Conformational Basis and Small Molecule Antagonists of E. coli Adhesion to the Urinary Tract

Urinary tract infections (UTIs) are one of the most prevalent infections, afflicting 15 million women per year in the United States with annual healthcare costs exceeding $2-3 billion. Uropathogenic Escherichia coli (UPEC) are the main etiological agent of UTIs and employ numerous virulence factors for host colonization. The most common adhesive mechanism by which UPEC mediate host-pathogen interactions is the chaperone-usher pathway (CUP), which is responsible for the assembly of proteinaceous surface appendages termed pili. Generally, CUP pili function in adherence or invasion of host tissues and in biofilm formation on medical devices and body habitats. CUP pili are highly abundant and diverse among a wide variety of Gram-negative pathogens, with 38 distinct pilus types in Escherichia species alone, mediating a considerable range of biological tropisms through adhesins at the distal pilus tip. Typically, these adhesins have a lectin domain, which recognizes a specific carbohydrate receptor, and a pilin domain to anchor the adhesin to the pilus. This thesis specifically examines the structural, dynamic, and allosteric properties of distinct E. coli CUP pilus adhesins that govern interactions critical for pilus function at the host-pathogen interface during UTI. The type 1 pilus adhesin FimH is a critical virulence factor necessary for bacterial attachment to mannosylated receptors on the bladder epithelium during UTI. I determined through molecular and computational biophysics that FimH natively exists in a two-state conformational equilibrium in solution, composed of one low-affinity tense (T) and multiple high-affinity relaxed (R) conformations. I demonstrated that positively selected residues in FimH and ligand binding allosterically modulate this conformational equilibrium and that each of these conformational states engage mannose receptors through distinct binding modes. Mouse models of UTI indicate that FimH has evolved a ҭoderateӠmannose binding affinity through a balanced conformational equilibrium to optimize persistence in the bladder during UTI. Furthermore, I discovered novel small-molecule galactoside antagonists that inhibit the FimH-like adhesin FmlH from binding galactose-containing bladder and kidney epithelial receptors present during chronic UTI. Taken together, this thesis defines the biophysical basis of host receptor recognition and bacterial pathogenesis mediated by FimH and defines the atomic bases of distinct bacterial host tropisms mediated by FimH homologs, which were leveraged to spur the development of antibiotic-sparing, small-molecule glycomimetic antagonists as therapeutics for UTI and other infectious diseases

mAb Das-1 recognizes 3\u27-Sulfated Lewis A/C, which is aberrantly expressed during metaplastic and oncogenic transformation of several gastrointestinal epithelia

INTRODUCTION: Multiple previous studies have shown the monoclonal antibody Das-1 (formerly called 7E12H12) is specifically reactive towards metaplastic and carcinomatous lesions in multiple organs of the gastrointestinal system (e.g. Barrett\u27s esophagus, intestinal-type metaplasia of the stomach, gastric adenocarcinoma, high-grade pancreatic intraepithelial neoplasm, and pancreatic ductal adenocarcinoma) as well as in other organs (bladder and lung carcinomas). Beyond being a useful biomarker in tissue, mAb Das-1 has recently proven to be more accurate than current paradigms for identifying cysts harboring advanced neoplasia. Though this antibody has been used extensively for clinical, basic science, and translational applications for decades, its epitope has remained elusive. METHODS: In this study, we chemically deglycosylated a standard source of antigen, which resulted in near complete loss of the signal as measured by western blot analysis. The epitope recognized by mAb Das-1 was determined by affinity to a comprehensive glycan array and validated by inhibition of a direct ELISA. RESULTS: The epitope recognized by mAb Das-1 is 3\u27-Sulfo-Lewis A/C (3\u27-Sulfo-LeA/C). 3\u27-Sulfo-LeA/C is broadly reexpressed across numerous GI epithelia and elsewhere during metaplastic and carcinomatous transformation. DISCUSSION: 3\u27-Sulfo-LeA/C is a clinically important antigen that can be detected both intracellularly in tissue using immunohistochemistry and extracellularly in cyst fluid and serum by ELISA. The results open new avenues for tumorigenic risk stratification of various gastrointestinal lesions

Adaptation of arginine synthesis among uropathogenic branches of the Escherichia coli phylogeny reveals adjustment to the urinary tract habitat

Urinary tract infections (UTIs) are predominantly caused by uropathogeni

Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection

Significance The emergence of multidrug-resistant bacteria, including uropathogenic Escherichia coli (UPEC), makes the development of targeted antivirulence therapeutics a critical focus of research. During urinary tract infections (UTIs), UPEC uses chaperone–usher pathway pili tipped with an array of adhesins that recognize distinct receptors with sterochemical specificity to facilitate persistence in various tissues and habitats. We used an interdisciplinary approach driven by structural biology and synthetic glycoside chemistry to design and optimize glycomimetic inhibitors of the UPEC adhesin FmlH. These inhibitors competitively blocked FmlH in vitro, in in vivo mouse UTI models, and in ex vivo healthy human kidney tissue. This work demonstrates the utility of structure-driven drug design in the effort to develop antivirulence therapeutic compounds. </jats:p

Evolutionary fine-tuning of conformational ensembles in FimH during host-pathogen interactions

Positive selection in the two-domain type 1 pilus adhesin FimH enhances Escherichia coli fitness in urinary tract infection (UTI). We report a comprehensive atomic-level view of FimH in two-state conformational ensembles in solution, composed of one low-affinity tense (T) and multiple high-affinity relaxed (R) conformations. Positively selected residues allosterically modulate the equilibrium between these two conformational states, each of which engages mannose through distinct binding orientations. A FimH variant that only adopts the R state is severely attenuated early in a mouse model of uncomplicated UTI but is proficient at colonizing catheterized bladders in vivo or bladder transitional-like epithelial cells in vitro. Thus, the bladder habitat has barrier(s) to R state–mediated colonization possibly conferred by the terminally differentiated bladder epithelium and/or decoy receptors in urine. Together, our studies reveal the conformational landscape in solution, binding mechanisms, and adhesive strength of an allosteric two-domain adhesin that evolved “moderate” affinity to optimize persistence in the bladder during UTI

Structure-function correlates of fibrinogen binding by Acinetobacter adhesins critical in catheter-associated urinary tract infections

Multidrug-resistan

Antivirulence <i>C</i>‑Mannosides as Antibiotic-Sparing, Oral Therapeutics for Urinary Tract Infections

Gram-negative uropathogenic Escherichia coli (UPEC) bacteria are a causative pathogen of urinary tract infections (UTIs). Previously developed antivirulence inhibitors of the type 1 pilus adhesin, FimH, demonstrated oral activity in animal models of UTI but were found to have limited compound exposure due to the metabolic instability of the <i>O</i>-glycosidic bond (<i>O</i>-mannosides). Herein, we disclose that compounds having the <i>O</i>-glycosidic bond replaced with carbon linkages had improved stability and inhibitory activity against FimH. We report on the design, synthesis, and in vivo evaluation of this promising new class of carbon-linked <i>C</i>-mannosides that show improved pharmacokinetic (PK) properties relative to <i>O</i>-mannosides. Interestingly, we found that FimH binding is stereospecifically modulated by hydroxyl substitution on the methylene linker, where the <i>R</i>-hydroxy isomer has a 60-fold increase in potency. This new class of <i>C</i>-mannoside antagonists have significantly increased compound exposure and, as a result, enhanced efficacy in mouse models of acute and chronic UTI