Urinary tract infections (UTIs) are among the most common bacterial infections, affecting 150 million people worldwide each year. In the USA, community-acquired UTIs account for about 11 million cases each year that cost the U.S. public health budget $5 billion annually. Uropathogenic Escherichia coli (UPEC) accounts for up to 80% of UTIs. While UTIs are currently treated with antibiotics, the frequency of multi-drug resistance is increasing, portending a future of untreatable UTIs. A promising alternative to antibiotics is to directly target bacterial virulence factors that are critical for bacterial adhesion to and invasion of uroepithelial cells. These virulence factors include the bacterial adhesin FimH, expressed by the majority of uropathogenic E. coli strains. FimH binds to a specific glycoform, oligomannose-3, carried by an abundant membrane glycoprotein on uroepithelial cells. We have produced a recombinant protein targeting FimH. It is a molecular fusion of human protein, dipeptidyl peptidase 4 (DPP4) with IgG1 Fc (DPP4-Fc), carries eight mannose-containing N-glycans and binds in a mannose-specific manner to FimH. We have shown that that DPP4-Fc promotes killing of uropathogenic E. coli by complement. The purpose of this research project is to demonstrate the ability of DPP4-Fc to promote activation of complement on the surface of antibiotic-resistant uropathogenic E. coli thereby leading to increased bacterial killing by complement and polymorphonuclear cells (PMNs). We will produce three new variants of DPP4-Fc carrying different numbers of N-glycans per monomer and different glycoforms. We will modify the Fc of DPP4-Fc to improve C1q binding and thus increase activation of the classical complement pathway. Modifications will include point mutations and replacement of IgG1 Fc with IgG3 Fc. We will also produce a negative control DPP4-Fc in which the Fc contains two point mutations that abrogate C1q binding and thus has no opsonizing activity. We will test all these new DPP4-Fc variants for their ability to: i) bind to uropathogenic E.coli, Enterobacter and Klebsiella pneumonia strains, ii) inhibit bacterial attachment to urothelial cells, iii) promote serum mediated killing of bacteria and iv) promote opsonophagocytosis of bacteria. We expect that DPP4-Fc can be developed into a novel treatment for uropathogenic E. coli, Enterobacter and Klebsiella infections. Future research projects will test the activity of these molecules in animal models and later in human clinical trials.
Public Health Relevance Statement: Urinary tract infections are among the most common bacterial infections, of which up to 80% are caused by E. coli. At increasing frequency, these pathogens are resistant to most or even all last line antibiotics. We have devised a hybrid protein combining a protein fragment that can block bacterial cell attachment with an immune defense-activating portion of human antibody. We will demonstrate the ability of this hybrid to facilitate killing multi-drug resistant uropathogenic E. coli, Enterobacter and Klebsiella pneumoniae.
Project Terms: Adhesions; Affect; Animal Model; Antibiotic Resistance; Antibiotics; Antibodies; Bacteria; Bacterial Adhesins; Bacterial Adhesion; Bacterial Attachment Site; Bacterial Infections; bacterial resistance; Binding; Binding Proteins; Biological Assay; Budgets; C3bi; Cell Surface Receptors; Cell-Matrix Junction; Cells; Classical Complement Pathway; Clinical Trials; community-acquired UTI; Complement; Complement 1q; Complement 3b; Complement Activation; Complement Membrane Attack Complex; Complement-Dependent Cytotoxicity; cost; Dipeptidyl Peptidases; Enterobacter; Enterobacteriaceae; Escherichia coli; Escherichia coli Infections; Frequencies; Future; Galactose; glycosylation; Goals; Human; hybrid protein; Hybrids; IgG1; IgG3; Immune; Immunoglobulin G; improved; In Vitro; in vitro activity; Klebsiella; Klebsiella Infections; Klebsiella pneumoniae; Macrophage-1 Antigen; Mannose; Mediating; Membrane; Membrane Glycoproteins; Modification; Molecular; monomer; Multi-Drug Resistance; N-terminal; neutrophil; novel; pandemic disease; pathogen; pathogenic bacteria; Phagocytes; Phagocytosis; Phase; Plants; Point Mutation; Polysaccharides; Protein Fragment; Proteins; Public Health; Recombinant Proteins; Recombinants; Research Project Grants; Resistance; resistant strain; Serum; Small Business Innovation Research Grant; Surface; Testing; type 1 fimbriae; Urinary tract infection; Uropathogenic E. coli; Urothelial Cell; Urothelium; Variant; Virulence Factors
