Conformational inactivation induces immunogenicity of the receptor-binding pocket of a bacterial adhesin

Abstract

Significance Antibodies targeting the receptor-binding pocket of viral and bacterial adhesins are highly protective against infection. However, functional site epitopes in the antigens are not always highly immunogenic, possibly because of the binding epitope-masking effects of natural ligand–receptor interactions. By using the mannose-specific fimbrial adhesin of Escherichia coli , FimH, we demonstrate that locking the adhesin in a low-binding conformation abrogates its capability to interact with mannose but, at the same time, facilitates the immune response against binding-pocket epitopes and production of adhesion-inhibitory antibodies. We believe our findings provide insight into strategies to elicit neutralizing and functional antibodies against a broad spectrum of receptor-binding proteins.

Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain: Engineering a minimalistic allosteric system

“…6 and 7) revealed an altered protein conformation in the ligand-free state that was converted to a conformation similar to WT FimH LD upon mannoside addition. In contrast to previously suggested lock in the low-affinity conformation of these mutants (5,38,40), this demonstrates for the first time a considerable conformational change of the isolated FimH LD in the absence of the regulatory pilin domain. For the mutant V67K, designed to destabilize the helical ␣-switch region of the highaffinity FimH LD , binding affinity and kinetics were only mildly affected, suggesting solely a weak coupling between the ␣-switch region and the binding pocket.…”

“…Interestingly, such a conformation accurately resembles the medium-affinity state observed in the crystal structure of the mannoside-bound FimH FL (23). Indirectly, this is evidenced by the observation that an inhibitory antibody raised against the FimH LD mutant V27C/L34C, with an epitope in the binding pocket, strongly binds to FimH in the high-affinity state (40), which displays a nearly identical geometry of the binding pocket compared with the medium-affinity state (23). However, the exact conformation of V27C/L34C and R60P in the bound state remains to be confirmed, e.g.…”

“…Five FimH LD variants were generated with single-or doublepoint mutations within the four allosteric regions assumed to stabilize or even lock FimH in the low-affinity state (5,38,40). The A10P mutation is located in the pocket zipper/clamp loop; R60P and V67K are part of the ␤-bulge and ␣-switch regions, respectively; and A119L and V27C/L34C are located within the interdomain loops (Fig.…”

“…Furthermore, bacterial adhesion is enhanced in the presence of mAb21, which stabilizes the highaffinity state (21). In contrast, a monoclonal blocking antibody directed against the FimH LD mutant V27C/L34C, presumably locked in the low-affinity state, protected mice from bacterial infection, thus demonstrating the relevance of considering all conformational states for the design of therapeutic FimH antagonists (40). Despite the intensive studies of the FimH variants mentioned above, structural, thermodynamic, and kinetic data regarding the low-affinity conformation were not reported so far.…”

Immunogenicity of a prototype enterotoxigenic Escherichia coli adhesin vaccine in mice and nonhuman primates

“…Notably, the actual proteins used in these studies consisted of either a natural adhesion-competent truncate of FimH or a bimolecular complex of the adhesin and its cognate chaperone for both FimH and PapG, unlike our use here of a donor strand complemented derivative of CfaE. Interestingly, recent studies of FimH have demonstrated the existence of low-and high-affinity binding conformations and suggest that locking FimH into its low-affinity state results in enhancement of adhesion-neutralizing antibodies (30,31). To date, structural evidence indicates that dscCfaE exists in a low-affinity conformational state, while a high-affinity state has been postulated but not proven (16,17,32).…”