Many chronic/recurrent human infectious disease states are recognized to involve bacterial biofilms. These include urinary tract infections (UTIs), chronic skin infections, otitis media and cystic fibrosis lung infections. In each, bacterial communities form, encased in an extracellular matrix where they are shielded from host immune mechanisms and from the action of currently used antibiotics. This greatly complicates the clearance of these organisms and the ability to cure these infections. Amyloid fibers called curli are critical in the formation of a biofilm extracellular matrix by uropathogenic Escherichia coli (UPEC). This fiber is made up of the major subunit CsgA, and a minor subunit CsgB. Using Thioflavin T fluorescence, circular dichroism and atomic force microscopy the in vitro mechanism by which CsgB nucleates CsgA fibrillization has been elucidated. The methods used in these studies will serve as a model for examining interactions between other components of the curli system. In vivo, the formation of curli amyloid fibers is directed by specific chaperone-like proteins (CsgE and CsgF) to occur extracellularly at the CsgG assembly site in the outer membrane. These proteins also serve to restrict premature, intracellular amyloid fibrillization, which otherwise would be toxic. In this proposal we utilize a combination of biophysical, biochemical, structural, genetic and chemical genetic methodologies to elucidate the structure, function and mechanism of action of the chaperone-like proteins CsgE, CsgF and the outer membrane assembly protein CsgG in facilitating the CsgB-templated fibrillization of CsgA. These studies will elucidate basic principles of curli formation and more generally amyloid formation. Then, using a thiazole ring-fused 2-pyridone scaffold we will investigate the mechanism of action of small molecule inhibitors (""""""""curlicides"""""""") of curli assembly and curli-mediated biofilm formation. Curlicides will be used as molecular scalpels to dissect the details of the protein-protein interactions required for curli biogenesis. Curli promote biotic and abiotic surface colonization, stabilize cell-cell contacts allowing cell aggregation and thickening of the biofilm layer and confer resistance to the biofilm against environmental stresses and biocides. Thus, curlicides will be tested for their efficacy in preventing the formation of UPEC biofilms on silicone catheter tubing both in vitro and in a murine model of catheter- associated urinary tract infection. This work will provide key insights into processes necessary for the formation of medically important biofilms and will serve as a model to better understand amyloid plaques important in neurodegenerative disorders.

Public Health Relevance

Curli are important constituents of Escherichia coli biofilms, promoting both adhesion and strength to the extracellular matrix that helps to shield bacteria within the biofilm from immune and antibiotic clearance mechanisms. This proposal focuses on elucidating the structure, function and mechanism of action of the chaperone-like proteins necessary for assembly of these amyloid fibers and proposes inhibitors of curli/amyloid formation.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Korpela, Jukka K
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Washington University
Schools of Medicine
Saint Louis
United States
Zip Code
Klein, Roger D; Shu, Qin; Cusumano, Zachary T et al. (2018) Structure-Function Analysis of the Curli Accessory Protein CsgE Defines Surfaces Essential for Coordinating Amyloid Fiber Formation. MBio 9:
Wang, Hanliu; Shu, Qin; Rempel, Don L et al. (2017) Understanding Curli Amyloid-Protein Aggregation by Hydrogen-Deuterium Exchange and Mass Spectrometry. Int J Mass Spectrom 420:16-23
Jain, Neha; Ådén, Jörgen; Nagamatsu, Kanna et al. (2017) Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin. Proc Natl Acad Sci U S A 114:12184-12189
Wang, Hanliu; Shu, Qin; Frieden, Carl et al. (2017) Deamidation Slows Curli Amyloid-Protein Aggregation. Biochemistry 56:2865-2872
Shu, Qin; Krezel, Andrzej M; Cusumano, Zachary T et al. (2016) Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation. Proc Natl Acad Sci U S A 113:7130-5
Hufnagel, David A; Evans, Margery L; Greene, Sarah E et al. (2016) The Catabolite Repressor Protein-Cyclic AMP Complex Regulates csgD and Biofilm Formation in Uropathogenic Escherichia coli. J Bacteriol 198:3329-3334
Van Gerven, Nani; Klein, Roger D; Hultgren, Scott J et al. (2015) Bacterial amyloid formation: structural insights into curli biogensis. Trends Microbiol 23:693-706
Wang, Hanliu; Shu, Qin; Rempel, Don L et al. (2015) Continuous and pulsed hydrogen-deuterium exchange and mass spectrometry characterize CsgE oligomerization. Biochemistry 54:6475-81
Goyal, Parveen; Krasteva, Petya V; Van Gerven, Nani et al. (2014) Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG. Nature 516:250-3
Engström, Patrik; Krishnan, K Syam; Ngyuen, Bidong D et al. (2014) A 2-pyridone-amide inhibitor targets the glucose metabolism pathway of Chlamydia trachomatis. MBio 6:e02304-14

Showing the most recent 10 out of 14 publications