Escherichia coli and other enteric bacteria are a major cause of human diseases. Biofilm formation contributes greatly to bacterial persistence and antimicrobial resistance in the host. Many enteric bacteria, including E. coli, produce functional amyloid fibers called curli as a major proteinaceous component of their extracellular matrix. It is now clear that functional amyloids are widespread, with examples found throughout cellular life. The curli system in E. coli provides a rich and high throughput genetic and biochemical toolbox for the study of amyloid formation. We want to learn how E. coli controls curli amyloid formation with protein inhibitors and how we can interrogate curli amyloid formation with rationally-designed chemical chaperones. We hypothesize that E. coli utilizes periplasmic proteins to shuttle the curli subunits through the periplasmic space and to avoid inappropriate amyloid formation in the periplasm. We have identified two chaperone-like proteins in the E. coli periplasm for which we will characterize the molecular mechanism of their amyloid inhibitory activity. We will also use rationally-designed small molecules to investigate the biochemical stage(s) at which curli amyloid formation can be stopped. Knowledge gained from the following experiments will have implications for microbial pathogenesis, general protein folding, and amyloid biogenesis, thus paving the way for new therapies that rationally target these critical biological processes. Our previous discoveries have contributed to a curli assembly model where the main fiber component CsgA and the minor subunit CsgB are secreted through the outer membrane via the lipoprotein CsgG. CsgB attaches to the surface of the cell and templates the folding of CsgA into an amyloid fiber. CsgE, an accessory protein with chaperone-like activity against CsgA, is also required for curli subunit secretion. CsgA subunits that might inappropriately polymerize in the periplasm are inhibited from amyloid accumulation via CsgC. In order to rationally develop therapeutics against amyloid assembly and amyloid-dependent biofilm formation, we must better understand curli biogenesis and its function in biofilm development.
In Aim 1 the coordinated roles of the chaperone-like protein CsgE and the outer membrane lipoprotein CsgG in directing efficient CsgA transport through the periplasm will be explored. We will also determine not only how CsgE interacts with CsgA and other amyloidogenic proteins but also the consequences of these interactions.
In Aim 2 we will characterize the interaction and specificity of the potent anti-amyloid factor CsgC. We will determine how CsgC functions to inhibit CsgA and ?-synuclein polymerization at low stoichiometric ratios. Finally, in Aim 3 we will characterize small molecules with amyloid-inhibiting capabilities. In collaboration with Fredrik Almqvist at Ume University in Sweden, we have already identified molecules that discourage CsgA polymerization. We will further characterize how these peptidomimetic compounds inhibit amyloid polymerization, assess their specificity, and test for their ability to inhibit biofilm formation.

Public Health Relevance

Escherichia coli and related enteric bacteria are a major causative agent of infections worldwide. During pathogenesis, E. coli enters into a protective biofilm state that can resist host immune defenses and antibiotic therapies. We propose to determine how E. coli assembles and utilizes a critical biofilm determinant called curli to inspire new approaches for combating infectious disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM118651-02
Application #
9244843
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Wehrle, Janna P
Project Start
2016-04-01
Project End
2020-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Smith, Daniel R; Price, Janet E; Burby, Peter E et al. (2017) The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of Escherichia coli. Biomolecules 7:
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
Hufnagel, David A; Price, Janet E; Stephenson, Rachel E et al. (2017) Thiol Starvation induces redox-mediated dysregulation of Escherichia coli biofilm components. J Bacteriol :
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
Cremers, Claudia M; Knoefler, Daniela; Gates, Stephanie et al. (2016) Polyphosphate: A Conserved Modifier of Amyloidogenic Processes. Mol Cell 63:768-80
Floyd, Kyle A; Mitchell, Courtney A; Eberly, Allison R et al. (2016) The UbiI (VisC) Aerobic Ubiquinone Synthase Is Required for Expression of Type 1 Pili, Biofilm Formation, and Pathogenesis in Uropathogenic Escherichia coli. J Bacteriol 198:2662-72
Horowitz, Scott; Koepnick, Brian; Martin, Raoul et al. (2016) Determining crystal structures through crowdsourcing and coursework. Nat Commun 7:12549
Taylor, Jonathan D; Hawthorne, William J; Lo, Joanne et al. (2016) Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones. Sci Rep 6:24656