Escherichia coli's literal link to infection: Exploring mutagenesis of the FimH adhesin to allosterically "lock" in low-affinity

Abstract

Expressed by most commensal and uropathogenic <italic>Escherichia coli (E.coli)</italic>, the protein FimH mediates adhesion via catch–bonds to ligand mannose and has become a significant antigen for antiadhesive therapies targeted at urinary tract infections (UTIs). A possible barrier to therapies is that the FimH crystal structure shows a substantial allosteric conformational change between a low and high–affinity state and it is uncertain which regions contribute to the change. Additionally, previous work strongly suggests that the FimH ligand–binding lectin domain’s (LD’s) stability depends on its interaction with the fimbrial–anchoring pilin domain (PD). This may explain evidence of antibodies raised against the isolated LD biasing the high–affinity state and allosterically <italic>enhancing</italic> FimH adhesion to urothelial cells. My thesis is devoted to the investigation of a “locked” low–affinity FimH for future antiadhesive therapies. First, we used RosettaDesign and human insight to synthesize 10 point mutants in 4 regions that we predicted would stabilize either state. By measuring the binding to mannosyl substrates and preferred conformation with a high–affinity–binding monoclonal antibody (MAb), we demonstrated that each region investigated was part of the FimH allosteric pathway. Second, one particular low–affinity variant, V27C–L34C or “locked-low”, was characterized in new forms that physically altered the PD’s interaction with the LD in order to assess the LD’s independence. Locked–low may be unlocked to increase binding via reducing agent dithiothreitol (DTT). Under native conditions (without soluble ligand and DTT), our investigation discovered that locked–low pili containing an altered PD, FocH, and its isolated LD were unrecognized by high–affinity–binding MAbs and weakly bound mannosyl substrates. Our study strongly suggests the native LD may independently stabilize a low–affinity state. This is also the first LD isolate that switches states via DTT to be useful in future biophysical characterization. Third, we generated mouse–MAbs from the LD isolate and preliminary data indicate these MAbs significantly (p < 0.05) reduce the adhesion of wildtype FimH–expressing <italic>E.coli</italic> to urothelial cells <italic>in vitro</italic>. This work will aide future development of novel antiadhesive therapies designed to inhibit the adhesion of uropathogenic <italic>E.coli</italic>.