicating the pocket anchored by these three residues as the putative receptor-binding domain. To determine the role in hemagglutination of individual residues surround the binding site, we further made twelve mutations involving residues that are either invariant (fully conserved) or subclass-specific for the Class 5 ETEC fimbrial adhesins. As a result of this analysis, we found that all positively charged residues (R181, R182, R67) are absolutely required for receptor binding, whereas those surrounding residues display altered interactions with red-blood cells and several show discriminatory behavior to either human type-A or bovine red cell species. We have also determined subunit structures of the major pilin (CfaB). For the first time, we elucidate atomic structures of an ETEC major pilin subunit, CfaB from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for two morphological forms of CFA/I fimbriae that are both observed in vivo, the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between the helical and extended forms of CFA/I fimbriae. Our findings affirm the strong structural similarities seen between Class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and Class 1 pili (from bacteria that cause urinary, respiratory and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Lastly, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related Class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein. To understand the pilus assembly, we have begun to study functional components in the pilus assembly, namely CfaA and CfaC. We have successfully obtained crystals of CfaA, and obtained complexes formed between CfaA and either CfaE or CfaB. Also, we have begun structural investigation of CfaC by over-expressing this large bacterial outer membrane protein.
