~80% of urinary tract infections (UTIs) are caused by uropathogenic E. coli (UPEC). Adhesion proteins on the pilus-covered surface of UPEC are required for attachment to host cells for colonization and disease progression. The primary adhesion proteins associated with UPEC virulence are FimH and FmlH, which bind D-mannose or D-galactose (respectively) on uroepithelial/kidney cells. Our goal is to develop UTI treatments that prevent the attachment and subsequent internalization of UPEC in host cells, eliminating bacteria from the urinary tract. However, it remains challenging to design long-lived inhibitors that reach the urinary tract without disrupting adhesion in commensal gut bacteria. We are using mirror-image phage display to identify D-peptide inhibitors of UPEC adhesion. Towards this goal, we have synthesized both FimH and FmlH using native chemical ligation of three peptide segments. Both adhesion proteins are densely populated with hydrophobic and negatively charged amino acids, particularly in FimH, making synthesis impractical without the use of removable, solubilizing “Helping Hand” (HH) tags developed by our lab that can be added at Lys or Glu positions. We have developed a folding protocol and confirmed that folded synthetic FimH and FmlH retain the same structure and function, using circular dichroism and sugar binding assays, as their natural counterparts. We are now synthesizing both proteins using D-amino acids to provide D-targets to screen in mirror-image phage display and identify D-peptide inhibitors. These D-peptides will be used to prevent and treat lower and upper UTIs.
