Many of the adhesins of Gram-negative bacteria are incorporated into heteropolymeric fibers assembled by the chaperone/usher pathway. These fibers range in morphology from composite pili assembled by members of the FGS (F1G1 short) subfamily of chaperones, to fibrous capsule-like structures assembled by the FGL (F1G1 long) subfamily of chaperones. Here we propose to build on our work on P and type 1 pili, which has provided a paradigm for the assembly of hundreds of virulence fibers in diverse Gram-negative organisms. Using biochemistry, genetics, electron microscopy and X-ray crystallography, we have defined the structure and mechanism of action of the periplasmic chaperones in significant detail. We have solved the structure of the PapD periplasmic chaperone in complex with the tip adaptor subunit, PapK, and with the major tip subunit, PapE. We have also solved the structure of the PapE subunit in complex with the N-terminal extension of PapK and the structure of the receptor binding domain of the PapG adhesin with and without its host carbohydrate ligand. Pilus chaperones are comprised of two immunoglobulin (Ig)-like domains. These structures showed that pilin subunits also have an Ig-like fold, but they are missing their seventh (G) strand, thus exposing the hydrophobic core. In a process we termed donor-strand complementation, the chaperone's G1 strand serves as the pilin's seventh strand, catalyzing the folding of the subunit and preventing non-productive subunit aggregation. At the usher, pilus assembly occurs by donor-strand exchange, in which the G1 strand of the chaperone is replaced by an N-terminal extension that is present on every subunit and exposed on incoming chaperone-subunit complexes. The pilus subunit then undergoes a topological transition that triggers the closure of its groove, cementing its neighbor's NTE as part of its own Ig fold. Thus, the final pilus structure is a series of Ig-like domains, each of which is formed from parts of two individual subunit monomers. Each subunit has distinct specificity for other interactive subunits and distinct roles in pilus assembly. We propose to expand our knowledge of the structures of the pilus adhesins and further study the structure of the subunits and assembly machinery of two such assembly systems: the prototypical FGS P pilus system of uropathogenic Escherichia coli and the Saf FGL system of Salmonella. In addition, we will perform structural studies on anti-chaperone compounds.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI049950-10
Application #
8077256
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Korpela, Jukka K
Project Start
2001-06-01
Project End
2012-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
10
Fiscal Year
2011
Total Cost
$311,172
Indirect Cost
Name
Washington University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
O'Brien, Valerie P; Hannan, Thomas J; Nielsen, Hailyn V et al. (2016) Drug and Vaccine Development for the Treatment and Prevention of Urinary Tract Infections. Microbiol Spectr 4:
Spaulding, Caitlin N; Hultgren, Scott J (2016) Adhesive Pili in UTI Pathogenesis and Drug Development. Pathogens 5:
O'Brien, Valerie P; Hannan, Thomas J; Schaeffer, Anthony J et al. (2015) Are you experienced? Understanding bladder innate immunity in the context of recurrent urinary tract infection. Curr Opin Infect Dis 28:97-105
Kostakioti, Maria; Hadjifrangiskou, Maria; Hultgren, Scott J (2013) Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era. Cold Spring Harb Perspect Med 3:a010306
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Chorell, Erik; Pinkner, Jerome S; Bengtsson, Christoffer et al. (2012) Design and synthesis of fluorescent pilicides and curlicides: bioactive tools to study bacterial virulence mechanisms. Chemistry 18:4522-32
Cusumano, Corinne K; Pinkner, Jerome S; Han, Zhenfu et al. (2011) Treatment and prevention of urinary tract infection with orally active FimH inhibitors. Sci Transl Med 3:109ra115

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