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Structural and Functional Characterization of Outer Membrane Usher Activation in Uropathogenic E. coli
Author(s) -
Omattage Natalie,
Deng Zengqin,
Yuan Peng,
Hultgren Scott J.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.950.4
Subject(s) - periplasmic space , pilus , bacterial outer membrane , chaperone (clinical) , bacterial adhesin , microbiology and biotechnology , biology , protein subunit , escherichia coli , fimbriae proteins , biochemistry , chemistry , gene , medicine , pathology
Gram‐negative bacteria assemble extracellular fibers termed chaperone‐usher pathway (CUP) pili to adhere to host tissues and promote colonization. This has been best studied in the establishment of urinary tract infections (UTIs), which affect 60% of women in their lifetime. Uropathogenic Escherichia coli (UPEC) is the causative agent of 85% of community‐acquired UTIs. UPEC utilize the type 1 pilus, via its tip‐located FimH adhesin, to bind and adhere to mannosylated receptors on the surface of bladder epithelium. Type 1 pilus assembly is an intricate molecular process that requires the passage and polymerization of hundreds of subunits across both the inner and outer membranes of the bacterium. To accomplish this, the dedicated periplasmic chaperone, FimC, binds to and facilitates folding and maintenance of pilus subunits in a high‐energy conformation suitable for polymerization; and the outer membrane usher, FimD, serves as a multi‐domain assembly platform to catalyze subunit polymerization. Pilus assembly is initiated by the high‐affinity binding of the chaperone‐adhesin complex (FimCH) to the N‐terminal periplasmic domain of FimD. By some unknown mechanism, this binding event results in the “activation” of the usher for its catalytic activity. Usher activation results in marked rearrangements within the transmembrane and periplasmic FimD domains, but it remains unclear how chaperone‐adhesin binding to FimD triggers these conformational changes. We have conducted extensive structure‐function studies to gain insight into the molecular mechanism by which the chaperone‐adhesin primes the FimD usher for activation. Using mutagenesis and in vivo functional assays, we have identified interaction interfaces important for usher activation. We have developed systems for large‐scale expression and purification of the FimD usher and for reconstitution of pilus assembly intermediates in vitro. We are currently characterizing the structural basis for chaperone‐adhesin complex engagement of the N‐terminal domain of the FimD usher using X‐ray crystallography, single particle cryo‐electron microscopy (cryo‐EM) and small angle X‐ray scattering (SAXS) methods. Taken together, these biochemical and biophysical studies will provide unprecedented insight into how allostery primes the usher and drives pilus biogenesis. Ultimately, these studies have the potential to inform the development of innovative small molecules that block events at the outer membrane usher and prevent assembly of one of the bacterium's major virulence factors. Support or Funding Information NSF GRFP DGE‐1143954 (NSO) and NIH R01 AI029549‐24 (SJH)