MP24-04 THE UROPATHOGENIC ESCHERICHIA COLI PILUS USHER CONTROLS PROTEIN INTERACTIONS VIA DOMAIN MASKING AND IS FUNCTIONAL AS AN OLIGOMER

Abstract

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I1 Apr 2016MP24-04 THE UROPATHOGENIC ESCHERICHIA COLI PILUS USHER CONTROLS PROTEIN INTERACTIONS VIA DOMAIN MASKING AND IS FUNCTIONAL AS AN OLIGOMER Glenn Werneburg, Hemil Chauhan, Nadine Henderson, and David Thanassi Glenn WerneburgGlenn Werneburg More articles by this author , Hemil ChauhanHemil Chauhan More articles by this author , Nadine HendersonNadine Henderson More articles by this author , and David ThanassiDavid Thanassi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.760AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urinary tract infections (UTIs), the majority of which are caused by E. coli, are among the most common and costly human infections. Uropathogenic E. coli strains use the chaperone/usher (CU) pathway to construct surface structures termed pili, adhesive organelles that facilitate binding to bladder or kidney epithelial cells, thus promoting colonization and infection. The CU pilus biogenesis pathway requires a periplasmic chaperone protein and an outer membrane protein, the usher. We sought to gain a mechanistic understanding of how the usher protein catalyzes the assembly of pili and identify new opportunities for therapeutics designed to target specific steps of the process. METHODS We monitored pilus subunit interactions with various usher domains using fluorescence approaches and various biochemical and genetic techniques. RESULTS The usher (FimD), an oligomer, has 5 domains: a periplasmic N-terminal domain (N), a transmembrane channel domain gated by an internal plug domain, and two periplasmic C-terminal domains (C1, C2). We identified the C domains as the high affinity binding site of FimD, and confirmed that the N domain provides an additional, lower affinity, binding site. We show that the C domains are ″masked″ by the plug domain prior to FimD activation. Upon activation by pilus subunit binding to the usher′s N domain, the plug domain undergoes a drastic positional shift, freeing the high affinity C domains for subunit binding. We demonstrate that a lectin domain on a pilus subunit is necessary and sufficient for plug expulsion. Our data indicate that the pilus subunit is then handed off from the N to the (now free) C domains in a process driven by differential affinity. Concurrent with this handoff, the growing pilus fiber is then secreted to an extracellular position where it can adhere to the urinary tract and facilitate UTI. Further, in vivo we show that FimD is functional in trans, suggesting a basis for function of the usher oligomer. CONCLUSIONS Our study identified novel and discrete aspects of pilus assembly, each of which could potentially be targeted by a novel pharmaceutical strategy. A small molecule inhibitor that effectively prevents usher activation, N-to-C handoff, or affinity differential, could serve as an anti-infective drug. This therapeutic would prevent infection by disrupting cell adhesion, but without killing bacteria, and thus would not select for antibiotic-resistant strains. Our fluorescence assays are poised for adaptation as tools for screening large libraries of molecules to identify such a drug. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e271 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Glenn Werneburg More articles by this author Hemil Chauhan More articles by this author Nadine Henderson More articles by this author David Thanassi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...