The CFA/I assembly consists of 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 the pili. 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). In order to understand how the CFA/I pili are assembled, we have obtained structures for their complexes in various forms such as the minor and major pilin complex CfaEB, the complexes between major pilins such as CfaBB and CfaBBB. Based on these structures, we identified the receptor-binding site for the adhesive subunit, which was subsequently confirmed with site-directed mutagenesis and we obtained a putative model for the pilus assembly.
| Li, Yong Fu; Poole, Steven; Rasulova, Fatima et al. (2009) Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriae. Acta Crystallogr Sect F Struct Biol Cryst Commun 65:242-7 |
